Routine Laboratory Conditions Research Articles

Rapid detection of tomato spotted wilt virus by real-time RT-LAMP and in-field application

Tomato spotted wilt virus (TSWV) is considered one of the most threatening viruses worldwide for different economically important agricultural crops. In this scenario, it is important to perform an early detection by laboratory tests to prevent TSWV spread. A rapid and sensitive TSWV detection protocol based on real time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed in this work, also using cost-effective and simplified sample preparation procedure, to assess the suitability of the RT-LAMP assay in field conditions on tomato and pepper samples. A set of six primers was designed within the nucleotide sequence region coding for the nucleocapsid protein (N) of segment S, targeting a 220-nucleotide sequence. Sensitivity, specificity, accuracy, and in-field application of the real-time RT-LAMP assay were evaluated. The developed real-time RT-LAMP assay proved to be one thousand and one hundred times more sensitive than end-point RT-PCR and real-time RT-PCR methods, respectively, detecting a total of 9.191 × 101 genome copies as minimum target, and no cross-reactivity were detected with other viruses belonging to Tospoviridae and Bromoviridae families used as outgroup. In addition, the in-field application of the assay using the rapid sample preparation gave adequate and reliable results within 60 minutes, with an acceptable reaction delay when compared to canonical RNA extraction. The in-field analyses showed an increase of TSWV-positive samples (37%) detection compared with end-point RT-PCR and real-time RT-PCR (32% and 29%, respectively), particularly on asymptomatic samples, confirming that the real-time RT-LAMP assay can be implemented as a routine test both in-field and laboratory conditions as a rapid and sensitive technique for TSWV detection.

The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

Is the automated Elecsys tacrolimus assay on the Roche cobas e 602 analyzer an acceptable replacement for a liquid chromatography-tandem mass spectrometry-based assay?

This study evaluated the suitability and accuracy of the automated Roche Elecsys tacrolimus electrochemiluminescence immunoassay (ECLIA) by comparing it with a current laboratory-developed test by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The tacrolimus ECLIA was evaluated for precision, linearity, interference, and postextraction stability. Accuracy was compared with LC-MS/MS. The tacrolimus ECLIA assay is precise, exhibits a measuring range of 0.75 to 30 ng/mL, and is tolerant of significant interferences (plasma indices: hemolysis <2306, icterus <55, lipemia <1427, and biotin <1200 ng/mL). Comparison with LC-MS/MS revealed a 26% proportional bias in patient samples evaluated for tacrolimus concentration (y = 1.26x + 0.08; r2 = 0.97; Sy/x = 0.94; n = 43) and an absolute mean (SD) bias of 2.2 (1.7) ng/mL. Postextraction studies confirmed that samples were stable for up to 30 minutes under routine laboratory conditions. The 2 major challenges for implementation of the tacrolimus ECLIA assay are the postextraction sample stability and the significant proportional bias observed compared with the LC-MS/MS reference method. The 30-minute window for analysis of extracted samples is a practical challenge to the routine workflow of the core laboratory. In addition, disagreement between the immunoassay and LC-MS/MS methods can lead to discordant clinical interpretations and ultimately affect patient care.

Identification and characterization of a strong constitutive promoter stnYp for activating biosynthetic genes and producing natural products in streptomyces

BackgroundStreptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest.ResultsA strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain.ConclusionsThe promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2-3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions.

The principle of pooled calibrations delivers full correspondence between uncertainties of measurements of Na, Mg and Ni when determined using HR-CS FAAS

Methods for determination of elements in various types of samples are generally considered to be very precise and highly accurate. For reliable analysis of elements Na, Mg and Ni in food samples is it worthwhile to perform an extensive method validation of high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS) according to the principle of pooled calibrations (PoPC). Under routine laboratory conditions of analysis, elevated levels of relative uncertainty of measurement reaching values of more than 50% were detected, which jeopardized the validity of results, even when the measurements in the present study were performed with samples of tap water and borehole water. Comparison of relative uncertainties with corresponding literature results indicates that sample-signal differences may be due to detector noise rather than specimen variations.

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen.

The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions.

Performance evaluation of the Elecsys PIVKA-II and Elecsys AFP assays for hepatocellular carcinoma diagnosis.

Background and AimsProthrombin induced by vitamin K absence‐II (PIVKA‐II) is a serum biomarker linked to hepatocellular carcinoma (HCC), showing superiority to alpha‐fetoprotein (AFP) for early disease detection. We aimed to assess the clinical and analytical performance of the Elecsys® PIVKA‐II immunoassay in diagnosing HCC and evaluate PIVKA‐II's technical performance.MethodsSerum samples from adult cases (i.e. patients with a first‐time HCC diagnosis; n = 168) and disease controls (i.e. patients without HCC with an at‐risk condition; n = 208) were assessed. An AFP cut‐off of 20 ng/mL was used to differentiate between HCC cases and disease controls. Clinical performance of the Elecsys PIVKA‐II assay was compared with that of comparator assays (Lumipulse G PIVKA‐II, μTASWako DCP, ARCHITECT PIVKA‐II) using receiver operating characteristic curve analysis to determine the area under the curve (AUC) values.ResultsThe Elecsys PIVKA‐II assay compared favorably with comparator assays. Using a 28.4 ng/mL cut‐off, the Elecsys PIVKA‐II assay detected HCC with 86.9% sensitivity and 83.7% specificity. Clinical performance of the Elecsys PIVKA‐II assay (AUC: 90.8%) was equivalent to that of comparator assays (AUC: 88.3–89.6%). Relatively high PIVKA‐II concentrations were observed for cholangiocarcinoma and pancreatic cancer with the Elecsys assay in specificity panel analyses, indicating that high PIVKA‐II concentrations should not be used alone in the absence of other clinical data.ConclusionsThe Elecsys PIVKA‐II assay showed good analytical performance under routine laboratory conditions, comparing favorably with comparator assays. These findings support the suitability of the Elecsys PIVKA‐II assay as an aid in HCC diagnosis.

Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma

Asymmetric dimethylguanidino valeric acid (ADGV), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are three arginine metabolites which have utility in the assessment of cardiovascular disease, renal disease and non-alcoholic fatty liver disease (NAFLD). Translation of these research metabolomic markers into routine clinical use requires the development of robust assays with appropriately assessed preanalytical variables and traceable clinical reference intervals. A hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADGV, ADMA and SDMA was developed. Sample stability and collection conditions were scrutinised to determine any preanalytical factors that could affect quantification under routine laboratory conditions. Patient samples from 120 males and 120 females were used to derive preliminary reference intervals. All three analytes were quantifiable in human plasma using unique MS/MS transitions. The analytes were stable for up to a week once separated from red cells, though reduced stability was observed upon extraction of the analytes from plasma. The assay was linear for concentration of ADGV between 1.6nmol/L and 200nmol/L and for ADMA and SDMA between 0.1μmol/L and 4.0μmol/L. The accuracy for all analytes was 97-103% and interday and intraday imprecisions (coefficients of variation) were less than 10%. ADGV concentrations were noted to be lower in the female reference population when compared to males. The analytical method shows excellent performance and is sufficiently robust to be used in the clinical investigation of cardiovascular disease and NAFLD.

Performance of Fully Automated Antimicrobial Disk Diffusion Susceptibility Testing Using Copan WASP Colibri Coupled to the Radian In-Line Carousel and Expert System.

ABSTRACTThe purpose of the present study was to assess the agreement at the categorical level between the Vitek 2 system and the Colibri coupled to the Radian under real routine laboratory conditions. The 675 nonduplicate clinical strains included in this study (249 Enterobacterales isolates, 198 Pseudomonas aeruginosa, 107 Staphylococcus aureus, 78 coagulase-negative staphylococci, 38 Enterococcus faecalis, and 5 Enterococcus faecium) were isolated from nonconsecutive clinical samples referred to our laboratory between June and November 2020. In addition, 43 carbapenemase-producing Enterobacterales (CPE) formerly identified and stored in our laboratory were added to the panel, for a total of 718 strains. The overall categorical agreements between the two compared methods were 99.3% (4,350/4,380; 95% CI 99% to 99.5%); 98.6% (2,147/2,178; 95% CI 98.0% to 99.0%); 99.4% (1,839/1,850; 95% CI 98.9% to 99.7%); and 99.4% (342/344; 95% CI 97.9% to 99.8%) for Enterobacterales, P. aeruginosa, Staphylococcus spp., and Enterococcus spp., respectively. The most important cause of the very major errors encountered on the Vitek 2 for P. aeruginosa (62%, 13/21) was related to the presence of heteroresistant populations. Among the 43 CPE included in this study, one OXA-48-like, and one OXA-181-like were missed by the Vitek 2, even by rigorously applying the CPE screening cutoffs defined by EUCAST. The Colibri coupled to the Radian provide a fully automated solution for antimicrobial disk diffusion susceptibility testing with an accuracy that is equal to or better than that of the Vitek 2 system.

Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures.

BackgroundLeptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C).Methodology/Principal findingsL. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203.Conclusions/SignificanceInvestigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

An easy method for developing fusion enabled SARS-CoV2 virus fusion mimic (SCFM), bypassing the need of Bio Safety Level (BSL) facility

ABSTRACT Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory. Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Comparison of five different laboratory techniques for the rabies diagnosis in clinically suspected cattle in Brazil

The direct-fluorescent antibody test (dFAT) is considered the “gold standard” assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

On the influence of surface coating on tissue biomechanics \u2013 effects on rat bones under routine conditions with implications for image-based deformation detection

BackgroundBiomechanical testing using image-based deformation detection techniques such as digital image correlation (DIC) offer optical contactless methods for strain and displacement measurements of biological tissues. However, given the need of most samples to be speckled for image correlation using sprays, chemical alterations with impact on tissue mechanicals may result. The aim of this study was to assess the impact of such surface coating on the mechanical properties of rat bones, under routine laboratory conditions including multiple freeze-thaw cycles.MethodsTwo groups of rat bones, highly-uniform and mixed-effects, were assigned to six subgroups consisting of three types of surface coating (uncoated, commercially-available water- and solvent-based sprays) and two types of bone conditions (periosteum attached and removed). The mixed-effects group had undergone an additional freeze-thaw cycle at − 20 degrees. All bones underwent a three-point bending test ranging until material failure.ResultsCoating resulted in similar and non-significantly different mechanical properties of rat bones, indicated by elastic moduli, maximum force and bending stress. Scanning electron microscopy showed more pronounced mechanical alterations related to the additional freeze-thaw cycle, with fewer cracks being present in a bone from the highly-uniform group.ConclusionsThis study has concluded that surface coating with water- or solvent-based sprays for enhancing image correlation for DIC and having an additional freeze-thaw cycle do not significantly alter mechanical properties of rat bones. Therefore, this method may be recommended as an effective way of obtaining a speckled pattern.

Reliable, rapid and simple method for the analysis of phthalates in sediments by ultrasonic solvent extraction followed by head space-solid phase microextraction gas chromatography mass spectrometry determination

In this work, a new reliable, simple and fast method for the determination of six PAEs in sediments, based on ultrasonic solvent extraction (USE) followed by head space solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry determination (GC–MS), is proposed. The extraction parameters were studied, and the most favourable conditions were selected. The analytical features of the method were calculated: matrix effect, accuracy (ranged from 90% to 111%), repeatability and intermediate precision (RSD <10%), detection and quantification limits of the method (ranged from 0.001µgg−1 (DOP) to 0.142µgg−1 (DEP)), and satisfactory results were obtained. Major advantages of this approach are low consumption of reagents and solvents, no clean-up or evaporation steps were required and minimum sample manipulation. In addition, cross contamination from glassware, solvents and samples is minimized, thus procedural blanks are keeping to a minimum.. The applicability of the proposed method was demonstrated analysing sediment samples from Galician coast (NW Spain). The proposed method allows the application in routine laboratory conditions and its implementation in environmental monitoring studies under the European Water Framework Directive (WFD) and Marine Strategy Framework Directive (MSFD).

MALDI-TOF-Based Dermatophyte Identification.

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100% for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.

A broader look at ammonia production, excretion, and transport in fish: a review of impacts of feeding and the environment.

For nearly a century, researchers have studied ammonia production and excretion in teleost fish. Stemming from past investigations a body of knowledge now exists on various aspects including biochemical mechanisms of ammonia formation and specific routes and tissues used for ammonia transport, culminating in a current detailed theoretical model of branchial transport, including the molecular identities of the moieties involved. However, typical studies examining ammonia balance use routine laboratory conditions and fasted fish. While avoiding additional variables that influence nitrogen balance, these studies are arguably idealistic and do not reflect the natural variety of conditions that fish encounter. Further studies have revealed the impacts of extrinsic factors (e.g. salinity, pH, temperature) on ammonia handling in fasted fish whereas others have explored intrinsic factors, such as life history and developmental impacts. One routine challenge for ammonia balance that fish encounter is feeding and digestion. Fortunately, many new studies have revealed the impact of feeding and digestion on several aspects of ammonia balance; from production to excretion and to transport, and several have done so incorporating supplemental extrinsic and/or intrinsic factors. Together, these complex studies reveal realistic responses to multifactorial challenges encountered by animals in the wild and begin to provide a holistic view of ammonia balance in freshwater teleost fish.

Colchicine measurement using LC-MS/MS with ESI in serum with liquid liquid extraction

Familial Mediterranean fever (FMF) is an inherited autoinflammatory disorder characterized by recurring attacks of fever accompanied by intense pain in the abdomen, chest, or joints. Colchicine, is an important drug which is used in diseases such as FMF and acute gout attacks. However, it has significant side effects such as diarrhea, nausea, vomiting and fatigue. Some of the patients does not respond to treatment due to resistance. Therefore it is important to measure the serum levels of colchicine. The expected serum levels of colchicine is about 5-15 ng/ml. Correct measurement of this level with the classical analytical methods are not easy. Phase extraction and sample concentration of colchicine in routine laboratory conditions is time-consuming, so advanced analytical instruments such as LC-MS/MS and easy sample preparation methods are needed.

Harnessing Intracellular Biochemical Pathways for In Vitro Synthesis of Designer Tellurium Nanorods.

Synthesizing nanomaterials of desired properties is a big challenge, which requires extremely harsh conditions and/or use of toxic materials. More recently developed in vivo methods have brought a different set of problems such as separation and purification of nanomaterials made in vivo. Here, a novel approach that harnesses cellular pathways for in vitro synthesis of high-quality tellurium nanorods with tunable lengths and optical properties is reported. It is first demonstrated that in vivo biochemical pathways could be used to synthesize Te nanorods via the intracellular reduction of TeO3(2-) in living Staphylococcus aureus cells. The pathways to set up a quasi-biological system for Te precursor formation are then utilized, which could further synthesize Te nanorods in vitro. This allows to successfully synthesize in vitro, under routine laboratory conditions, Te nanorods with uniform and tunable lengths, ranging from about 10 to 200 nm, and controllable optical properties with high molar extinction coefficients. The approach here should open new avenues for controllable, facile, and efficient synthesis of designer nanomaterials for diverse industrial and biomedical applications.