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MethodsX 2015The C26 adenocarcinoma tumor line is frequently used to establish peripheral tumors in mice for the study of cancer cachexia and cancer-related fatigue. Recently, we...
The C26 adenocarcinoma tumor line is frequently used to establish peripheral tumors in mice for the study of cancer cachexia and cancer-related fatigue. Recently, we have noticed a progressive decline in the effects of tumor growth on our biological and behavioral measures in the tumor-bearing mice. Therefore, we compared effects of three aliquots of the C26 tumor cell line that differed in storage condition and number of passages on cytokine secretion, tumor growth, weight loss and fatigue behavior. Three aliquots of the C26 tumor line were selected as alpha (α), beta (β), and gamma (γ). Aliquot α was an original C26 stock line that had been stored at -80°C. Aliquot β was stored in liquid nitrogen. Aliquot γ was taken from aliquot β and passaged three times. The three aliquots of the C26 tumor line showed differences in IL-6 mRNA and protein secretion , with aliquot β showing the greatest IL-6 secretion. These differences were mirrored . Plasma IL-6 levels were elevated in all tumor bearing mice but was greatest in group β mice. Carcass weight was decreased in all three tumor groups. Brain expression of IL-1β mRNA was greatest in group β and group β demonstrated the greatest decline in running activity at day 19. Storage conditions and number of passages influence C26 tumor cell secretion of cytokines.Variations in C26 aliquots may explain differences observed between laboratories using the same cell line.We recommend always storing cell lines in liquid nitrogen and limiting the number of passages before use in experiments.
PubMed: 25709898
DOI: 10.1016/j.mex.2015.02.001 -
Nature Communications Jan 2019The success of lab-on-a-chip systems may depend on a low-cost device that incorporates on-chip storage and fluidic operations. To date many different methods have been...
The success of lab-on-a-chip systems may depend on a low-cost device that incorporates on-chip storage and fluidic operations. To date many different methods have been developed that cope separately with on-chip storage and fluidic operations e.g., hydrophobic and capillary valves pneumatic pumping and blister storage packages. The blister packages seem difficult to miniaturize and none of the existing liquid handling techniques despite their variety are capable of proportional repeatable dispensing. We report here on an inexpensive robust and scalable micro-dispenser that incorporates long-term storage and aliquoting of reagents on different microfluidics platforms. It provides long-term shelf-life for different liquids enables precise dispensing on lab-on-a-disc platforms and less accurate but proportional dispensing when operated by finger pressure. Based on this technology we introduce a method for automation of blood plasma separation and multi-step bioassay procedures. This micro-dispenser intends to facilitate affordable portable diagnostic devices and accelerate the commercialization of lab-on-a-chip devices.
PubMed: 30643146
DOI: 10.1038/s41467-018-08091-z -
Journal of Endodontics Nov 2014Eliminating and/or inhibiting bacterial growth within the root canal system has been shown to play a key role in the regenerative outcome. The aim of this study was to...
INTRODUCTION
Eliminating and/or inhibiting bacterial growth within the root canal system has been shown to play a key role in the regenerative outcome. The aim of this study was to synthesize and determine in vitro both the antimicrobial effectiveness and cytocompatibility of bimix antibiotic-containing polydioxanone-based polymer scaffolds.
METHODS
Antibiotic-containing (metronidazole [MET] and ciprofloxacin [CIP]) polymer solutions (distinct antibiotic weight ratios) were spun into fibers as a potential mimic to the double antibiotic paste (DAP, a MET/CIP mixture). Fiber morphology, chemical characteristics, and tensile strength were evaluated by scanning electron microscopy, Fourier transform infrared spectroscopy, and tensile testing, respectively. Antimicrobial efficacy was tested over time (aliquot collection) against Enterococcus faecalis (Ef), Porphyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn). Similarly, cytotoxicity was evaluated in human dental pulp stem cells. Data were statistically analyzed (P < .05).
RESULTS
Scanning electron microscopy and Fourier transform infrared spectroscopy confirmed that electrospinning was able to produce antibiotic-containing fibers with a diameter mostly in the nanoscale. The tensile strength of 1:1MET/CIP scaffolds was significantly (P < .05) higher than pure polydioxanone (control). Meanwhile, all other groups presented similar strength as the control. Aliquots obtained from antibiotic-containing scaffolds inhibited the growth of Ef, Pg, and Fn, except pure MET, which did not show an inhibitory action toward Pg or Fn. Antibiotic-containing aliquots promoted slight human dental pulp stem cell viability reduction, but none of them were considered to be cytotoxic.
CONCLUSIONS
Our data suggest that the incorporation of multiple antibiotics within a nanofibrous scaffold holds great potential toward the development of a drug delivery system for regenerative endodontics.
Topics: Anti-Bacterial Agents; Cell Survival; Ciprofloxacin; Dental Pulp; Electrochemical Techniques; Enterococcus faecalis; Fusobacterium nucleatum; Humans; Materials Testing; Metronidazole; Microscopy, Electron, Scanning; Nanofibers; Polydioxanone; Porphyromonas gingivalis; Regeneration; Root Canal Therapy; Spectroscopy, Fourier Transform Infrared; Stem Cells; Surface Properties; Tensile Strength; Tissue Engineering; Tissue Scaffolds
PubMed: 25201643
DOI: 10.1016/j.joen.2014.07.017 -
Nepalese Journal of Ophthalmology : a... Jul 2021The main purpose of this survey was to find out what technique for bevacizumab injection is practiced by ophthalmologists in Nepal and to evaluate which is the best...
INTRODUCTION
The main purpose of this survey was to find out what technique for bevacizumab injection is practiced by ophthalmologists in Nepal and to evaluate which is the best technique of drug dispensing and what possible hindrances are there in following it.
MATERIALS AND METHODS
This was an online survey using google forms.
RESULTS
There were a total of 34 participants in the survey. Most of the participants (58.8%) followed the same vial, multiple prick, multiple days method for giving intravitreal bevacizumab.. Majority of participants said they thought that aliquoting the drug and using it same day would be the best technique to prevent post injection endophthalmitis. Cost and unsuitability for small hospitals were the main factor preventing surgeons from practicing the best method.
CONCLUSION
Risk of endophthalmitis can be reduced by following proper drug dispensing techniques. Aliquoting bevacizumab in smaller syringes under aseptic conditions can reduce the risk of endophthalmitis.
Topics: Angiogenesis Inhibitors; Bevacizumab; Endophthalmitis; Eye Infections, Bacterial; Humans; Intravitreal Injections; Nepal; Retrospective Studies; Vascular Endothelial Growth Factor A
PubMed: 35996794
DOI: 10.3126/nepjoph.v13i2.32408 -
Journal of Immunological Methods Jul 2024There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of...
There is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines against SARS-CoV-2 variants. We describe the generation of reference reagents comprised of post-vaccination sera from recipients of different primary vaccines with or without different vaccine booster regimens in order to allow standardized characterization of SARS-CoV-2 neutralization in vitro. We prepared and pooled serum obtained from donors who received a either primary vaccine series alone, or a vaccination strategy that included primary and boosted immunization using available SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), replication-incompetent adenovirus type 26 vaccine (Ad26.COV2·S, Johnson and Johnson), or recombinant baculovirus-expressed spike protein in a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). No subjects had a history of clinical SARS-CoV-2 infection, and sera were screened with confirmation that there were no nucleocapsid antibodies detected to suggest natural infection. Twice frozen sera were aliquoted, and serum antibodies were characterized for SARS-CoV-2 spike protein binding (estimated WHO antibody binding units/ml), spike protein competition for ACE-2 binding, and SARS-CoV-2 spike protein pseudotyped lentivirus transduction. These reagents are available for distribution to the research community (BEI Resources), and should allow the direct comparison of antibody neutralization results between different laboratories. Further, these sera are an important tool to evaluate the functional neutralization activity of vaccine-induced antibodies against emerging SARS-CoV-2 variants of concern. IMPORTANCE: The explosion of COVID-19 demonstrated how novel coronaviruses can rapidly spread and evolve following introduction into human hosts. The extent of vaccine- and infection-induced protection against infection and disease severity is reduced over time due to the fall in concentration, and due to emerging variants that have altered antibody binding regions on the viral envelope spike protein. Here, we pooled sera obtained from individuals who were immunized with different SARS-CoV-2 vaccines and who did not have clinical or serologic evidence of prior infection. The sera pools were characterized for direct spike protein binding, blockade of virus-receptor binding, and neutralization of spike protein pseudotyped lentiviruses. These sera pools were aliquoted and are available to allow inter-laboratory comparison of results and to provide a tool to determine the effectiveness of prior vaccines in recognizing and neutralizing emerging variants of concern.
Topics: Humans; SARS-CoV-2; Antibodies, Viral; COVID-19; Antibodies, Neutralizing; COVID-19 Vaccines; 2019-nCoV Vaccine mRNA-1273; BNT162 Vaccine; Neutralization Tests; Spike Glycoprotein, Coronavirus; Reference Standards; Immunization, Secondary; Vaccination; Ad26COVS1
PubMed: 38823574
DOI: 10.1016/j.jim.2024.113698 -
PloS One 2017The blood to anticoagulant ratio is standardized according to the physiological calcium concentration in blood samples conventionally used for hemostasis testing....
BACKGROUND
The blood to anticoagulant ratio is standardized according to the physiological calcium concentration in blood samples conventionally used for hemostasis testing. Specifically, one fixed volume of 0.109 mmol/L sodium citrate is added to 9 volumes of blood. Since little is known about the impact of hypercalcemia on the calcium-binding capacity of citrate, this study was planned to investigate the effect of experimental hypercalcemia on routine hemostasis testing.
METHODS
Fifteen pooled citrated plasmas with matching lithium-heparin pooled plasma from patients with different values of prothrombin time (PT) were divided in three aliquots of 0.6mL each. The first paired aliquots of both citrate and lithium-heparin plasma were supplemented with 60μL of saline, the second paired aliquots with 30μL of saline and 30μL of calcium chloride and the third paired aliquots with 60μL of calcium chloride. Total and ionized calcium was measured in all aliquots of citrate and lithium-heparin plasma, whereas PT, activated partial thromboplastin time (APTT) and fibrinogen were measured in citrate plasma aliquots.
RESULTS
Total calcium concentration gradually increased in both lithium-heparin and citrate plasma aliquots 2 and 3 compared to baseline aliquot 1. The concentration of ionized calcium also gradually increased in lithium-heparin plasma aliquots 2 and 3, whereas it remained immeasurable (i.e., <0.10 mmol/L) in all citrate plasma aliquots. No significant differences were observed for values of PT, APTT and fibrinogen in citrate plasma aliquots 2 and 3 compared to the baseline aliquot 1, with a mean bias was always comprised within the desirable quality specifications derived from biological variability data.
CONCLUSION
Hypercalcemia, up to severe hypercalcemia does not generate significant bias in results of first-line coagulations tests, so that hypothetical consideration of adjusting citrate-blood ratio is unjustified in hypercalcemic patients.
Topics: Anticoagulants; Blood Coagulation; Citric Acid; Female; Fibrinogen; Hemostasis; Heparin; Humans; Hypercalcemia; Lithium; Male; Partial Thromboplastin Time; Prothrombin Time
PubMed: 28362859
DOI: 10.1371/journal.pone.0175094 -
Biomedicines Sep 2023The composition, viability and metabolic functionality of intestinal microbiota play an important role in human health and disease. Studies on intestinal microbiota are... (Review)
Review
The composition, viability and metabolic functionality of intestinal microbiota play an important role in human health and disease. Studies on intestinal microbiota are often based on fecal samples, because these can be sampled in a non-invasive way, although procedures for sampling, processing and storage vary. This review presents factors to consider when developing an automated protocol for sampling, processing and storing fecal samples: donor inclusion criteria, urine-feces separation in smart toilets, homogenization, aliquoting, usage or type of buffer to dissolve and store fecal material, temperature and time for processing and storage and quality control. The lack of standardization and low-throughput of state-of-the-art fecal collection procedures promote a more automated protocol. Based on this review, an automated protocol is proposed. Fecal samples should be collected and immediately processed under anaerobic conditions at either room temperature (RT) for a maximum of 4 h or at 4 °C for no more than 24 h. Upon homogenization, preferably in the absence of added solvent to allow addition of a buffer of choice at a later stage, aliquots obtained should be stored at either -20 °C for up to a few months or -80 °C for a longer period-up to 2 years. Protocols for quality control should characterize microbial composition and viability as well as metabolic functionality.
PubMed: 37893032
DOI: 10.3390/biomedicines11102658 -
Frontiers in Veterinary Science 2023The use of shipping canine semen for artificial insemination has bloomed over the last 20 years. This allows for the spread of genetic material while overcoming...
The use of shipping canine semen for artificial insemination has bloomed over the last 20 years. This allows for the spread of genetic material while overcoming geographical or time-related challenges. The optimal sperm concentration for cooled semen transport in the dog is unknown. Often canine semen is extended 1:3-5 vol:vol without standardized sperm concentrations for cooled shipment. We compared different sperm concentrations for cooled storage and hypothesized that lower concentrations would result in better semen quality. Semen was collected from healthy client-owned dogs ( = 8). Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 ×10 sperm/ml (C200), then serially diluted to 100, 50, and 25 ×10 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24 h and then centrifuged and re-extended. Sperm concentration, plasma membrane integrity (PMI, %), motility (subjective total, STM; computer-assisted sperm analysis (CASA) total and progressive, TM, PM; %), and normal morphology (NM, %) were assessed in raw semen (T0), post-extension (T1), after 24 h of cooling (T2), and after processing at 24 h (T3). Cooling resulted in significant declines in STM and NM for all groups and in decreased PMI for CON and C25-50. After cooling (at T2), PMI was significantly lower for C25 compared with all the groups and higher for CON compared with C25-100 ( ≤ 0.038). Processing and re-extension after cooling further decreased the spermiogram parameters. At T3, PMI for CON was similar to C200 but significantly higher than C25-100, while C25 had the lowest PMI. For motility parameters and NM, C25 performed worse than all or most of the other groups. Comparing CON at T3 with C25-200 at T2, PMI, STM, and NM for CON were significantly lower than C25-200, C200, and C100-200, respectively. In conclusion, our results show that cooling canine semen for 24 h at 200 ×10 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on the highest PMI. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24 h of cooled storage.
PubMed: 38347887
DOI: 10.3389/fvets.2023.1339840 -
Journal of Laboratory Physicians Dec 2020The present COVID-19 pandemic resulted in an increased need for molecular diagnostic testing. Delay in the specimen processing and suboptimal storage of suspected...
The present COVID-19 pandemic resulted in an increased need for molecular diagnostic testing. Delay in the specimen processing and suboptimal storage of suspected samples in laboratories leads to degradation of SARS-CoV-2 viral RNA. Viral lysis buffers from RNA extraction kits have the potential to stabilize RNA. Hence, this study aimed to investigate the stability of SARS-CoV-2 RNA in viral lysis buffer at different temperatures and time periods. Aliquots of samples with known SARS-CoV-2 RNA were processed in viral lysis buffers simultaneously, stored separately at 2 to 8°C and 22 to 28°C for 24 hours, 48 hours and 72 hours. SARS-CoV-2 viral RNA was extracted from each aliquot and analyzed using multiplex real-time PCR. SARS-CoV-2 RNA in samples placed in viral lysis buffer was stable for 48 hours at both 2 to 8°C and 22 to 28°C temperatures. Slight decline in the viral RNA quantity was found on aliquots tested after 48 hours of both the temperatures. Viral lysis buffer maintains the integrity of SARS-CoV-2 RNA for up to 48 hours even at room temperature and supports delayed diagnosis with an overwhelming sample load in testing laboratories.
PubMed: 33390676
DOI: 10.1055/s-0040-1722551 -
Hormone and Metabolic Research =... Jul 2022We assessed the impact of intact parathyroid hormone (iPTH) and adjusted calcium analyses on Abbott, Roche and Siemens analytical platforms in the diagnosis of...
We assessed the impact of intact parathyroid hormone (iPTH) and adjusted calcium analyses on Abbott, Roche and Siemens analytical platforms in the diagnosis of normocalcaemic primary hyperparathyroidism (NCPHPT). These assays are used by over 85% of clinical laboratories in the UK. Over five months, consecutive serum samples from outpatients with NCPHPT in the laboratory with Abbott assays were identified, aliquoted and stored at -80°C. Frozen aliquots were transported monthly to the other two laboratories. After thawing, samples were mixed and analysed immediately for calcium, albumin and iPTH in the laboratories with Abbott, Roche and Siemens analytical platforms. Adjusted calcium was calculated using the equation used in the respective laboratory. Diagnostic concordance of iPTH and adjusted calcium were assessed using manufacturer-provided assay-specific reference intervals and the pathology harmony reference interval respectively. Fifty-five patients with NCPHPT were identified using Abbott assays. Of these, 16 (29.1%) and 11 (20.0%) had NCPHPT, 9 (16.4%) and 13 (23.6%) had hypercalcaemic primary hyperparathyroidism, and 30 (54.6%) and 31 (56.4%) patients had normal results when analysed in laboratories with Roche and Siemens assays, respectively. The diagnosis of NCPHPT was strikingly different depending on the commercial assay used. There is a pressing need for iPTH assay harmonisation and robust reference intervals. Reference intervals may become invalid if an assay drifts, as exemplified by adjusted calcium in this study.
Topics: Calcium; Humans; Hypercalcemia; Hyperparathyroidism, Primary; Laboratories; Parathyroid Hormone
PubMed: 35835142
DOI: 10.1055/a-1856-4900