Potential applications of CRISPR/Cas for next-generation biomonitoring of harmful algae blooms: A review.

Research on harmful algal and cyanobacterial blooms (HABs and CHABs) has risen dramatically due to their increasing global distribution, frequency, and intensity. These blooms jeopardize public health, ecosystem function, sustainability and can have negative economic impacts. Numerous monitoring programs have been established using light microscopy, liquid chromatography coupled to mass spectrometry (LC-MS), ELISA, and spectrophotometry to monitor HABs/CHABs outbreaks. Recently, DNA/RNA-based molecular methods have been integrated into these programs to replace or complement traditional methods through analyzing environmental DNA and RNA (eDNA/eRNA) with techniques such as quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), sandwich hybridization assay (SHA), isothermal amplification methods, and microarrays. These have enabled the detection of rare or cryptic species, enhanced sample throughput, and reduced costs and the need for visual taxonomic expertise. However, these methods have limitations, such as the need for high capital investment in equipment or detection uncertainties, including determining whether organisms are viable. In this review, we discuss the potential of newly developed molecular diagnosis technology based on Clustered Regularly Interspaced Short Palindromic Repeats/Cas proteins (CRISPR/Cas), which utilizes the prokaryotic adaptative immune systems of bacteria and archaea. Cas12 and Cas13-based platforms can detect both DNA and RNA with attomolar sensitivity within an hour. CRISPR/Cas diagnostic is a rapid, inexpensive, specific, and ultrasensitive technology that, with some further development, will provide many new platforms that can be used for HABs/CHABs biomonitoring and research.

Atomised lidocaine for airway topical anaesthesia in the morbidly obese: 1% compared with 2%.

Airway anaesthesia using atomised lidocaine for awake oral fibreoptic intubation in morbidly obese patients was evaluated using two doses of local anaesthetic. In this randomised, blinded prospective study, 40 ml of atomised 1% (n = 11) or 2% (n = 10) lidocaine was administered with high oxygen flow as carrier. Outcomes included time for intubation, patient tolerance to airway manipulation, haemodynamic parameters, the bronchoscopist's overall satisfaction, and serial serum lidocaine concentrations. Patients receiving lidocaine 1% had a longer mean (SD) time from the start of topicalisation to tracheal tube cuff inflation than those receiving lidocaine 2% (8.6 (0.9) min vs 6.9 (0.5) min, respectively; p < 0.05). Patients in the 1% cohort demonstrated increased responses to airway manipulation (p < 0.0001), reflecting lower bronchoscopist's satisfaction scores (p < 0.03). Haemodynamic responses to topicalisation and airway manipulation were similar in both groups. Peak plasma concentration was lower in the 1% group (mean (SD) 1.4 (0.3) and 3.8 (0.5) microg.ml(-1), respectively; p < 0.001). Airway anaesthesia using atomised lidocaine for awake oral fibreoptic intubation in the morbidly obese is efficacious, rapid and safe. Compared with lidocaine 1%, the 2% dose provides superior intubating conditions.

High pentamidine levels associated with hypoglycemia and azotemia in a patient with Pneumocystis carinii pneumonia.

We report on a patient who presented with a Pneumocystis carinii pneumonia. Intravenous pentamidine (4 mg/kg/day) was given for 14 days without the occurrence of adverse effects. During this treatment, the mean (+/- SD) serum pentamidine trough concentration was 94 +/- 16 ng/ml. Three days later, the patient was admitted because of fever, and pentamidine (4 mg/kg/day) was again started. Fasting hypoglycemia and azotemia then occurred; the mean serum trough pentamidine level was 190 +/- 10 ng/ml during this week of treatment. We conclude that the occurrence of hypoglycemia and azotemia during pentamidine therapy may not be idiosyncrasic, but seemed associated in our patient with high levels of serum pentamidine.

The in vitro effect of drugs on biochemical parameters determined by a SMAC system.

1. We have studied the in vitro effect of 39 drugs on 17 biochemical parameters determined by a SMAC System. Only two drugs were found to interfere: ascorbic and theophyline. 2. The ascorbic acid lowers the glucose and the bilirubine values; it increases the creatinine and the uric acid concentration. At concentration smaller than 5 mg/dl of this drug, these effects are negligible. 3. We have found a new drug interference: theophylline inhibits the alkaline phosphatase and LDH activities. This effect is not negligible on alkaline phosphatase for therapeutic levels of this drug; the action on LDH can be ignored at normal therapeutic range. 4. For a given drug, we have found different interference with biochemical parameters determined with various commercial lyophlised control sera or a liquid pool of sera. This indicates that the type of sera used in drug interference studies must be described.

EMIT antiepileptic drug assays adapted to an Abbott-VP Analyzer and compared with a gas-chromatographic procedure.

The Abbott-VP Bichromatic Analyzer was used for the determination of four antiepileptic drugs (phenobarbital, phenytoin, carbamazepine, primidone) in serum by means of a modified EMIT homogeneous enzyme immunoassay procedure. The main objective of the work was to examine the precision and the accuracy of the results obtained with this system and its cost effectiveness in comparison to the manual method. Day-to-day precision for all four drugs is excellent with coefficients of variation averaging around 5% in the therapeutic range of concentrations. Results for sera analyzed by this procedure and by a gas chromatographic method do not show any significant difference for phenytoin and phenobarbital. There are however slight differences between the two methods for carbamazepine and primidone. These small differences do not modify significantly the clinical interpretation of the results. The procedure is simple and rapid, and requires only one third of the reagents needed in the recommended manual EMIT procedure using the Gilford Stasar III, thus greatly reducing the operation costs.

Stability of PO2, PCO2, and pH in fresh blood samples stored in a plastic syringe with low heparin in relation to various blood-gas and hematological parameters.

OBJECTIVE: To assess the stability of PO2, PCO2 and pH in fresh individual blood samples drawn in a plastic syringe with low heparin and stored at 22 degrees C or on ice in relation to various biochemical and hematological parameters of the samples. METHODS: PO2, PCO2, and pH were measured at determined times in samples kept at 22 degrees C or on ice. The magnitude of change in blood-gas parameters over time for each group of samples was determined. The change in PO2 was examined according to its initial value, the oxygen content (CaO2), the hemoglobin concentration, the leukocyte and platelet counts of each sample. RESULTS: The changes in PO2, PCO2, and pH over time were about three times lower in samples stored on ice than in the ones at 22 degrees C. Nevertheless, rapid increases in PO2 were observed for samples stored on ice with an initial PO2 between 50 and 250 mm Hg. Samples with PO2 over 250 mmHg exhibited a decrease in PO2 over time. No correlation was observed between the change in PO2 and the oxygen content, the hemoglobin concentration or the leukocyte and platelet counts for both groups of samples stored at 22 degrees C or on ice. CONCLUSION: PO2 in samples with an initial PO2 between 50 and 250 mm Hg stored on ice should be analyzed within 30 min. However, PCO2 and pH didn't exhibit clinically significant changes within 60 min under similar conditions. The magnitude of change in PO2 for samples kept on ice was dependent on their hemoglobin capacity for buffering oxygen, which is in converse relation with the oxygen saturation level of hemoglobin (SatHbO2). It is believed that the alteration in PO2 for samples stored on ice is produced predominantly by diffusion of oxygen through the plastic wall of the syringe, whereas it would be mainly of metabolic origin in samples at room temperature.

Clinical usefulness of high-pressure liquid chromatographic determination of serum pentamidine in AIDS patients.

We have developed a reproducible HPLC method to determine serum pentamidine, which demonstrates good chromatographic performance, and is sensitive enough to measure therapeutic doses. Pentamidine is first extracted from serum by passage through a C-18 extraction cartridge. Potential interfering substances are then removed by washing with 100% methanol. Pentamidine is eluted from the extraction cartridge with 1-heptanesulfonic acid. The extract is chromatographed on a highly deactivated column for basic compounds in the presence of minimal concentrations of 1-heptanesulfonic acid as the pairing agent. Detection is by fluorescence. The method can determine serum pentamidine levels in the range of 15-600 ng/mL free of interference from other drugs. In monitoring pentamidine levels in AIDS patients with Pneumocystis carinii, we found that trough serum levels over 100 ng/mL were associated with toxicity (hypoglycemia or azotemia) in 100% of patients. With levels under 100 ng/mL, signs of toxicity were observed in only 29% of the patients. We conclude that dose adjustment based on serum levels reduces the incidence of toxicity and enhances pentamidine therapy.

The penetration of oral ciprofloxacin into the aqueous humor, vitreous, and subretinal fluid of humans.

We examined ciprofloxacin levels in the aqueous humor, vitreous, or subretinal fluid in 40 patients undergoing cataract extraction, vitrectomy, or scleral buckling. Ciprofloxacin, 750 mg, was administered orally an average of 17 1/2 and 5 1/2 hours preoperatively. We obtained mean ciprofloxacin levels of 0.53 microgram/ml in aqueous humor, 0.51 microgram/ml in vitreous, and 0.71 microgram/ml in subretinal fluid. These vitreous levels exceed the minimum inhibitory concentration (MIC)90 of Staphylococcus epidermidis, Propionibacterium species, Pseudomonas aeruginosa, Proteus mirabilis, and Haemophilus influenzae, as well as the MIC70 of S. aureus and Bacillus cereus. Therefore, ciprofloxacin may have a role in the management and prevention of endophthalmitis.

Airway topicalisation in morbidly obese patients using atomised lidocaine: 2% compared with 4%.

We evaluated the technique of airway anaesthesia using atomised lidocaine for awake oral fibreoptic intubation in morbidly obese patients using two doses of local anaesthetic. Morbidly obese patients were allocated to receive either 2% or 4% lidocaine (40 ml) for oral airway anaesthesia using an atomiser with high oxygen flow. Patients were carefully sedated using midazolam and fentanyl. Outcomes included patient tolerance to airway manipulation, haemodynamic parameters, and serial plasma lidocaine concentrations. In all, 27 patients were enrolled in the study (2% cohort n = 14, 4% cohort n = 13). Patient characteristics and time for topicalisation and airway management were similar. Haemodynamic parameters did not change significantly. Tolerance to insertion of the Ovassapian airway, bronchoscopy, and tracheal tube positioning was excellent (12 vs 12 patients, 12 vs 12 patients, and 8 vs 12 patients had no response, respectively, 2% vs 4%). Differences did not reach statistical significance. Peak plasma lidocaine concentration was significantly lower in the 2% group (2.8 (0.8) microg.ml(-1) compared with 6.5 (1.0) microg.ml(-1), p < 0.05). Airway anaesthesia using atomised lidocaine for awake fibreoptic intubation in the morbidly obese is efficacious, rapid, and safe. Compared with 4% lidocaine, the 2% dose provides acceptable intubating conditions in most cases and produces lower plasma lidocaine levels.

Column deactivation in analysis for underivatized tricyclic antidepressants by gas chromatography with use of a nitrogen detector.

I describe deactivation treatment of the OV-17 chromatographic column to minimize adsorption of tricyclic antidepressant drugs on the solid support of the column. The procedure involves heat treatment at 399 degrees C under a low flow of nitrogen, with bleeding of OV-17 liquid phase from the injector tube into the column. The column is then conditioned with vapors of phenyldiethanolamine succinate, added to the carrier gas stream by bleeding from a coated injector glass tube. This deactivation process much improves the chromatographic performance of the column, allowing a sensitivity at the nanogram level with a nitrogen-sensitive detector. Determinations of tricyclic antidepressants in plasma with such a deactivated column results in a low CV and a linear calibration curve, reflecting the effectiveness of the deactivation.

An evaluation of three new blood-gas analyzer systems.

Three new pH-blood-gas analyzer systems have been evaluated. These are the ABL-1, the Croning 165, the Il 513. Because of automation of analysis, accuracy, and speed of analysis, this generation of instruments represents great progress over the previous one. The ABL-1 is the most automated system: all analyses are computer controlled. The pH, P02 and PCO2 values are accurate. Calibration can not be adjusted. The Corning 165 is the least automated system. Its pH determinations are the most accurate and it is the one which uses the smallest volume of blood (125 mul). However it is the least stable and takes the longest time for completion of the analysis. The IL 513 is probably the best balanced system and its calibration can be modified. Its operation is well automated and its results are accurate.

An enzymic assay for the specific determination of methanol in serum.

This method for the specific determination of methanol in serum is based on the following two reactions: (formula; see text) Alcohol oxidase is not specific: it converts all lower alcohols to their corresponding aldehydes; however, formaldehyde dehydrogenase is specific and thus the transformation of NAD+ to NADH (which is used to monitor the reaction) proceeds only if methanol is originally present in the sample. The method was automated with a Roche COBAS FARA centrifugal analyzer. The calibration curve is linear between 0.6 and 12 mmol/L. The detection limit is about 0.6 mmol/L. The CV is 4.6% for a concentration of 3 mmol/L. When 55 serum specimens known to be free of methanol were supplemented with known amounts of methanol and analyzed by the enzymatic method, the results correlated well (r = 0.987) with the true values, the regression equation being: y = 1.016x + 0.661, where x represents the true values. Results are not affected by other alcohols that may be present in serum, by methanol metabolites, or by some commonly prescribed drugs. The major advantage of this new assay is that it can be used 24 h a day in any clinical chemistry laboratory.

Improved determination of serum theophylline by gas chromatography with use of a nitrogen-phosphorus detector.

We describe a method for the 7-min determination of theophylline in less than 100 muL of serum. The procedure requires no centrifugation or solvent evaporation after extraction. Butylation is done on the gas-chromatographic column by injecting the serum extract followed by a butylating mixture which contains 1-iodobutane as the alkylating agent. The method is precise, accurate, and free of interference. Results correlate well with those by ultraviolet spectrophotometry.

Enzymic assay for serum theophylline.

We describe an original procedure for determination of theophylline in serum. The drug is extracted from 0.4 mL of serum at pH 7.4 with chloroform/isopropanol (20/1 by vol) and back-extracted into sodium hydroxide (1 mmol/L). The inhibition of beef-liver alkaline phosphatases by theophylline in this alkaline phase is measured at 25 degrees C, with p-nitrophenyl phosphate as substrate, in 2-amino-2-methyl-1-propanol buffer, pH 9.4- The reciprocal of enzyme activity and theophylline concentration are linearly related in the range 2 to 60 mg/L. The maximum interference to be expected from 3-methylxanthine would increase apparent theophylline concentration by no more than 1 mg/L. The method is accurate, free of interference by other xanthines and often-coadministered drugs, and results correlate well with those by the immunoenzymic assay. Major advantages are reagent stability, low cost, and simplicity of instrumentation.

In vivo higher glucuronidation of mycophenolic acid in male than in female recipients of a cadaveric kidney allograft and under immunosuppressive therapy with mycophenolate mofetil.

Mycophenolate mofetil (MMF), an immunosuppressant drug used in organ transplantation to prevent rejection, is being used increasingly in association with cyclosporine and tacrolimus. Mycophenolic acid (MPA) is primarily metabolized in the liver to its 7-O-glucuronide (MPAG) derivative. The concentrations of MPAG in serum are many times the concentrations of MPA. Although MPAG has not shown immunosuppressant activity, it was postulated that it could displace MPA from its binding sites on albumin and hence increase the biologic effects of MPA. This effect could be important for patients with acute renal failure; under this condition, MPAG was shown to accumulate. The goal of this study was to document the MPAG/MPA concentration ratio in 100 renal transplant patients under a mixed immunosuppressive therapy. Further, the study addressed the question of whether MPAG can displace MPA in vivo from bound albumin in a representative renal transplant patient population under immunosuppressive therapy. Levels of MPAG and MPA were measured by high-performance liquid chromatography. The distribution of the ratios was not parametric as it tailed toward elevated values. After a square root transformation of the data, parametric analysis was possible. The average MPAG/MPA ratio was 15.0 +/- 2.2 for men versus 7.7 +/- 0.9 for women. Men treated with MMF and tacrolimus showed a lower ratio than patients treated with MMF and cyclosporine, confirming that tacrolimus inhibits glucuronidation of MPA. Further, it was determined that at physiologic concentrations, MPAG does not increase the amount of free MPA. Because MPAG can favor the elimination of MPA, it can be concluded that gender differences and cotreatment with tacrolimus must be taken into consideration when MMF is being administered.

Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence.

To measure the concentration of nitrites and nitrates by chemiluminescence, we examined the efficiency of five reducing agents [V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(III)] to reduce nitrite (NO2-) and (or) nitrate (NO3-) to nitric oxide (NO). The effect of each reducing agent on the conversion of different amounts of NO2- and (or) NO3- (100-500 pmol, representing concentrations of 0.4 to 2 mu molar) to NO was determined at 20 degrees C for NO2- and at 80 degrees C for NO3-. The effect of temperature from 20 to 90 degrees C on the conversion of a fixed amount of NO2- or NO3- (400 pmol or 1.6 mu molar) to NO was also determined. These five reducing agents are similarly efficient for the conversion of NO2- to NO at 20 degrees C. V(III) and Mo(VI) + Fe(II) can completely reduce NO3- to NO at 80 degrees C. NaI and Cr(III) were unable to convert NO3- to NO. Increased temperature facilitated the conversion of NO3- to NO, rather than that of NO2- to NO. We evaluated the recovery of NO2- and NO3- from plasmas of pig and of dog. Recovery from plasma of both animals was reproducible and near quantitative.