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.

mRNA-1273 but not BNT162b2 induces antibodies against polyethylene glycol (PEG) contained in mRNA-based vaccine formulations.

Two messenger RNA (mRNA)-based vaccines are widely used globally to prevent coronavirus disease 2019 (COVID-19). Both vaccine formulations contain PEGylated lipids in their composition, in the form of polyethylene glycol [PEG] 2000 dimyristoyl glycerol for mRNA-1273, and 2 [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide for BNT162b2. It is known that some PEGylated drugs and products for human use which contain PEG are capable of eliciting immune responses that lead to to detectable PEG-specific antibodies in serum. In this study, we determined if any of the components of mRNA-1273 or BNT162b2 formulations elicited PEG-specific antibody responses in serum by enzyme linked immunosorbent assay (ELISA). We detected an increase in the reactivity to mRNA vaccine formulations in mRNA-1273 but not BNT162b2 vaccinees' sera in a prime-boost dependent manner. Furthermore, we observed the same pattern of reactivity against irrelevant lipid nanoparticles from an influenza virus mRNA formulation and found that the reactivity of such antibodies correlated well with antibody levels against high and low molecular weight PEG. Using sera from participants selected based on the vaccine-associated side effects experienced after vaccination, including delayed onset, injection site or severe allergic reactions, we found no obvious association between PEG antibodies and adverse reactions. Overall, our data shows a differential induction of anti-PEG antibodies by mRNA-1273 and BNT162b2. The clinical relevance of PEG reactive antibodies induced by administration of the mRNA-1273 vaccine, and the potential interaction of these antibodies with other PEGylated drugs remains to be explored.

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.

1H qNMR-Based Metabolomics Discrimination of Covid-19 Severity.

The coronavirus disease 2019 (Covid-19), which caused respiratory problems in many patients worldwide, led to more than 5 million deaths by the end of 2021. Experienced symptoms vary from mild to severe illness. Understanding the infection severity to reach a better prognosis could be useful to the clinics, and one study area to fulfill one piece of this biological puzzle is metabolomics. The metabolite profile and/or levels being monitored can help predict phenotype properties. Therefore, this study evaluated plasma metabolomes of 110 individual samples, 57 from control patients and 53 from recent positive cases of Covid-19 (IgM 98% reagent), representing mild to severe symptoms, before any clinical intervention. Polar metabolites from plasma samples were analyzed by quantitative 1H NMR. Glycerol, 3-aminoisobutyrate, formate, and glucuronate levels showed alterations in Covid-19 patients compared to those in the control group (Tukey's HSD p-value cutoff = 0.05), affecting the lactate, phenylalanine, tyrosine, and tryptophan biosynthesis and d-glutamine, d-glutamate, and glycerolipid metabolisms. These metabolic alterations show that SARS-CoV-2 infection led to disturbance in the energetic system, supporting the viral replication and corroborating with the severe clinical conditions of patients. Six polar metabolites (glycerol, acetate, 3-aminoisobutyrate, formate, glucuronate, and lactate) were revealed by PLS-DA and predicted by ROC curves and ANOVA to be potential prognostic metabolite panels for Covid-19 and considered clinically relevant for predicting infection severity due to their straight roles in the lipid and energy metabolism. Thus, metabolomics from samples of Covid-19 patients is a powerful tool for a better understanding of the disease mechanism of action and metabolic consequences of the infection in the human body and may corroborate allowing clinicians to intervene quickly according to the needs of Covid-19 patients.

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay.

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.

Reverse graft suturing to avoid Descemet's membrane detachment of glycerol-preserved donor cornea used for therapeutic penetrating keratoplasty during COVID-19 to overcome the tissue shortage - A novel surgical technique.

To overcome tissue shortage during pandemic, we switched to 100% glycerol preservation of the donor cornea, which is economical and provides longer duration of storage than the short and intermediate storage mediums we normally use like McCAREY Kaufman (MK) or cornisol. During our initial few cases of therapeutic penetrating keratoplasty using glycerol preserved donor cornea, we faced spontaneous Descemet's detachments resistant to air tamponade. We tried reverse graft suturing and successfully reinforced Descemet's attachment along with air tamponade, in one of the cases after multiple failed air injections. In the subsequent two cases of infective keratitis needing therapeutic penetrating Keratoplasty, we took eight reverse sutures in between the eight cardinals, to anchor the Descemet's membrane of the graft. Both the grafts showed attached Descemet's and maintained good graft clarity. The reverse corneal suturing technique has not been described to the best of our knowledge and hope this helps our corneal fraternity.

Intermediate outcomes of therapeutic penetrating keratoplasty for severe microbial keratitis using glycerol-preserved donor corneas during the COVID-19 pandemic.

Purpose: To report the intermediate outcomes of therapeutic penetrating keratoplasty (TPK) performed for severe microbial keratitis using glycerol-preserved corneas during the Corona virus diseases of 2019 (COVID-19). Methods: Retrospective non-comparative case series from April to August 2020 in a network of tertiary eye care centers. Glycerol-preserved tissues were used for therapeutic keratoplasty (TPK). We reviewed the demographics, microbiology, surgical outcomes such as wound integrity, recurrence, graft melt, epithelialization, and complications. Results: A total of 49 eyes that underwent TPK with glycerol-preserved corneal tissues were analyzed. The primary indication was severe microbial keratitis in 47 eyes. The majority was a fungal infection in 33 eyes (67.3%). The mean age was 53.8 ± 12.2 years, with male predominance (3:1). The corneas were stored for an average of 85.5 ± 53 days prior to transplant. The median donor age was 65 years. The grafts were tectonically stable in 32/36 eyes (88.9%) at 1 month and 20/24 eyes (83.3%) at 3 months. The graft melt was noted in three eyes at 1 and 3 months. The recurrence of the infection was noted in four eyes and all were of fungal etiology. The graft epithelialization was delayed with a mean duration of 48.9 ± 25 days after surgery. Post-TPK, raised intra-ocular pressure (>21 mm Hg) was noted in 51.2% at 1 week, 17.4% at 1 month, and 11.8% at 3 months. Conclusion: Glycerol preservation is a reliable alternative with good therapeutic outcomes in the short and interim postoperative period. Delayed epithelialization and secondary glaucoma were the commonest postoperative complications.

Inactivation of SARS-CoV-2 by Ozonated Glycerol.

We evaluated the SARS-CoV-2-inactivation activity of ozonated glycerol (OG). When a viral solution with 1% fetal bovine serum (FBS) was mixed with test solutions at a ratio of 1:19 and incubated for 20 s, OG with ozone concentrations of over 1000 ppm inactivated ≥ 94.38% of the virus. Extension of the reaction time to 1 h led to the inactivation of ≥ 99.82% of the virus (the viral titer was below the detection limit). Extension to 24 h resulted in concentrations over 200 ppm OG inactivating ≥ 99.87% of the virus (the viral titers were below the detection limit). Next, viral solutions with 1, 20, and 40% FBS were mixed with test solutions at a ratio of 1:19 and incubated for 5 min. Whereas the virucidal activity of 500 ppm OG was very limited in the presence of 1% FBS (79.47% inactivation), it increased in the presence of 20 and 40% FBS (95.13 and 97.95% inactivation, respectively; the viral titers were not below the detection limit). Meanwhile, over 1000 ppm OG inactivated ≥ 99.44% of the virus regardless of the FBS concentration (the viral titers were below the detection limit). Extension of the reaction time to 1 h led to 500 ppm OG inactivating ≥ 99.91 and ≥ 99.95% of the virus with 20 and 40% FBS, respectively (the viral titers were below the detection limit). These results suggested that OG might be useful as a virucidal agent against SARS-CoV-2.

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.

Lopinavir/ritonavir, a new galenic oral formulation from commercial solid form, fine-tuned by nuclear magnetic resonance spectroscopy.

OBJECTIVES: The lopinavir/ritonavir combination is one of the first antiretroviral drugs to be used in the treatment of COVID-19. In incapacitated patients, such as those in intensive care, an oral liquid formulation is needed. In Italy a marketed formulation is available, but only by importing it from other European countries. A galenic oral formulation prepared in the hospital pharmacy from lopinavir/ritonavir tablets was fine-tuned, evaluating the content of the active pharmaceutical ingredient (API) and stability of the formulation by using nuclear magnetic resonance (NMR) spectroscopy. METHODS: To overcome the insolubility of lopinavir/ritonavir in water, ethanol and glycerol have been used as additional excipients. To define the best excipient proportion and best preparation method, three different formulations (ethanol 7.1-7.5%, glycerol 6-15%, and water) and two different preparation procedures (two step vs one step) have been studied. Each formulation has been compared with Kaletra oral solution (lopinavir 80 mg/mL, ritonavir 20 mg/mL) by NMR spectroscopy. API content and stability were measured. RESULTS: The presence of ethanol and glycerol as co-solvents is crucial both to improve solubilisation and promote the stability of the oral form. In the two-step preparation method, when crushed tablets were first dispersed in the ethanol/glycerol mixture and then in water, the content of solubilised active ingredients was equal or only slightly lower than the standard Kaletra (range 89-100%). The one-step method provided a comparable API content (65%) to that obtained by using water as the sole dispersing medium. CONCLUSIONS: The two-step setup method with final 7.1% ethanol and 11% glycerol concentration is an efficient procedure for extemporaneous preparation of lopinavir/ritonavir liquid formulations from crushed tablets. The method combines simplicity of preparation and reconstitution in the hospital ward with good solubilisation, comparable to the commercial solution, and stability of active ingredients over time.

Prospective study on hand dermatitis in nurses and doctors during COVID-19 pandemic and its improvement by use of adopted recommendations of the European Academy of Dermatology and Venereology Task Force on Contact Dermatitis.

Frequency of hand disinfection and adverse skin reactions among health care workers dramatically increased since the COVID-19 outbreak and consensus recommendations on hand hygiene were presented. The aim of the present study was to check the efficacy of the European Academy of Dermatology and Venereology (EADV) Task Force (TF) on Contact Dermatitis (CD) recommendations in a real life and to search if providing products mentioned in that recommendations may increase its efficacy. Doctors and nurses who worked with patients during COVID-19 pandemic and use hand disinfectants received adopted recommendations of the EADV TF on CD only or together with mentioned in that recommendations gel with ethanol and glycerin and emollient. Prevalence of adverse skin reactions on hand disinfectants at baseline was 80.21%. In a month significant improvement of health-related quality of life (HRQoL) and self-assessed improvement of hand skin (P < .01 for both) was reported in "products" group only. Number of participants that had no impact on their HRQoL became higher and the Dermatology Life Quality Index scores lower than in "recommendations only" group (P = .03 and P = .02, respectively). Our results showed that recommendations of the EADV TF on CD may significantly improve HRQoL and hand skin status in health care professionals but provision with products mentioned in that recommendations is crucial.

Synthesis and characterization of novel organo-hydrogel based agar, glycerol and peppermint oil as a natural drug carrier/release material.

The very recent Covid-19 pandemic has made the need to understand biocompatible polymers as support material in drug delivery systems and controlled release clearer, especially for organo-hydrogels. This study aims to synthesize various new polymeric materials called gels, hydrogels, and organo-hydrogels according to the monomer used and to investigate their use as drug release systems. The agar-glycerol (AG) pair was used to synthesize the polymers, N, N, methylene bisacrylamide (MBA, m) and glutaraldehyde (GA, g) were used as cross-linkers and peppermint oil (PmO) was included to obtain the organo-hydrogels. Therefore, one AG gel and two p (AG-m) and p (GA-g) hydrogels were synthesized within the scope of the study. Six different organo-hydrogels based on p(AG-m-PmO) or p (AG-g-PmO) were also synthesized by varying the amount of peppermint oil. Paracetamol and carboplatin were selected as the sample drugs. Synthesized gels, hydrogels and organo-hydrogels were characterized by FTIR and SEM analysis. Additionally, swelling behaviors of the synthesized gels were investigated in different media (ID water, tap water, ethanol, acetone, ethanol/ID water (1:1), acetone/ID water (1:1) and gasoline) and at different pHs. Moreover, it was determined that organo-hydrogels were blood compatible and had antioxidant properties based on hemolysis, blood clotting and antioxidant analysis. Therefore, the release of paracetamol (a known antipyretic-painkiller, recommended and used in the treatment of Covid-19) and carboplatin (widely used in cancer treatment) were studied. Evidently, as the amount of PMO oil increases, the -OH groups in organo-hydrogels will increase and the chemical and physical bonding rates will increase; therefore it was observed that increasing peppermint oil in the organo-hydrogels structure to 0.3 mL stimulated the release of the drugs. For instance, maximum paracetamol release amount from p(AG-g-PmO) and p(AG-m-PmO) organo-hydrogels was calculated to be 72.3% at pH 7.4 and 69.8% at pH 2.0, respectively. The maximum carboplatin release amount from p(AG-g-PmO) and p(AG-m-PmO) organo-hydrogels was calculated to be 99.7% at pH 7.4 and 100% at pH 7.4, respectively. It was concluded that the synthesized organo-hydrogels might easily be used as drug carrier and controlled drug release materials.

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.