The effect of cryopreservation media on the quality of ß-thalassemia mouse spermatozoa.

Background: The mouse model of human diseases is commonly used for biomedical study, including ß-thalassemia (ß-thal), an inherited hemoglobin disorder. Maintaining the mice strain by natural mating systems is costly and seems impractical, especially during the COVID-19 pandemic. Sperm-freezing is a cost-effective solution for ß-thal mouse colony management. Aim: To determine appropriate cryopreservation media for ß-thal mouse spermatozoa to establish a ß-thal mouse sperm bank. Methods: The epididymal spermatozoa of C57BL/6 wild-type (WT) and ß-globin gene knockout thalassemia (BKO) mice were frozen in four freezing media: I) raffinose-skim milk-monothioglycerol (MTG), II) raffinose-skim milk-glutamine, III) raffinose-egg yolk-glycerol, and IV) egg yolk-TES-Tris. The sperm quality was assessed prior to and following freeze-thawing. Results: Compared with WT counterparts, the viable spermatozoa before freezing exhibiting elevated levels of oxidative stress were significantly greater in BKO (p = 0.01). After thawing, the membrane integrity of BKO spermatozoa preserved in I was significantly lower (p = 0.001). The sperm viability and membrane integrity of BKO males were also inferior when media III and IV were used (p = 0.008-0.027). The amount of oxidative stress in the spermatozoon of BKO mice was significantly greater when preserved in I, III, and IV (p = 0.002-0.044). Comparing freezing media, the motility and acrosome integrity of WT and BKO spermatozoa preserved in IV were significantly higher than those in other media (p < 0.001 to p = 0.01). Spermatozoa with the highest mitochondrial membrane potential were observed in I in both genotypes (p = 0.012 to p > 0.05). The viability, membrane integrity, and oxidative stress of post-thaw BKO spermatozoa did not significantly differ among freezing solutions. Conclusion: Irrespective of freezing media, spermatozoa of BKO males are rather more sensitive to cryopreservation than those of WT. Raffinose-skim milk-MTG/glutamine, raffinose-egg yolk-glycerol, and egg yolk-TES-Tris can all be used to preserve BKO mouse spermatozoa. However, with slightly better sperm characteristics, egg yolk-TES-Tris may be a diluent of choice for BKO mouse sperm cryopreservation. The addition of a reducing agent to thawing media is also strongly recommended to efficiently prevent oxidative stress and therefore improve frozen-thawed sperm survival.

Anti-adipogenic and Pro-lipolytic Effects on 3T3-L1 Preadipocytes by CX-4945, an Inhibitor of Casein Kinase 2.

Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 µM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug's anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug's pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug's ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL.

Inactivation of SARS-CoV-2 and HCoV-229E in vitro by ColdZyme® a medical device mouth spray against the common cold.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic calls for effective and safe treatments. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 actively replicates in the throat, unlike SARS-CoV, and shows high pharyngeal viral shedding even in patients with mild symptoms of the disease. HCoV-229E is one of four coronaviruses causing the common cold. In this study, the efficacy of ColdZyme® (CZ-MD), a medical device mouth spray, was tested against SARS-CoV-2 and HCoV-229E in vitro. The CZ-MD provides a protective glycerol barrier containing cod trypsin as an ancillary component. Combined, these ingredients can inactivate common cold viruses in the throat and mouth. The CZ-MD is believed to act on the viral surface proteins that would perturb their entry pathway into cells. The efficacy and safety of the CZ-MD have been demonstrated in clinical trials on the common cold. METHOD OF STUDY: The ability of the CZ-MD to inactivate SARS-CoV-2 and HCoV-229E was tested using an in vitro virucidal suspension test (ASTM E1052). RESULTS: CZ-MD inactivated SARS-CoV-2 by 98.3% and HCoV-229E by 99.9%. CONCLUSION: CZ-MD mouth spray can inactivate the respiratory coronaviruses SARS-CoV-2 and HCoV-229E in vitro. Although the in vitro results presented cannot be directly translated into clinical efficacy, the study indicates that CZ-MD might offer a protective barrier against SARS-CoV-2 and a decreased risk of COVID-19 transmission.

Synergistic effects of anionic surfactants on coronavirus (SARS-CoV-2) virucidal efficiency of sanitizing fluids to fight COVID-19.

Our surrounding environment, especially often-touched contaminated surfaces, plays an important role in the transmission of pathogens in society. The shortage of effective sanitizing fluids, however, became a global challenge quickly after the coronavirus disease-19 (COVID-19) outbreak in December 2019. In this study, we present the effect of surfactants on coronavirus (SARS-CoV-2) virucidal efficiency in sanitizing fluids. Sodium dodecylbenzenesulfonate (SDBS), sodium laureth sulfate (SLS), and two commercial dish soap and liquid hand soap were studied with the goal of evaporation rate reduction in sanitizing liquids to maximize surface contact time. Twelve fluids with different recipes composed of ethanol, isopropanol, SDBS, SLS, glycerin, and water of standardized hardness (WSH) were tested for their evaporation time and virucidal efficiency. Evaporation time increased by 17-63% when surfactant agents were added to the liquid. In addition, surfactant incorporation enhanced the virucidal efficiency between 15 and 27% according to the 4-field test in the EN 16615:2015 European Standard method. Most importantly, however, we found that surfactant addition provides a synergistic effect with alcohols to inactivate the SARS-CoV-2 virus. This study provides a simple, yet effective solution to improve the virucidal efficiency of commonly used sanitizers.

Glycerol-preserved corneal tissue in emergency corneal transplantation: An alternative for fresh corneal tissue in COVID-19 crisis.

PURPOSE: Due to the COVID-19 pandemic, most of the eye banks have limited/stopped corneal collection, as this is a highly contagious disease. This has led to shortage of donor corneas worldwide. Glycerol preservation of tissue remains a viable option in this scenario. The objective is to compare fresh corneal tissue (FCT) with glycerol-preserved cornea (GPC) in emergency corneal transplantation. METHODS: This was a retrospective cohort study conducted in a tertiary care centre of Uttarakhand. Medical records of the patients who underwent therapeutic penetrating keratoplasty (TPK) were reviewed. FCT group included patients who underwent TPK with fresh corneal tissue and GPC group included patients who underwent TPK with glycerol preserved cornea. The indications and outcomes of TPK in the terms of therapeutic success were analysed and compared between both the groups. RESULTS: A total of 94 eyes of 91 patients underwent TPK from October 2011 to August 2017. FCT group included 60 eyes of 57 patients and GPC group included 34 eyes of 34 patients. The primary indication of TPK was infectious keratitis in both the groups (FCT-81.6%; GPC - 91.2%) There was no significant difference in the therapeutic success in both the groups (P = 0.741, Odds ratio- 1.59 with 95% CI- 0.39-6.44). Complications included glaucoma (FCT-21.7%; GPC- 35.2%) graft infection (FCT- 18.33% GPC- 2.9%); graft rejection (FCT-11.66%, GPC- 0%); and graft failure (FCT-88.33%, GPC-100%). CONCLUSION: The GPC is comparable to FCTs in therapeutic transplant and can be a useful interim procedure in saving the eyes in cases of infective keratitis in the time of crisis.