Dynamics of Organic Acids during the Droplet-Vitrification Cryopreservation Procedure Can Be a Signature of Oxidative Stress in Pogostemon yatabeanus.

Cryopreservation in liquid nitrogen (LN, -196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60-62%) > regrowth medium (40-64%) > no osmoprotection, cryopreservation in vials (28-30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials.

Influence of Intramuscular Injection Sites on Pharmacokinetics of Amoxicillin in Olive Flounder (Paralichthys olivaceus) and Its Implication for Antibacterial Efficacy.

This study aimed to investigate the effects of different injection sites, including dorsal, cheek, and pectoral fin muscles, on the pharmacological properties of amoxicillin (AMOX) in olive flounder (Paralichthys olivaceus) after a single intramuscular (IM) injection of 40 mg/kg. The AMOX concentration was measured using high-performance liquid chromatography-tandem mass spectrometry, followed by a non-compartmental model analysis. The peak serum concentrations (Cmax) achieved 3 h after dorsal, cheek, and pectoral fin IM injections were 202.79, 203.96, and 229.59 µg/mL, respectively. The area under the concentration-time curve (AUC) was 1697.23, 2006.71, and 1846.61 µg/mL·h, respectively. The terminal half-life (t1/2λZ) was prolonged for cheek and pectoral fin IM injections (10.12 and 10.33 h, respectively) compared to dorsal IM injection (8.89 h). In the pharmacokinetic-pharmacodynamic analysis, a higher T > minimum inhibitory concentration (MIC) and AUC/MIC values were observed after AMOX was injected into the cheek and pectoral fin muscles compared to the dorsal muscle. Muscle residue depletion was below the maximum residue level from day 7 after IM injection at all three sites. These findings suggest that the cheek and pectoral fin sites provide advantages regarding systemic drug exposure and prolonged action compared with the dorsal site.

Alternative Plant Vitrification Solution A3-80% and Initial Ammonium-Free Regrowth Medium Enable Cryobanking of Chrysanthemum Germplasm.

Cryopreservation, storing biological material in liquid nitrogen (LN, -196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L-1 gibberellic acid (GA3) and 1 mg L-1 benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method.

Vigorous Growing of Donor Plantlets by Liquid Overlay in Subcultures Is the Key to Cryopreservation of Endangered Species Pogostemon yatabeanus.

Cryopreservation is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. However, the wide application of cryopreservation for the protection of wild flora is hampered by some reasons: limits of source material available, difficulties in in vitro propagation, needs to re-optimize protocol steps for new species, etc. In this study, using an endemic and endangered Korean species, Pogostemon yatabeanus, we investigated subculture medium and supplements on in vitro growth of donor plants: medium strength, gelling agents, liquid overlay, plant hormones, and activated charcoal. Subculture conditions of each cycle tested significantly impacted on height and dry weight of subcultured donor plantlets. Among the treatments tested, the overlay of the liquid medium on top of gellan gum-gelled medium significantly increased the growth of shoots and roots. In the droplet-vitrification procedure, the survival and regeneration of cryopreserved shoot tips were critically impacted by the dry weight of donor plantlets (CORELL = 0.85~0.95) which was affected by the following subculture conditions. Moreover, every subsequent subculture cycle before cryopreservation positively or negatively impacted post-cryopreservation regeneration. This study highlights the vigor of donor plantlets for post-cryopreservation regeneration and provides practices for the revitalization of donor plants during subcultures.

In Vitro Multiplication and Cryopreservation of Penthorum chinense Shoot Tips.

This study provides alternative approaches toward ex situ conservation by means of in vitro seed germination and the multiplication of Penthorum chinense Pursh using nodal explants. An overlay of a liquid medium on top of a gelled medium significantly increased the growth of shoots and roots, while the presence of activated charcoal or growth regulators (benzyl adenine and α-naphthaleneacetic acid) decreased the growth. Shoot tips of in vitro plantlets were cryopreserved using a droplet-vitrification method. The standard procedure included preculture with 10% sucrose for 31 h and with 17.5% sucrose for 17 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 20 min, cryoprotection with alternative plant vitrification solution (PVS) A3-70% (29.2% glycerol + 11.7% DMSO + 11.7% EG + 17.4% sucrose, w/v) at 0 °C for 30 min, cooling the samples in liquid nitrogen using aluminum foil strips and rewarming by plunging into pre-heated (40 °C) unloading solution (35% sucrose) for 40 min. A three-step regrowth procedure starting with ammonium-free medium followed by ammonium-containing medium with and without growth regulators was essential for the regeneration of cryopreserved shoot tips. The species was found to be very sensitive to the chemical cytotoxicity of permeating cryoprotectants during cryoprotection and to ammonium-induced oxidant stress during initial regrowth steps. Improvement of donor plant vigor by using apical sections and liquid overlay on top of the solid medium for propagation, improved shoot tip tolerance to osmotic stress and increased post-cryopreservation regeneration up to 64% were observed following PVS B5-85% (42.5% glycerol + 42.5% sucrose) treatment for 60 min. The systematic approach used in this study enables fast optimization of the in vitro growth and cryopreservation procedure for a new stress-sensitive wild plant species.

First Report of Enterocytozoon hepatopenaei Infection in Giant Freshwater Prawn (Macrobrachium rosenbergii de Man) Cultured in the Republic of Korea.

In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns.

Sensitive Affinity-Based Biosensor Using the Autocatalytic Activation of Trypsinogen Mutant by Trypsin with Low Self-activation.

Self-propagating autocatalytic reactions of proteases that can provide high signal amplification have not been applied to affinity-based biosensors owing to the limited number of fast autocatalytic proteolytic reactions available and the self-activation of protease proenzymes. Here, we report that a self-propagating autocatalytic reaction based on the autocatalytic activation of the trypsinogen mutant by trypsin facilitates high signal amplification and a low background level, resulting in a low detection limit for prostate-specific antigen (PSA). A commercially available trypsinogen mutant minimizes the self-activation of trypsinogen by trypsinogen. Trypsin, which is used as a catalytic label in a sandwich-type immunosensor, converts the trypsinogen mutant into trypsin; the generated trypsin then further converts the trypsinogen mutant into trypsin. The autocatalytically produced trypsin proteolytically cleaves the peptide bond of a trypsin substrate, resulting in the liberation of electrochemically active 4-aminophenol (AP). The electrochemical oxidation of AP at a modified indium tin oxide (ITO) electrode induces electrochemical-chemical redox cycling involving the ITO electrode, AP, and a reductant. The triple combination of autocatalytic activation, proteolytic cleavage, and redox cycling results in a high electrochemical signal level. The detection limit for PSA obtained using a trypsin label and trypsinogen (∼7 pg/mL) is lower than that obtained using a trypsin label alone (∼100 pg/mL). This study demonstrated that autocatalytically activating a proenzyme is a very useful method for highly amplifying signals.

Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators.

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, Pogostemon yatabeanus. This approach consisted of a standard procedure pre-selected based on material type and size, which was complemented with 11 additional treatments to reveal the most impactful conditions. Effect of ammonium nitrate at various protocol steps was also tested. The highest shoot tip survival (92%) and plant regeneration (90%) after cryopreservation were achieved using preculture with 10% sucrose followed by 40 min osmoprotection and 60 min treatment with vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose) on ice. A three-step regrowth procedure starting with ammonium-free medium with 1 mg/L GA3 and 1 mg/L BA followed by ammonium-containing medium with and without growth regulators was essential for the development of healthy plants from cryopreserved shoot tips. This approach enables fast optimization of the cryopreservation procedure for new osmotic stress-sensitive plant species.

Astrocytes phagocytose adult hippocampal synapses for circuit homeostasis.

In the adult hippocampus, synapses are constantly formed and eliminated1,2. However, the exact function of synapse elimination in the adult brain, and how it is regulated, are largely unknown. Here we show that astrocytic phagocytosis3 is important for maintaining proper hippocampal synaptic connectivity and plasticity. By using fluorescent phagocytosis reporters, we find that excitatory and inhibitory synapses are eliminated by glial phagocytosis in the CA1 region of the adult mouse hippocampus. Unexpectedly, we found that astrocytes have a major role in the neuronal activity-dependent elimination of excitatory synapses. Furthermore, mice in which astrocytes lack the phagocytic receptor MEGF10 show a reduction in the elimination of excitatory synapses; as a result, excessive but functionally impaired synapses accumulate. Finally, Megf10-knockout mice show defective long-term synaptic plasticity and impaired formation of hippocampal memories. Together, our data provide strong evidence that astrocytes eliminate unnecessary excitatory synaptic connections in the adult hippocampus through MEGF10, and that this astrocytic function is crucial for maintaining circuit connectivity and thereby supporting cognitive function.

Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System.

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called "cFork (see fork)", includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.

Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions.

Postsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

Clinical Characteristics Associated with Electrocardiographic Left Ventricular Hypertrophy in Clinical Normotensives without a History of Hypertension: a Cross-Sectional Study.

BACKGROUND: This study evaluated factors independently associated with electrocardiographic left ventricular hypertrophy (ECG-LVH) in subjects who were normotensive on clinical measurement and had no prior history of hypertension. METHODS: This cross-sectional study analyzed cases and controls in the Comprehensive Medical Examination Center of Hallym University Sacred Heart Hospital. Eligible case participants presented ECG-LVH according to the Sokolow-Lyon or Cornell criteria, were normotensive on clinical measurement, and had never received a diagnosis of hypertension. The control group comprised subjects with normal sinus rhythm who were normotensive on clinical measurement with no history of hypertension. RESULTS: A multiple logistic regression model showed male sex, age and systolic blood pressure to be positively related to the presence of ECG-LVH. A positive relation of smoking and regular exercise; an inverse relation of pulse rate to the presence of ECG-LVH were found only in men. An inverse relation of uric acid level was found only in women. Detailed analyses of relatively healthy and young men according to whether or not to exercise regularly showed that positive relations of age and systolic blood pressure; an inverse relation of obesity to the presence of ECG-LVH were apparent in the non-regular exercise group but not in the regular exercise group. In the regular exercise group, only pulse rate showed significant (inverse) association with the presence of ECG-LVH. CONCLUSION: The varying risk factor profiles associated with ECG-LVH according to sex and the participation in regular exercise may help to elucidate the ECG-LVH in clinical normotensives with no prior history of hypertension.

Anterior cingulate cortex and its input to the basolateral amygdala control innate fear response.

Prefrontal brain areas are implicated in the control of fear behavior. However, how prefrontal circuits control fear response to innate threat is poorly understood. Here, we show that the anterior cingulate cortex (ACC) and its input to the basolateral nucleus of amygdala (BLA) contribute to innate fear response to a predator odor in mice. Optogenetic inactivation of the ACC enhances freezing response to fox urine without affecting conditioned freezing. Conversely, ACC stimulation robustly inhibits both innate and conditioned freezing. Circuit tracing and slice patch recordings demonstrate a monosynaptic glutamatergic connectivity of ACC-BLA but no or very sparse ACC input to the central amygdala. Finally, our optogenetic manipulations of the ACC-BLA projection suggest its inhibitory control of innate freezing response to predator odors. Together, our results reveal the role of the ACC and its projection to BLA in innate fear response to olfactory threat stimulus.

Association of Time to First Morning Cigarette and Chronic Obstructive Pulmonary Disease Measured by Spirometry in Current Smokers.

BACKGROUND: Time to first cigarette after waking is an indicator of nicotine dependence. We aimed to identify the association between time to first cigarette and spirometry-proven obstructive respiratory impairment, especially chronic obstructive pulmonary disease, in current smokers. METHODS: We included 392 subjects who visited the comprehensive medical examination center of Hallym University Sacred Heart Hospital between July 2014 and September 2015. Subjects with lung disease or anemia were excluded. Obstructive pulmonary impairment was defined as <70% of the predicted value of forced expiratory volume in 1 second/forced vital capacity. Subjects were classified into the early (≤30 minutes) and late (>30 minutes) groups based on the time to first cigarette. Logistic regression and linear regression analyses were used for data analysis. RESULTS: Ninety-eight subjects (25%) were classified into the early group. After adjusting for smoking behaviors (cigarettes per day and smoking duration), socioeconomic status (education and income), age, and physical activity, an early time to first cigarette was found to be associated with an increased risk of obstructive pulmonary impairment measured using spirometry (adjusted odds ratio, 2.84; 95% confidence interval, 1.22-6.61). CONCLUSION: Compared to current smokers with a late time to first cigarette, those with an early time to first cigarette had a higher risk of obstructive pulmonary impairment, especially chronic obstructive pulmonary disease. Classifying smoking-related behaviors, especially time to first cigarette, may help target clinical screening for chronic obstructive pulmonary disease.

Antioxidant effects of Geranium nepalense ethanol extract on H2O2-induced cytotoxicity in H9c2, SH-SY5Y, BEAS-2B, and HEK293.

Oxidative damage leads to many diseases. In this study, we evaluated the antioxidant effects of 70% ethanol extract of Geranium nepalense (GNE) on hydrogen peroxide-induced cytotoxicity in cell lines: H9c2, SH-SY5Y, HEK293, and BEAS-2B. We determined the free radical scavenging activity of GNE using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and superoxide dismutase-like activities, as well as the polyphenol and flavonoid contents of GNE. The results showed that GNE scavenged DPPH and ABTS radicals in a dose-dependent manner. In addition, it contained abundant contents of total polyphenol and flavonoid contents and strongly suppressed cellular reactive oxygen species, thereby protecting H2O2-induced cytotoxicity in H9c2, SH-SY5Y, HEK293, and BEAS-2B cell lines. The powerful antioxidant activity exhibited by GNE, both in vitro and in cell systems, was attributed to its free radical scavenging activity. Therefore, GNE may be useful in preventing oxidative stress-induced diseases including Alzheimer's disease, respiratory inflammatory disease, and chronic kidney diseases.