Alkaline Protocol Research Articles

DNA integrity under alkaline conditions: An investigation of factors affecting the comet assay.

The effect of pH on DNA integrity was assessed using a three-step approach. The comet assay was used on a whole genome level, with three different protocols: neutral (no alkaline unwinding), flash (pH 12.5 with 2.5min unwinding), and the conventional alkaline protocol (pH>13 with 40min unwinding). Real-time quantitative PCR (RT-qPCR) was then used to study the isolated DNA, revealing that gene amplification decreased with increasing pH, indicating DNA degradation. Specially designed molecular beacons were used to examine DNA at the molecular level, with or without alkali-labile site (ALS) insertions. At pH 12.5, fluorescence in the hairpins with ALS started to increase after 30min, while at pH>13, this increase was already observed after 5min, indicating a significant increase in DNA strand breaks. Liquid chromatography analysis was also used, demonstrating that the hairpins remained intact up to pH 10, even after 1h exposure, whereas, at pH 12.5, partial conversion into strand breaks occurred after 30min. At pH>13, the hairpins were almost completely degraded after 30min. The flash protocol effectively detects DNA single- and double-strand breaks and identified these damages after 2.5min of alkaline treatment at pH 12.5. When the hairpins were exposed to pH 12.5 for 60min, ALS were converted to strand breaks, demonstrating the sensitivity of this approach to detect changes in DNA structure. These findings indicate that pH poses a substantial risk to DNA integrity, leading to significantly higher background levels of DNA damage compared to conditions closer to neutrality. Our study demonstrates the importance of understanding the influence of pH on DNA stability and provides insights into risks associated with alkaline environments, especially at pH>13.

Microplastic extraction protocols can impact the polymer structure

Although microplastics are ubiquitous in today’s natural environments, our understanding of the materials, quantities, and particle sizes involved remains limited. The recovery of microplastics from different types of environmental matrices requires standardized matrix digestion protocols that allow inter-laboratory comparisons and that have no effect on the polymers themselves. A number of commonly used digestion methods rely on oxidation with concentrated hydrogen peroxide solutions to remove organic matter from the matrix. However, this can alter the nature of polymers through hydrolysis and often does not lead to a complete matrix removal. We have therefore investigated the use of two altered matrix digestion protocols, an acidic (Fenton) protocol and a new alkaline (Basic Piranha) protocol, focusing mainly on the effect on biodegradable polymers (polylactide, polybutylene adipate terephthalate, polybutylene succinate) and polymers with known degradation pathways via hydrolysis (thermoplastic polyurethanes, polyamide). Comparing the initial surface textures, chemical compositions, and particle size distributions with those obtained after digestion revealed that the Fenton protocol left most of the polymers unchanged. The ferrous residue that remains following Fenton digestion had no effect on either the polymer composition or the particle size distribution, but could disturb further analytics (e.g. Raman microscopy due to auto-fluorescence). While increasing the chance of complete matrix removal, the more powerful Basic Piranha protocol is also more likely to affect the polymer properties: Polylactide polymers in particular showed signs of degradation under alkaline digestion (reduced polylactide content, holes in the polymer matrix), indicating the unsuitability of the Basic Piranha protocol in this specific case. Polyamide, however, remained stable during the Basic Piranha treatment, and the surface chemistry, the particle size as well as the molar mass distribution of the investigated thermoplastic polyurethanes were also not affected. Hence, this protocol offers a powerful alternative for microplastic analysis with focus on particle size in more complex environmental matrices (e.g. removal of cellulose in soil), while avoiding ferrous Fenton residue. Unexpectedly, also tire rubber, a frequent target analyte in microplastic monitoring, was found to be susceptible to artefact structures by both oxidation protocols. In summary, controls for the specific combination of polymer and sample preparation protocol are highly recommended to select the most fitting protocol. Here selected suitable combinations are reported.

Standardized Supportive Care Documentation Improves Safety of High-Dose Methotrexate Treatment.

BackgroundHigh‐dose (HD) methotrexate (MTX) is an essential component of treatment protocols in acute lymphoblastic leukemia, aggressive lymphoma, and osteosarcoma. However, delayed MTX clearance may lead to life‐threatening toxicities. Administration of supportive therapy for HD‐MTX is complex, and insufficient supportive care increases the risk of MTX toxicity. To improve patient safety, we investigated the implementation of a checklist and urine alkalinization protocol in addition to standard supportive care during HD‐MTX therapy.Materials and MethodsThe intervention included individualized patient checklists for control of adequate supportive care for every HD‐MTX treatment cycle and a urine alkalinization protocol for documentation and guidance during urine alkalinization therapy. The impact of these tools on the rate of adverse events (acute renal injury, delayed MTX clearance) was retrospectively assessed in patients treated from April 2017 to April 2019 (intervention group) and compared with patients treated from January 2015 to March 2017 who received standard supportive care for HD‐MTX according to a standard operating procedure (SOP).ResultsIn total, 118 patients received 414 HD‐MTX cycles in the intervention group compared with 108 patients with 332 treatment cycles in the SOP group. Delayed MTX clearance was observed in 2.6% of treatment cycles in the intervention cohort opposed to 15.2% of cycles in the SOP group. The rate of acute kidney injury was also significantly reduced in the intervention group (6.2%. vs. 0.7%). The use of carboxypeptidase as rescue treatment for severe renal impairment and insufficient MTX clearance was necessary in five cases in the SOP group and in only two cycles within the intervention group.ConclusionThe use of standardized documentation for supportive care during HD‐MTX therapy is recommended to minimize the risk of adverse events.Implications for PracticeHigh‐dose methotrexate (HD‐MTX) is a commonly used treatment in several cancer types. Distinct supportive measures are necessary to minimize the risk of HD‐MTX side effects, which can be life‐threatening. Supportive care consists of certain examinations and interventions before starting HD‐MTX and permanent alkalinization of the urine, as this greatly increases the elimination of MTX and decreases the risk of kidney injury. After implementing a checklist for control of supportive care and a urine alkalinization protocol to optimize urine alkalinization, a significant decrease of side effects was observed in comparison to the standard of care; therefore, the use of a safety checklist and alkalinization protocol is recommended for all patients who receive HD‐MTX.

Synergistic Action of Alkalis Improve the Quality Hemicellulose Extraction from Sugarcane Bagasse for the Production of Xylooligosaccharides

The present study demonstrates an effective alkaline protocol for the extraction of quality hemicellulose using NaOH and NH4OH combination from sugarcane bagasse for the production of xylooligosaacharides (XOs). For achieving maximum recovery of hemicellulose, the statistical optimization technique was adapted, while considering alkali concentration and temperature, as important process parameters, for all of the alkaline tested protocols like individual or mixed alkalis. The comparative study exposed that mixed alkalis promoted higher recovery of hemicellulose (68% wt xylose), which is relatively higher (up to 1.3-times) than the individual alkali protocols, such that it contained predominantly the xylose (xyl/ara ratio was 3.94) in the form of xylan. In perception, the synergistic action of different alkalis (weak and strong bases) have strongly influenced the selective cleavage of lignin-carbohydrate linkages, thereby enabling the higher release of hemicellulose under the modest reaction conditions (10% alkali conc. and 120 °C). Moreover, the analytical characterization witnessed that it is composed of majorly xylose with less or no undesired residual biomass constituents, including lignin. Upon evaluating the resultant hemicelluloses for XOs production via acidic hydrolysis, the hemicellulose obtained through the mixed alkalis protocol exhibited exceptional, resulting in ~ 13% wt XOs yield with a high degree of polymerization (2–4 units); it is relatively ~ 2.8-times higher than the result of other hemicelluloses. In addition, the formation of gaseous ammonia during the reaction of the mixed alkalis could potentially contribute to the reduction of overall processing cost through recovery and reuse strategy during the large-scale XOs production.

Evaluation of methotrexate clearance with an enteral urine alkalinization protocol for patients receiving high-dose methotrexate.

High-dose methotrexate is a cytotoxic agent used to treat several malignancies. Urine alkalinization with sodium bicarbonate and hyperhydration are given with methotrexate to prevent drug precipitation in the kidneys. Due to a nationwide intravenous sodium bicarbonate shortage, an enteral-based urine alkalinization protocol was instituted. This study compared outcomes and adverse effects between the previously used intravenous and newly implemented enteral protocols. Single center retrospective cohort study comparing parenteral and enteral urine alkalinization for patients that received methotrexate doses ≥ 500 mg/m2 between 1 April 2016 and 1 October 2018. The primary endpoint was time to methotrexate clearance. Secondary outcomes included length of stay, time to administration of methotrexate, amount of sodium bicarbonate utilized, toxicities of methotrexate, and protocol-associated adverse effects. There were 67 patients included in the study for a total of 195 infusions. The average time to methotrexate clearance between the two cohorts was similar (parenteral 88 h vs. enteral 98 h p = 0.06). Likewise, length of stay was not different between the two cohorts (p = ns). The enteral cohort methotrexate's doses were initiated faster and received significantly less intravenous sodium bicarbonate when compared to the parenteral cohort (p = 0.04). Rates of acute kidney injury, neutropenia, hepatotoxicity, and mucositis were similar between the two groups. There were higher rates of diarrhea and low serum bicarbonate values in the enteral cohort. This study supports the ability to conserve intravenous sodium bicarbonate by using an enteral-based urine alkalization regimen for HD methotrexate, with no difference in outcomes or toxicity.

Evaluation of decellularization protocols for production of porcine small intestine submucosa for use in abdominal wall reconstruction.

Porcine-derived acellular biologic grafts are increasingly used in abdominal wall reconstruction and other soft tissue repairs. In a previous work, we have shown porcine small intestine submucosa (PSIS) exhibits clear advantages over porcine pericardium (PPC) and porcine acellular dermal matrix (PADM) in repairing full-thickness abdominal wall defects. In the present study, we aim to determine, quantify, and compare the effects of two most commonly used decellularization protocols on biomechanical and biocompatible properties of PSIS. After mechanical preparation, PSIS was treated with either alkaline and acid (AA) protocol or sodium dodecyl sulfate (SDS) protocol. Cellular content removal, preservation of matrix components, micro- and ultra- structures, and mechanical properties were compared. The host responses were evaluated using PSIS for repairing rat abdominal wall defects. With regard to the absence of cellular contents, neatly arranged collagen fiber structures, better retention of growth factors, better mechanical strength, lower degrees of local and systemic inflammatory responses, higher degree of vascularization and tissue ingrowth, alkaline and acid protocol exhibits clear advantages over SDS protocol for the preparation of PSIS extracellular matrix.

Biochemical and molecular tolerance of Carpobrotus acinaciformis L. halophyte plants exposed to high level of NaCl stress

Carpobrotus acinaciformis L. plant is a kind of halophyte that is able to survive in high salt conditions. It is important to determine its physiological, biochemical and molecular limit of NaCl stress if one aims to use it for phytoremediation purpose. In this study, the alkaline protocol of the modified plant comet assay were used for rapid detection of DNA damage in C. acinaciformis L. plants exposed to a series of NaCl stress concentrations (0-, 50-, 100-, 200-, 300-, 400 and 500 mmol L-1) in hydroponic conditions for 2 weeks. DNA damage was measured as the values of percentage of DNA in tails and tail length. The halophyte C. acinaciformis L. did not show any dose response up to 400 mmol L-1 NaCl level in terms of DNA damages. DNA integrity measured via comet assay showed that DNA preserved its original shape up to 400 mmol L-1 NaCl level. However, the very high concentrations of NaCl (400 and 500 mmol L-1) caused DNA damages. When physiological and biochemical parameters such as proline, chlorophyll a and b, peroxidase (POX), catalase (CAT), H2O2, malondialdehyde (MDA) contents were examined, oxidant molecules such as H2O2 (0.912-3.72 µmol g-1 Fwt) and MDA (7.1-34 nmol g-1 Fwt) gradually increased along with the increase of NaCl concentrations, p<0.05. On the other hand, antioxidant enzyme POX and an osmolyte molecule proline slightly increased up to 400 mmol L-1 NaCl level then slightly decreased after that. Similar issues were obtained from that of protease enzyme which indicates the power of protein hydrolysis in which a slight decrease (182-95 Unit mg-1 protein) over a dose of NaCl was evident. Chlorophyll contents and CAT activity were not affected upon increase of NaCl concentrations. This study showed that the halophyte C. acinaciformis L. can be easily used to remove salt up to 400 mmol L-1 NaCl concentrations from a saline-affected soil. Measuring DNA damage is concluded as a very useful parameter to find out what level of NaCl could be tolerated if a halophyte plant is aimed to remediate the saline soils.

Safety and efficacy of a urine alkalinization protocol developed for high-dose methotrexate patients during intravenous bicarbonate shortage.

Urinary alkalinization with intravenous sodium bicarbonate is standard during high-dose methotrexate administration. Due to a national intravenous sodium bicarbonate shortage, a urinary alkalinization protocol involving hyperhydration with intravenous fluids, oral bicarbonate, and intravenous or oral acetazolamide was utilized from 10 April to 30 May 2017 ("shortage protocol"). This study compared outcomes between protocols. A single-center, retrospective chart review was conducted for adults who received methotrexate ≥500 mg/m2 on ≥ two occasions, at least once during each protocol, between 19 February and 19 July 2017. Eighteen patients (50% male), median age 65 years, received 76 total high-dose methotrexate cycles. Shortage protocol was used in 37 cycles (48.7%). Mean time to methotrexate clearance did not differ between groups (p = ns). Mean time to urinary alkalinization and duration of hospitalization were not statistically different (p = 0.49 and 0.23, respectively). Average total bicarbonate administered per 24 hours was higher in standard protocol (p < 0.05), but hydration rates were similar (p = 0.73). Creatinine clearance and urine output on days 1 and 2 post-high-dose methotrexate did not significantly differ (creatinine clearance day 1, p = 0.27; creatinine clearance day 2, p = 0.55; urine output day 1, p = 0.62; urine output day 2, p = 0.60). Interruptions in alkalinization were significantly higher during shortage (0.41 ± 0.75 instances of urine pH < 7 during standard vs. 1.3 ± 1.7 under shortage, p < 0.05).

Nanomaterials induce DNA-protein crosslink and DNA oxidation: A mechanistic study with RTG-2 fish cell line and Comet assay modifications

Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs.

Extraction of arabinoxylan from corncob through modified alkaline method to improve xylooligosaccharides synthesis

A modified alkaline protocol involving a combination of NaOH and NH4OH was employed for the isolation of hemicellulose from corncob. It was possible to obtain a hemicellulose fraction consisted of nominal branching constituents, such as arabinose, uronic acid and lignin. During the extraction of hemicellulose, alkaline reagents promoted selective cleavage of ester and ether linkages in corncob biomass depending on its nature and severity. Based on modeling analysis, reaction parameters, such as alkali concentration and temperature significantly influenced the amount of total hemicellulose extracted. Subsequent hydrolysis of isolated hemicellulose in the presence of H2SO4 resulted in better conversion (69 wt%) with enriched XOs conc. (73.68% with DP up to 4) than other fractions under milder conditions. Advantageously, gas phase NH3 formation was achieved during the reaction, where NaOH and NH4OH mix was used at an equal ratio that could significantly help in reducing the overall processing cost of XOs production (through recovery and reuse) during large-scale manufacture.

Identification of polymorphisms in the myostatin and leptin genes of santa inês breed and crossbreed sheep and association with carcass traits

The objective of this study was to identify polymorphisms in the myostatin and leptin genes in Santa Ines (SI) and crossbreed (SI x Dorper) sheep, to verify the effect of these polymorphisms on carcass traits. We evaluated seventy sheep of 8-month-old at the Federal Institute of Espirito Santo, Brazil. Data collected were slaughter weight (SW), hot carcass weight (HCW), cold carcass weight (CCW), loin weight, tenderloin weight and fat thickness (FT). The hot carcass yield (HCY) was calculated by the formula (HCW/SW) x 100. We collected hairs from each animal for DNA extraction by the alkaline protocol. The animals were genotyped for the G>A mutation in nucleotide 9827 of the myostatin gene and for three polymorphisms in exon 3 of the leptin gene, by the PCR-RFLP technique. The amplicons the myostatin and leptin gene were cleaved with restriction enzyme for allelic discrimination. The alleles were recorded for each animal and analysis of variance was performed to check the influence of the mutations on the carcass traits. The mutant allele of the myostatin gene showed association with increased measures of CCW, FT and with reduced HCY. Among the three alleles of the leptin gene, only one showed an effect (increased CCW). The other alleles were not associated with any traits.

DNA damage in potato plants induced by cadmium, ethyl methanesulphonate and γ-rays

We have calibrated the alkaline protocol of the plant comet (Single Cell Gel Electrophoresis) assay as a method for detecting the extent of induced DNA damage in potato plants ( Solanum tuberosum L. cultivar Korela). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd 2+), a dose–response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd 2+-induced DNA damage in roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd 2+ treatments, but continuous Cd 2+ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic and apoptotic DNA fragmentation, as the affected plants had inhibited growth and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd 2+ genotoxicity in leaves after short-term treatments.

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay - the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops.

Induced DNA Damage Measured by the Comet Assay in 10 Weed Species

For most plant species growing in polluted areas no mutagenicity assays are available. We have studied the possibility of using the alkaline protocol of the Comet assay as a method for detecting induced DNA damage in wildly growing weeds. The monofuctional alkylating agent ethyl methanesulphonate (EMS) was applied on leaves of 10 weed species (ordered according to the diameter of the nuclei): Arabidopsis thaliana, Convolvulus arvensis, Bellis perennis, Urtica dioica, Lamium album, Chenopodium rubrum, Plantago media, Poa annua, Taraxacum officinale, and Agropyron repens. With increasing concentrations of EMS (2 to 10 mM) the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all weeds studied. Using the Head Extent parameter of the Komet version 3.1, we have measured the diameter size of the nuclei of the 10 weed species either immediately after the isolation of the nuclei or after 20 or 45 min of treatment with alkaline buffer (pH > 13). According to the increase of the diameter of the nuclei (including the formed halo) resulting from the to alkaline buffer treatment, electrophoretic conditions (unwinding and electrophoresis time) for the Comet assay can be selected for the individual weed species.

Monitoring DNA damage in wood small-reed (Calamagrostis epigejos) plants growing in a sediment reservoir with substrates from uranium mining.

For most plant species growing in polluted areas genotoxicity assays are not available. We have studied the possibility of using the alkaline protocol of the Comet assay as a method for detecting induced DNA damage in a grass Calamagrostis epigejos, growing wild in highly polluted areas. To calibrate the Comet assay for C. epigejos, two model mutagens were applied: the monofunctional alkylating agent ethyl methanesulfonate (EMS) and gamma-rays. With increasing concentrations of EMS (0 to 10 mM, 18 h treatment at 26 degrees C) applied on excised leaves, the DNA damage, as expressed by the tail moment (TM) values, increased from 4.7 +/- 0.9 to 60.8 +/- 2.7 microns. After gamma irradiation (0 to 30 Gy) the TM value increased from 4.2 +/- 0.2 to 48.1 +/- 1.7 microns. A 24 to 72 h recovery of leaves after EMS treatment in an EMS-free medium did not result in a significant change in the induced EMS damage. By contrast, a 24 h recovery after gamma-irradiation led to a complete repair of DNA damage measurable by the Comet assay. We have measured the DNA damage in nuclei of leaves of C. epigejos plants growing in the area of a sediment reservoir with substrates from uranium mining, where the ore was exploited through leaching with sulfuric acid. The average specific activity of natural radionuclides measured in the substrate was for 226Ra = 11,818 Bq kg-1, for 232Th = 66 Bq kg-1 and for 40K = 75 Bq kg-1. No significant increase in the DNA damage in plants growing on the sediment substrate above the DNA damage in control plants was detected by the Comet assay.

Alkalinization-Induced K+ Current of the Mouse Megakaryocyte

We have recently found that mouse megakaryocytes responded to extracellular alkalinization to pH >8.0, generating a K+ current under voltage-clamped conditions with the whole cell recording mode of the patch-clamp technique. The purpose of this study was to physiologically and pharmacologically characterize the alkaline-dependent K+ conductance of the megakaryocyte membrane. The alkalinization-induced K+ current (IALK) did not seem to be Ca2+-dependent since IALK was allowed to be generated under intracellularly Ca2+-buffered conditions with 10 mM EGTA, which completely prevented the generation of caffeine-induced Ca2+-activated currents of mouse megakaryocytes; and no [Ca2+]i elevation was evoked by the alkalinization protocol in contrast to a significant increase in [Ca2+]i in response to caffeine when [Ca2+]i was measured with a fura 2 ratiometry. IALK was strongly suppressed with tetraethylammonium (TEA), 4-aminopyridine (4-AP) and streptomycin (SM), but was completely resistant to quinidine (QND). The values of IC50 for the suppression of IALK with TEA, 4-AP and SM were 5.6, 0.47 and 1.5 mM, respectively. Voltage-gated K+ currents (IK) of the same megakaryocyte preparation were weakly suppressed with TEA and 4-AP, while they were significantly suppressed with either SM or QND. These results suggest that mouse megakaryocytes possess K+ conductance that was activated by extracellular alkalinization and that probably differs from conventional K+ conductance in its pharmacological properties.