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Current Protocols Jul 2022Influenza is an infectious respiratory disease with significant morbidity and mortality rates among people of all ages. Influenza viruses spread and evolve rapidly in...
Influenza is an infectious respiratory disease with significant morbidity and mortality rates among people of all ages. Influenza viruses spread and evolve rapidly in the human population. Different immune histories, given by previous exposures to influenza virus infections and/or vaccinations, result in a great diversity of humoral and cellular immune responses. Understanding protective immune responses induced against circulating virus strains and potential pandemic strains is vital for infection prevention and disease mitigation. Vaccine formulations for seasonal influenza must be reformulated annually to stay abreast of occurring virus mutations. Assays to measure the capacity of antibodies to neutralize influenza viruses provide a good estimate of protection against future infections with strains similar or identical to those used in the assay. Here, we describe a detailed protocol of our standard in vitro microneutralization assay to assess the neutralization activity of polyclonal sera or purified monoclonal antibodies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Microneutralization assay to assess virus inhibition by serum or monoclonal antibodies Support Protocol 1: Preparation of cDMEM Support Protocol 2: Preparation and aliquoting of TPCK-treated trypsin Support Protocol 3: Inactivation of serum samples by RDE treatment.
Topics: Antibodies, Monoclonal; Antibodies, Viral; Humans; Influenza Vaccines; Influenza, Human; Neuraminidase; Orthomyxoviridae
PubMed: 35848945
DOI: 10.1002/cpz1.465 -
Animals : An Open Access Journal From... Oct 2023Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common...
Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.
PubMed: 37893927
DOI: 10.3390/ani13203203 -
Arthritis Research & Therapy Jan 2022Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex...
BACKGROUND
Synovial fluid (SF) is commonly used for diagnostic and research purposes, as it is believed to reflect the local inflammatory environment. Owing to its complex composition and especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. In this study, we investigated the importance of homogenization of the total SF sample before subsequent analysis.
METHODS
SF was obtained from the knee of 29 arthritis patients (26 rheumatoid arthritis, 2 osteoarthritis, and 1 juvenile idiopathic arthritis patient) as part of standard clinical care. Synovial fluid was either treated with hyaluronidase as a whole or after aliquoting to determine whether the concentration of soluble mediators is evenly distributed in the viscous synovial fluid. Cytokine and IgG levels were measured by ELISA or Luminex and a total of seven fatty acid and oxylipin levels were determined using LC-MS/MS in all aliquots. For cell analysis, synovial fluid was first centrifuged and the pellet was separated from the fluid. The fluid was subsequently treated with hyaluronidase and centrifuged to isolate remaining cells. Cell numbers and phenotype were determined using flow cytometry.
RESULTS
In all patients, there was less variation in IgG, 17-HDHA, leukotriene B (LTB), and prostaglandin E (PGE) levels when homogenization was performed before aliquoting the SF sample. There was no difference in variation for cytokines, 15-HETE, and fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Between 0.8 and 70% of immune cells (median 5%) remained in suspension and were missing in subsequent analyses when the cells were isolated from untreated SF. This percentage was higher for T and B cells: 7-85% (median 22%) and 7-88% (median 23 %), respectively.
CONCLUSIONS
Homogenization of the entire SF sample leads to less variability in IgG and oxylipin levels and prevents erroneous conclusions based on incomplete isolation of synovial fluid cells.
Topics: Arthritis, Rheumatoid; Chromatography, Liquid; Humans; Hyaluronoglucosaminidase; Synovial Fluid; Tandem Mass Spectrometry
PubMed: 34998422
DOI: 10.1186/s13075-021-02696-4 -
Veterinary Sciences Feb 2023This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After...
This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After collection (n = 40 ejaculates), the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of each ejaculate was extended and cooled for 48 h; a second aliquot was cushion-centrifuged and cooled for 48 h; and a third aliquot was processed and then frozen. The total motility (TM) and progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP) were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The second ejaculate had a lower gel-free semen volume ( = 0.026). The sperm concentration was greater in the first than in the second ejaculate ( < 0.001). The sperm morphology was similar between the ejaculates ( > 0.05). Cushion-centrifugation prevented a reduction in the TM, PM, and PMI over time ( < 0.05). The TM, PM, and PMI decreased after freezing but not between the ejaculates ( > 0.05). The first and second ejaculates of the season, which were collected 1 h apart, varied in quantity but not in quality after cooling and freezing.
PubMed: 36977212
DOI: 10.3390/vetsci10030173 -
Clinical Hemorheology and... 2018Timolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability...
Timolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability (ED), increased activity of erythrocyte acetylcholinesterase (AChE), increased values of nitrosoglutathione (GSNO) and nitic oxide (NO) and decreased plasma levels of NO metabolites, were described in primary open angle glaucoma patients. In healthy human red blood cells (RBCs), timolol is an inhibitor of AChE and induces NO efflux and GSNO efflux from that blood component in lower concentration than those obtained in presence of the natural AChE substrate, acetylcholine (ACh). The signal transduction pathway in RBCs described for NO in dependence of AChE-ACh active complex involves Gi protein, protein tyrosine kinase (PTK like Syk and p53/56Lyn), protein tyrosine phosphatase (PTP) and adenylyl cyclase (AC).The aim of this in vitro study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like nitrite (NO2-), nitrate (NO3-, peroxynitrite (-ONOO) and GSNO under the presence of PTK, PTP, AC and guanylyl cyclase (GC) enzyme proteins inhibitors.Blood samples from healthy donors were each one divided and were performed aliquots in absence (control aliquots) and presence of timolol or timolol plus each inhibitor and Gi protein uncoupling. No significant differences in erythrocyte NO efflux, GSNO, peroxynitrite, nitrite and nitrate concentrations in response to timolol when compared with the untreated blood samples aliquots were obtained.It was observed an increase in erythrocyte deformability at high shear stresses induced by the simultaneous presence of timolol and band 3 protein dephosphorylation by PTK syk inhibitor. No significant differences where verified in peroxynitrite levels in the blood aliquots in presence of timolol plus each enzyme inhibitor and Gi protein uncoupling in relation to the control aliquots. No variation of GSNO concentration occurs under the presence of timolol and AMGT (PTK lyn inhibitor) besides the significant higher values observed with each one of the other inhibitors. Nitrate concentration increases significantly in all aliquots with timolol plus each one of the inhibitors. The same was observe with nitrite levels with exception of the aliquots with timolol plus AMGT or timolol plus Gi protein uncoupling showing no significant values in relation to the control aliquots.Besides the changes in NO derivative molecules and NO efflux from RBCs obtained in this study with blood samples of healthy donors under the effect of timolol plus each inhibitor of the proteins participants in NO signal transduction mechanism, further analogue studies must be promoted with blood samples of patients with glaucoma or any other inflammatory vascular disease.
Topics: Adrenergic beta-Antagonists; Erythrocyte Deformability; Humans; Nitric Oxide; Timolol
PubMed: 29630536
DOI: 10.3233/CH-189110 -
Biopreservation and Biobanking Aug 2021The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year....
The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year. During 1 year, semen from 10 adult Gabon bucks was collected by electroejaculation every 2 weeks. Before and after freezing, samples were selected by density gradient centrifugation, and after sperm selection, the sample was divided into two aliquots. One aliquot was CF with an extender based on Tris, citric acid, and glucose (TCG) +6% yolk +5% glycerol, and maintained at 5°C for 3 hours of equilibration before freezing. The other aliquot was frozen using an UF method with an extender based on TCG +6% yolk +100 mM sucrose, and maintained at 5°C for 30 minutes. The evaluations included the percentages of motile sperm, sperm with progressive motility, quality of sperm motility, and the percentages of sperm with functional membrane, live sperm, sperm with morphoabnormalities, and sperm with intact acrosome. The percentage of sperm with intact acrosome was higher using the conventional freezing method ( < 0.05). After thawing and at pre- and postselection stages, the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome were greater using CF than UF ( < 0.005). Conventional freezing was more effective than UF to cryopreserve sperm from Gabon bucks, at least in our experimental conditions. Most differences in favor of CF were observed in the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome during long periods of the year, or even remained throughout it.
Topics: Acrosome; Cryopreservation; Cryoprotective Agents; Freezing; Gabon; Humans; Male; Semen Preservation; Sperm Motility; Spermatozoa
PubMed: 33751902
DOI: 10.1089/bio.2020.0155 -
The Journal of Maternal-fetal &... Dec 2022The Academy of Breastfeeding Medicine published a clinical protocol for Human Milk storage, recommending refrigeration at a temperature of 4 °C up to 4 d as the...
BACKGROUND
The Academy of Breastfeeding Medicine published a clinical protocol for Human Milk storage, recommending refrigeration at a temperature of 4 °C up to 4 d as the optimal conditions for the safety and bactericidal capacity of Human Milk. However, few studies were conducted to evaluate the change in milk composition during this type of refrigeration storage.
AIM
To elucidate some uncertainties regarding the Human Milk composition and prolonged cold storage, we have investigated the effects of storage at 4 °C up to 96 h on an important category of oxidative stress markers: the Isoprostanes (F2-isoprostanes, F4-neuroprostanes and F3-isoprostanes).
MATERIAL AND METHOD
The experiment was repeated 3 times to ensure reproducibility of the results. We enrolled 3 donating healthy mothers for each time (total: 9 mothers). Milk was collected with standard extraction methods. Immediately after collection, each Human Milk sample from each mother was pooled and then divided into 5 aliquots. One aliquot (0 h) was immediately frozen at -80 °C until the analysis. The other aliquots (24 h, 48 h, 72 h, 96 h) were stored in a refrigerator at 4 °C respectively for 24, 48, 72 and 96 h, then immediately frozen at -80 °C until the analysis. Milk samples were then used to determine concentration of Isoprostanes in Liquid Chromatography - Mass Spectrometry and Liquid Chromatography - Tandem Mass Spectrometry.
RESULTS
Isoprostanes were detectable in all Human Milk samples. There was no significant trend of the concentration of the tested analytes over time.
DISCUSSION AND CONCLUSION
This study provides evidence of the presence in human milk of all the tested isoprostanes: in particular, F2-isoprostanes, F4-neuroprostanes and F3-isoprostanes. Refrigeration and storage of fresh Human Milk in controlled conditions for 96 h did not significantly affect its bioactivity and nutritional quality related with these biomarkers.
Topics: Humans; Refrigeration; Isoprostanes; F2-Isoprostanes; Milk, Human; Neuroprostanes; Reproducibility of Results; Biomarkers
PubMed: 34806531
DOI: 10.1080/14767058.2021.2006626 -
Lab on a Chip Feb 2023Developing automated platforms for point-of-need testing is a crucial global demand. Digital microfluidics is a promising solution for expanding integrated testing...
Developing automated platforms for point-of-need testing is a crucial global demand. Digital microfluidics is a promising solution for expanding integrated testing devices featuring ultimate control over the chemical and biological reactions in micro/nanoliter droplets. In this study, robotic digital microfluidics (RDMF) is introduced for the mechanical manipulation of the droplets precisely and inexpensively. A controllable and multifunctional arm equipped with several actuators is responsible for dispensing and manipulating droplets on a disposable superhydrophobic cartridge. The platform has been demonstrated with diverse functions, including droplet dispensing, transport, mixing, aliquoting, and splitting. Moreover, incorporating magnetic and heating modules into the system can realize particle manipulation and droplet heating. The liquid handling operations are investigated from both experimental and modeling perspectives. Handling a wide range of droplet sizes without needing high-voltage electric sources, integrability with different detection techniques, and ease of manufacturing are the main advantages of the RDMF platform compared to conventional digital microfluidic systems. The availability of a complete fluidic toolbox and multiple detection choices make RDMF promising for droplet-based total analysis technology. The system was applied for a urinalysis test to show its versatility in handling complex biochemical assays. The results entirely matched those obtained based on laboratory gold standard techniques.
PubMed: 36606624
DOI: 10.1039/d2lc00849a -
Endocrine May 2017Reactive oxygen species favor reproductive processes at low concentrations, but damage spermatozoa and decrease their fertilizing capacity at high concentrations. During...
Reactive oxygen species favor reproductive processes at low concentrations, but damage spermatozoa and decrease their fertilizing capacity at high concentrations. During infection and/or inflammation of the accessory sex glands reactive oxygen species overproduction may occur which, in turn, may negatively impact on sperm motility, sperm DNA fragmentation, and lipid peroxidation. A number of nutraceutical formulations containing antioxidant molecules have been developed to counteract the deleterious effects of the oxidative stress. A recent formulation containing zinc, D-aspartic acid, and coenzyme-Q10 is present in the pharmaceutical market. Based on these premises, the aim of the present study was to evaluate the effects of this combination on spermatozoa in vitro. The study was conducted on 24 men (32.2 ± 5.5 years): 12 normozoospermic men and 12 asthenozoospermic patients. Spermatozoa from each sample were divided into two control aliquots (aliquot A and B) and an aliquot incubated with zinc, D-aspartic acid, and coenzyme-Q10 (aliquot C). After 3 h of incubation, the following parameters were evaluated: progressive motility, number of spermatozoa with progressive motility recovered after swim-up, lipid peroxidation, and DNA fragmentation. Incubation with zinc, D-aspartic acid, and coenzyme-Q10 maintained sperm motility in normozoospermic men (37.7 ± 1.2 % vs. 35.8 ± 2.3 % at time zero) and improved it significantly in asthenozoospermic patients (26.5 ± 1.9 % vs. 18.8 ± 2.0 % at time zero) (p < 0.01). This resulted in a significantly higher (p < 0.01) number of spermatozoa with progressive motility recovered after swim-up in both normozospermic men (4.1 ± 0.9 vs. 3.3 ± 1.0 millions) and asthenozooseprmic patients (3.2 ± 0.8 vs. 1.6 ± 0.5 millions). Finally, a statistically significant lower sperm lipid peroxidation was found after incubation with zinc, D-aspartic acid, and coenzyme-Q10 (p < 0.05) in both normozospermic men (1.0 ± 0.4 % vs. 2.4 ± 0.9 %) and asthenozooseprmic patients (0.2 ± 0.1 % vs. 0.6 ± 0.2 %). No statistically significant effect was observed on sperm DNA fragmentation. This nutraceutical formulation may be indicated in vitro during the separation of the spermatozoa in the assisted reproduction techniques, during which the spermatozoa undergo an increased oxidative stress.
Topics: Adult; Asthenozoospermia; D-Aspartic Acid; DNA Fragmentation; Humans; Lipid Peroxidation; Male; Oxidative Stress; Sperm Motility; Spermatozoa; Ubiquinone; Zinc
PubMed: 27422792
DOI: 10.1007/s12020-016-1013-7 -
Cryobiology Dec 2022Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study...
Leptospires are preserved by frequent sub-culturing in semisolid media due to the challenge of low recovery by freezing or liquid nitrogen methods. The present study evaluated three liquid EMJH medium compositions (Medium A: Leptospira medium base EMJH, Leptospira enrichment EMJH, 5-fluorouracil (3%), rabbit serum (1%) and calf serum (1%); Medium B: same as Medium A but without 5-fluorouracil; Medium C: same as Medium B but with the addition of sodium pyruvate) for the revival of leptospires after storage at -80 °C. A total of 18 Leptospira serovars cultured in Medium A was aliquoted into cryogenic vials and directly stored at -80 °C. A hundred microlitre from each serovar culture stored at -80 °C was sub-cultured on a selected time over a period of 30 months into Media A, B and C. Regrowth on Media B and C showed a better and faster recovery (89-100%) (p-value <0.05) compared to Medium A (67-100%). Leptospires can be stored longer at -80 °C and a good recovery could be obtained when sub-cultured on EMJH medium without 5-fluorouracil.
Topics: Animals; Rabbits; Cryopreservation; Leptospira; Freezing; Fluorouracil; Nitrogen; Culture Media
PubMed: 36179819
DOI: 10.1016/j.cryobiol.2022.09.007