Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

Antiviral Activities of Asarones and Rhizomes of Acorus gramineus on Murine Norovirus.

Noroviruses (NVs) are a major cause of foodborne diseases worldwide. The rhizomes of Acorus gramineus (AGR) have been used as a traditional medicinal plant and a food additive. In this study, AGR and its bioactive components-α-asarone and ß-asarone-showed significant antiviral activities against murine NV (MNV) with pre-treatment, with more than two log reductions in viral plaques. They also demonstrated strong inhibition on binding to A- and O-type saliva by the recombinant P domain derived from human NV (HuNV) GII.4. Both α- and ß-asarones also inhibited the binding of the P domain to the receptor at 0.125-1 mM in a concentration-dependent manner and induced a marked reduction in Tm, suggesting that they may reduce structural stability and block receptor binding by the P domain. In simulated digestive conditions, the AGR extract, α-asarone, or ß-asarone further showed a significant reduction of MNV plaques by 1.5-2.8 logs. The asarones show a potential for development as a scaffold for anti-NV agents.

Relationship between Regular Green Tea Intake and Osteoporosis in Korean Postmenopausal Women: A Nationwide Study.

Mixed results have been reported regarding whether habitual tea intake affects bone health. This study investigated the relationship between green tea intake and bone mineral density (BMD) in postmenopausal Korean women. We used data from the Korean National Health and Nutrition Examination Surveys from 2008 to 2011 and divided the participants into three groups according to their frequency of green tea intake over the past 12 months. BMD of the lumbar spine, total femur, and femur neck was measured using dual-energy X-ray absorptiometry. The odds ratios (ORs) and 95% confidence intervals (CIs) of osteoporosis and osteopenia according to green tea consumption were analyzed. Participants who did not consume green tea or consumed less than one cup per day were more likely to have osteopenia of the lumbar spine or femur than those who consumed it once to three times a day (OR 1.81 and 1.85, 95% CI, 1.20-2.71; and 1.23-2.77). Moreover, ORs for osteoporosis were 1.91 (95% CI 1.13-3.23) and 1.82 (95% CI 1.09-3.05) in non-consumers and consumers who drank less than one cup per day, respectively, compared with the reference group. These results support that green tea consumption may have benefits on bone health.

Inhibitory Effects of Laminaria japonica Fucoidans Against Noroviruses.

Norovirus is the leading cause of nonbacterial foodborne disease outbreaks. Human noroviruses (HuNoVs) bind to histo-blood group antigens as the host receptor for infection. In this study, the inhibitory effects of fucoidans from brown algae, Laminaria japonica (LJ), Undaria pinnatifida and Undaria pinnatifida sporophyll, were evaluated against murine norovirus (MNoV), feline calicivirus (FCV) and HuNoV. Pretreatment of MNoV or FCV with the fucoidans at 1 mg/mL showed high antiviral activities, with 1.1 average log reductions of viral titers in plaque assays. They also showed significant inhibition on the binding of the P domains of HuNoV GII.4 and GII.17 to A- or O-type saliva and the LJ fucoidan was the most effective, reaching 54-72% inhibition at 1 mg/mL. In STAT1-/- mice infected with MNoV, oral administration of the LJ fucoidan, composed of mainly sulfated fucose and minor amounts of glucose and galactose, improved the survival rates of mice and significantly reduced the viral titers in their feces. Overall, these results provide the LJ fucoidan can be used to reduce NoV outbreaks.

Divalent cation-induced conformational changes of influenza virus hemagglutinin.

Divalent cations Cu2+ and Zn2+ can prevent the viral growth in mammalian cells during influenza infection, and viral titers decrease significantly on a copper surface. The underlying mechanisms include DNA damage by radicals, modulation of viral protease, M1 or neuraminidase, and morphological changes in viral particles. However, the molecular mechanisms underlying divalent cation-mediated antiviral activities are unclear. An unexpected observation of this study was that a Zn2+ ion is bound by Glu68 and His137 residues at the head regions of two neighboring trimers in the crystal structure of hemagglutinin (HA) derived from A/Thailand/CU44/2006. The binding of Zn2+ at high concentrations induced multimerization of HA and decreased its acid stability. The acid-induced conformational change of HA occurred even at neutral pH in the presence of Zn2+. The fusion of viral and host endosomal membranes requires substantial conformational changes in HA upon exposure to acidic pH. Therefore, our results suggest that binding of Zn2+ may facilitate the conformational changes of HA, analogous to that induced by acidic pH.

Author Correction: Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

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Supersaturation-controlled microcrystallization and visualization analysis for serial femtosecond crystallography.

Time-resolved serial femtosecond crystallography with X-ray free electron laser (XFEL) holds the potential to view fast reactions occurring at near-physiological temperature. However, production and characterization of homogeneous micron-sized protein crystals at high density remain a bottleneck, due to the lack of the necessary equipments in ordinary laboratories. We describe here supersaturation-controlled microcrystallization and visualization and analysis tools that can be easily used in any laboratory. The microcrystallization conditions of the influenza virus hemagglutinin were initially obtained with low reproducibility, which was improved by employing a rapid evaporation of hanging drops. Supersaturation-controlled microcrystallization was then developed in a vapor diffusion mode, where supersaturation was induced by evaporation in hanging drops sequentially for durations ranging from 30 sec to 3 min, depending on the protein. It was applied successfully to the microcrystal formation of lysozyme, ferritin and hemagglutinin with high density. Moreover, visualization and analysis tools were developed to characterize the microcrystals observed by light microscopy. The size and density distributions of microcrystals analyzed by the tools were found to be consistent with the results of manual analysis, further validated by high-resolution microscopic analyses. Our supersaturation-controlled microcrystallization and visualization and analysis tools will provide universal access to successful XFEL studies.

Conformational modulation of influenza virus hemagglutinin: characterization and in vivo efficacy of monomeric form.

Mutational changes that mostly occur at the head region of hemagglutinin (HA) lead to the emergence of new epidemic influenza viruses, whereas HA antigens have been modified to generate broadly neutralizing antibodies toward highly conserved epitopes in the HA stem. Interestingly, a recent analysis of serum antibody repertoires showed that broadly neutralizing antibodies bind to HA monomer at a conserved region occluded at the intermonomer interface of HA trimer and confer protection in animal models. We showed previously that the recombinant HA ectodomain from a pandemic strain A/Korea/01/2009 was monomeric in solution and crystal structure. In order to examine the potential antigenicity of a monomeric form, we designed HA monomer that incorporates mutations to destabilize trimer conformations. Starting with the HA trimer from a seasonal strain A/Thailand/CU44/2006, mutations were introduced at the intermonomer interface, Ser199 of HA1 and Gly47, Arg75, Phe88, Val91, and Arg106 of HA2. Two mutants, F88E and V91W, were characterized to form a monomer and their double mutant F88E/V91W monomer was selected as an antigen. Animal studies showed that the HA monomer induced protective immunity in vivo, comparable to the trimer, albeit low antibody titers in sera.

Antiviral Effects of Black Raspberry (Rubus coreanus) Seed and Its Gallic Acid against Influenza Virus Infection.

Influenza is a serious public health concern worldwide, as it causes significant morbidity and mortality. The emergence of drug-resistant viral strains requires new approaches for the treatment of influenza. In this study, Rubus coreanus seed (RCS) that is left over from the production of wine or juice was found to show antiviral activities against influenza type A and B viruses. Using the time-of-addition plaque assay, viral replication was almost completely abolished by simultaneous treatment with the RCS fraction of less than a 1-kDa molecular weight (RCSF1). One of the polyphenols derived from RCSF1, gallic acid (GA), identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against both influenza type A and B viruses, albeit at relatively high concentrations. RCSF1 was bound to hemagglutinin protein, inhibited hemagglutination significantly and disrupted viral particles, whereas GA was found to only disrupt the viral particles by using transmission electron microscopy. In BALB/c mice infected with influenza virus, oral administration of RCSF1 significantly improved the survival rate and reduced the viral titers in the lungs. Our results demonstrate that RCSF1 and GA show potent and broad antiviral activity against influenza A and B type viruses and are promising sources of agents that target virus particles.

Clinical significance of fatty liver disease induced by tamoxifen and toremifene in breast cancer patients.

BACKGROUND AND AIM: The aim of this study was to identify the effect of selective estrogen receptor modulator (SERM) on non-alcoholic fatty liver disease (NAFLD) in Asian women. METHODS: We retrospectively evaluated fatty liver development and/or serum alanine aminotransferase (ALT) elevation during SERM treatment in 1061 women who were diagnosed and treated with breast cancer in 2005 at Asan Medical Center. RESULTS: 45 of 618 SERM-treated patients with normal ALT at baseline experienced ALT elevation during SERM treatment. Among the 112 SERM-treated patients who underwent liver imaging test, fatty liver was observed in 47 and both fatty liver and ALT elevation developed in 16 of 102 SERM-treated patients with normal baseline ALT. The cumulative rates of ALT elevation (10.7 vs. 4.3%; P = 0.002), fatty liver (48.5 vs. 20.9%; P < 0.001), and both fatty liver and ALT elevation (17.7 vs. 7.1%; P = 0.02) at 60 months were significantly higher in the SERM group than non-SERM group. By multivariate analysis, SERM treatment increased the risk of ALT elevation (hazard ratio [HR], 2.20; P = 0.01), fatty liver development (HR, 3.59; P < 0.001), and both fatty liver and ALT elevation (HR, 4.98; P = 0.01). After discontinuation of SERM, elevated serum ALT normalized in 39 (92.9%) and there were no instances of liver-related death or progression to liver cirrhosis in patients who experienced fatty liver or ALT elevation. CONCLUSIONS: Although SERM treatment is significantly associated with NAFLD in Asian women, considering the tolerability and reversibility of NAFLD induced by SERM, it can be continued with liver function monitoring in relevant patients.

A comparative study of low-fluence 1,064 nm Q-Switched Nd:YAG laser with or without chemical peeling using Jessner's solution in melasma patients.

BACKGROUND: Although low-fluence 1,064-nm Q-switched Nd:YAG laser (QSNYL) is widely used for the treatment of melasma, multiple treatments are necessary for clinical improvement. Superficial chemical peeling using Jessner's solution has been used for treatment of melasma conventionally. OBJECTIVES: To evaluate the additional therapeutic effect and adverse effects of Jessner's peel when combined with 1,064 nm QSNYL for melasma patients in a double-blind, placebo-controlled design. METHODS: Total of 52 patients were included. Patients who received 10 sessions of 1,064 nm QSNYL plus chemical peeling with placebo (Group A) in a two-week intervals and those who received 10 sessions of 1,064 nm QSNYL plus chemical peeling with Jessner's solution (Group B) in a 2-week intervals were analyzed. Responses were evaluated using the Melasma Area and Severity Index (MASI) score, physician's global assessment (PGA) and subjective self-assessment. RESULTS: At 8 weeks, the mean MASI score decreased from 8.68 ± 4.06 to 8.60 ± 3.88 in Group A and from 8.98 ± 3.72 to 7.13 ± 2.57 in Group B, showing a significant difference (p < 0.001). But at 20 weeks, there was no significant difference on reduction of MASI, self-assessment, and PGA between the two groups. No serious adverse effects were reported with the additional Jessner's peeling. CONCLUSION: This study suggests Jessner's peel is a safe and effective method in the early course of treatment for melasma, when combined with low-fluence 1,064-nm QSNYL.

A comparative study of low-fluence 1064-nm Q-switched Nd:YAG laser with or without chemical peeling using Jessner's solution in melasma patients.

BACKGROUND: Although low-fluence 1064-nm Q-switched Nd:YAG laser (QSNYL) is widely used for the treatment of melasma, multiple treatments are necessary for clinical improvement. Superficial chemical peeling using Jessner's solution has been used for treatment of melasma conventionally. OBJECTIVES: To evaluate the additional therapeutic effect and adverse effects of Jessner's peel when combined with 1064-nm QSNYL for melasma patients in a double-blind, placebo-controlled design. METHODS: Total of 52 patients were included. Patients who received 10 sessions of 1064-nm QSNYL plus chemical peeling with placebo (group A) in a two-week interval and those who received 10 sessions of 1064-nm QSNYL plus chemical peeling with Jessner's solution (group B) in a two-week interval were analyzed. Responses were evaluated using the Melasma Area and Severity Index (MASI) score, physician's global assessment (PGA) and subjective self-assessment. RESULTS: At 8 weeks, the mean MASI score decreased from 8.68 ± 4.06 to 8.60 ± 3.88 in group A and from 8.98 ± 3.72 to 7.13 ± 2.57 in group B, showing a significant difference (p < 0.001). But at 20 weeks, there was no significant difference on reduction of MASI, self-assessment and PGA between the two groups. No serious adverse effects were reported with the additional Jessner's peeling. CONCLUSION: This study suggests Jessner's peel is a safe and effective method in the early course of treatment for melasma when combined with low-fluence 1064-nm Q-switched Nd:YAG laser.

Drug rash with eosinophilia and systemic symptoms syndrome following cholestatic hepatitis A: a case report.

Hepatitis A virus (HAV) infections occur predominantly in children, and are usually self-limiting. However, 75-95% of the infections in adults are symptomatic (mostly with jaundice), with the illness symptoms usually persisting for a few weeks. Atypical manifestations include relapsing hepatitis, prolonged cholestasis, and complications involving renal injury. Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome is a severe, drug-induced hypersensitivity reaction characterized by skin rash, fever, lymph-node enlargement, and internal organ involvement. We describe a 22-year-old male who presented with acute kidney injury and was diagnosed with prolonged cholestatic hepatitis A. The patient also developed DRESS syndrome due to antibiotic and/or antiviral treatment. To our knowledge, this is the first report of histopathologically confirmed DRESS syndrome due to antibiotic and/or antiviral treatment following HAV infection with cholestatic features and renal injury.