Determination and health risk assessment of carbamate pesticide residues in date palm fruits (Phoenix dactylifera) using QuEChERS method and UHPLC-MS/MS.

This study aimed to investigate carbamate pesticide residues in different varieties of date palm fruits in the UAE, utilizing UHPLC-MS/MS. For sample preparation and clean-up, the efficiency and performance of different QuEChERS dispersive solid-phase extraction kits were compared. Precision and recovery were assessed at 10 µg kg-1 for the three kits, revealing that Kit 2 demonstrated the best performance. The selected QuEChERS method was validated to detect 14 carbamate residues in 55 date samples. The method exhibited strong linearity with R2 > 0.999 and low LOD (0.01-0.005 µg kg-1) and LOQ (0.003-0.04 µg kg-1). Excellent accuracy (recovery: 88-106%) and precision (RSD: 1-11%) were observed, with negligible matrix effect (- 4.98-13.26%). All samples contained at least one carbamate residue. While most detected residues were below their MRLs, carbosulfan was found in 21 samples, propoxur in 2 samples, and carbofuran in 1 sample above their MRLs. The hazard index (HI) was calculated for carbosulfan, phenmedipham, carbaryl, propoxur, carbofuran, and methomyl to assess potential health risks for date consumers. All HI values were below the safety limit of 1.0, indicating that the consumption of dates does not pose a non-carcinogenic health risk for adults and children.

Recent Advances in the Chromatographic Analysis of Emerging Pollutants in Dairy Milk: A Review (2018-2023).

Emerging pollutants (EPs) encompass natural or synthetic substances found in the environment that pose potential risks, but which have only recently been recognized or monitored. EPs consist of various categories, including pesticides, pharmaceuticals, hormones, mycotoxins, and endocrine-disrupting chemicals (EDCs). Through several pathways, EPs can access food, potentially leading to health impacts when safe concentrations are exceeded. Milk, being a highly nutritious food product that is heavily consumed by many consumers of different ages, is a crucial food matrix where EPs should be regularly monitored. In the literature, a large number of studies have been dedicated to the determination of different EPs in dairy milk, employing different analytical techniques to do so. Chromatography-based techniques are the most prevalent means used for the analysis of EPs in milk, demonstrating significant efficiency, sensitivity, and accuracy for this specific purpose. The extraction of EPs from a complex matrix like milk is essential prior to performing chromatographic analysis. This review comprehensively covers relevant research papers on the extraction and subsequent detection and determination of EPs in milk using chromatographic methods from 2018 to 2023.

Detection of 11 carbamate pesticide residues in raw and pasteurized camel milk samples using liquid chromatography tandem mass spectrometry: Method development, method validation, and health risk assessment.

This study aimed to use ultra-high-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer to detect 11 carbamate pesticide residues in raw and pasteurized camel milk samples collected from the United Arab Emirates. A method was developed and validated by evaluating limits of detection, limits of quantitation, linearity, extraction recovery, repeatability, intermediate precision, and matrix effect. Due to the high protein and fat content in camel milk, a sample preparation step was necessary to avoid potential interference during analysis. For this purpose, 5 different liquid-liquid extraction techniques were evaluated to determine their efficiency in extracting carbamate pesticides from camel milk. The established method demonstrated high accuracy and precision. The matrix effect for all carbamate pesticides was observed to fall within the soft range, indicating its negligible effect. Remarkably, detection limits for all carbamates were as low as 0.01 µg/kg. Additionally, the coefficients of determination were >0.998, demonstrating excellent linearity. A total of 17 camel milk samples were analyzed, and only one sample was found to be free from any carbamate residues. The remaining 16 samples contained at least one carbamate residue, yet all detected concentrations were below the recommended maximum residue limits set by Codex Alimentarius and the European Union pesticide databases. Nonetheless, it is worth noting that the detected levels of ethiofencarb in 3 samples were close to the borderline of the maximum residue limit. To assess the health risk for consumers of camel milk, the hazard index values of carbofuran, carbaryl, and propoxur were calculated. The hazard index values for these 3 carbamate pesticides were all below 1, indicating that camel milk consumers are not at risk from these residues.

Cucurbituril-assisted sensitive fluorescence detection and quantitation of naproxen drug in wastewater samples: Guest-host characterization and HPLC investigation.

Sensitive spectrofluorometric and liquid chromatography with fluorescence detection methods have been developed for detection and determination of naproxen drug in the presence of cucurbit7uril (CB7). Fluorescence signals have been improved with the addition of CB7 to the drug aqueous solution. Fluorescence spectroscopy, mass spectrometry, 1H-NMR, and liquid chromatography with fluorescence detection were used to investigate the guest-host interaction of naproxen drug and cucurbiturils. Naproxen was found to form a supramolecular complex with CB7 that had a high formation constant. The optimal conditions for the interaction were discovered using spectroflurometry to be 0.2 mg/ml of CB7, 2.4 µg/ml of naproxen drug, and pH10. A 1:1 complex between naproxen and CB7 is revealed by proton NMR and tandem mass spectrometry. Using the standard addition calibration method, an HPLC with a fluorescence detector was used to detect naproxen in influent and effluent wastewater samples. Finally, it was discovered that the measured concentrations of naproxen in the influent and the effluent wastewater were 1.87 × 10-4 ppb and 2.1 × 10-5 ppb, respectively. This was done by sample enrichment, which reduced the 1000 mL into 1 ml.

Detection of SARS-CoV-2 in COVID-19 Patient Nasal Swab Samples Using Signal Processing.

This work presents an opto-electrical method that measures the viral nucleocapsid protein and anti-N antibody interactions to differentiate between SARS-CoV-2 negative and positive nasal swab samples. Upon light exposure of the patient nasal swab sample mixed with the anti-N antibody, charge transfer (CT) transitions within the altered protein folds are initiated between the charged amino acids side chain moieties and the peptide backbone that play the role of donor and acceptor groups. A Figure of Merit (FOM) was introduced to correlate the relative variations of the samples with and without antibody at two different voltages. Empirically, SARS-CoV-2 in patient nasal swab samples was detected within two minutes, if an extracted FOM threshold of >1 was achieved; otherwise, the sample wasconsidered negative.

Enantioseparation of Synthetic Cathinones Enantiomers with Tertiary Amine Structure in Urine and Plasma.

A sensitive and selective method for detection and quantitation of the enantiomers of 18 synthetic cathinones with tertiary amine structure using HPLC-UV-VIS has been developed. Two chiral columns, Astec Cellulose DMP and Amylose-based Chiralpak AS-H, have been examined separately. Mobile phase composed of hexane, isopropanol and triethylamine (99.0:1.0:0.1) was used under an isocratic elution mode. Three of these compounds were separated simultaneously after being spiked into urine and plasma samples. 2,3-Methylenedioxy pyrovalerone was used as an internal standard for the purpose of quantitation. The analytical method has been validated in terms of linearity, limits of detection (LOD), limits of quantitation (LOQ), recoveries and reproducibilities in urine and plasma matrices. The calibration curves exhibited correlation coefficients better than 0.99. It was found that the LODs of these cathinone derivatives in urine were in the range of 1.00-1.47 ppm; while in plasma, the LODs were in the range of 0.14-0.67 ppm. The LOQs in urine were in the range of 3.03-4.46 ppm and in plasma they were in the range of 0.42-2.04 ppm. The method recoveries in terms of percent error averaged 2.4% and 3.2% for the spiked plasma and urine samples, respectively; while interday and intraday reproducibilities reported at three different levels, 5, 100 and 200 ppm, in terms of coefficient of variance were in the range of (0.27-5.39)% in plasma and (0.47-3.12)% in urine which lies in the acceptable range.

Simultaneous Quantitative Determination of Synthetic Cathinone Enantiomers in Urine and Plasma Using GC-NCI-MS.

Development and validation of sensitive and selective method for enantioseparation and quantitation of synthetic cathinones is reported using GC-MS triple quadrupole mass spectrometry with negative chemical ionization (NCI) mode. Indirect chiral separation of thirty-six synthetic cathinone compounds has been achieved by using an optically pure chiral derivatizing agent (CDA) called (S)-(-)-N-(trifluoroacetyl)pyrrolidine-2-carbonyl chloride (L-TPC), which converts cathinone enantiomers into diastereoisomers that can be separated on achiral columns. As a result of using Ultra Inert 60 m column and performing slow heating rate (2°C/min) on the GC oven, an observed enhancement in enantiomer peak resolution has been achieved. An internal standard, (+)-cathinone, was used for quantitation of synthetic cathinones. Method validation in terms of linearities and sensitivity in terms of limits of detection (LODs), limits of quantitation (LOQs), recoveries, and reproducibilities has been obtained for fourteen selected compounds that examined simultaneously as a mixture after being spiked in urine and plasma. It was found that the LOD of the fourteen synthetic cathinones in urine was in the range of 0.26-0.76 µg/L, and in plasma, it was in the range of 0.26-0.34 µg/L. While the LOQ of the mixture in urine was in the range of 0.86-2.34 µg/L, and in plasma, it was in the range of 0.89-1.12 µg/L. Unlike the electron impact (EI) ion source, NCI showed better sensitivity by two orders of magnitude by comparing the obtained results with the recently published reports for quantitative analysis and enantioseparation of synthetic cathinones.

Intermolecular interactions between cucurbit[7]uril and pilocarpine.

The interactions between cucurbit[7]uril (CB7) macrocycles and pilocarpine (PIL) were investigated in aqueous solution by using (1)H NMR and circular dichroism (CD) spectroscopic techniques. The characterizations of the freeze-drying solid complex were conducted by electrospray ionization mass spectroscopy (ESI-MS), Fourier transform-infrared spectroscopy (FT-IR), thermogravimetry, and differential scanning calorimetry (DSC) techniques. The DSC and thermogravimetry confirmed the production of a thermally stable solid complex. The NMR, CD and ESI-MS measurements confirmed asymmetric induction during the complexation reaction, in which the γ-lactone ring of PIL (not the imidazole nucleus) has been fully encapsulated within the cavity of CB7. The stability of the drug has significantly enhanced as evidenced by the high-performance liquid chromatographic (HPLC) method. The results are discussed in the context of utilizing non-conventional supramolecular host-guest approaches to enhance the chemical stability in aqueous media of hydrophilic PIL drugs as model compounds. The non-classical stereospecific interactions between CB7 and PIL drugs are also highlighted.

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae).

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianusAhl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensisPerret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.

Mechanistic study of a diazo dye degradation by Soybean Peroxidase.

BACKGROUND: Enzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published. RESULTS: The present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway. CONCLUSION: We report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue.

Evaluation of the skin peptide defenses of the Oregon spotted frog Rana pretiosa against infection by the chytrid fungus Batrachochytrium dendrobatidis.

Population declines due to amphibian chytridiomycosis among selected species of ranid frogs from western North America have been severe, but there is evidence that the Oregon spotted frog, Rana pretiosa Baird and Girard, 1853, displays resistance to the disease. Norepinephrine-stimulated skin secretions were collected from a non-declining population of R. pretiosa that had been exposed to the causative agent Batrachochytrium dendrobatidis. Peptidomic analysis led to identification and isolation, in pure form, of a total of 18 host-defense peptides that were characterized structurally. Brevinin-1PRa, -1PRb, -1PRc, and -1PRd, esculentin-2PRa and -PRb, ranatuerin-2PRa, -2PRb, -2PRc, and -2PRe, temporin-PRb and -PRc were identified in an earlier study of skin secretions of frogs from a different population of R. pretiosa known to be declining. Ranatuerin-2PRf, -2PRg, -2PRh, temporin-PRd, -PRe, and -PRf were not identified in skin secretions from frogs from the declining population, whereas temporin-PRa and ranatuerin-2PRd, present in skin secretions from the declining population, were not detected in the current study. All purified peptides inhibited the growth of B. dendrobatidis zoospores. Peptides of the brevinin-1 and esculentin-2 families displayed the highest potency (minimum inhibitory concentration = 6.25-12.5 µM). The study provides support for the hypothesis that the multiplicity and diversity of the antimicrobial peptide repertoire in R. pretiosa and the high growth-inhibitory potency of certain peptides against B. dendrobatidis are important in conferring a measure of resistance to fatal chytridiomycosis.

A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids.

Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (XLB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (XBL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1 was present in low concentration. No peptides were identified in secretions of either XLB or XBL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis×X. muelleri F1 hybrids and no novel peptide expression.

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring.

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly(13)→Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.

Fluorescent studies of salicylaldehyde and other related carbonyl compounds for the selective and sensitive detection of zinc(II) ions in aqueous solution.

Salicylaldehyde was found to have a high selectivity for zinc ions with simultaneous enhancement of fluorescence in aqueous buffer solution at optimum pH 8.5. The stoichiometry of the complex was determined to be 1:1 with a K(a) value of 3.4 × 10(4) M(-1) at 298 K. The fluorescence of the complex is not affected by common anions and Zn(2+) binds preferentially to salicylaldehyde in the presence of alkali, alkaline earth and heavy metal cations (Hg(2+), Cd(2+), Cr(3+) and Ni(2+)). This property is not observed with related phenolic compounds bearing a carbonyl group such as esters, amides, carboxylic acids and ketones.

Caerulein-and xenopsin-related peptides with insulin-releasing activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus amieti (Pipidae).

Caerulein-related peptides were identified in norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis and the octoploid frog Xenopus amieti using negative ion electrospray mass spectrometry and their primary structures determined by positive ion tandem (MS/MS) mass spectrometry. X. borealis caerulein-B1 (pGlu-Gln-Asp-Tyr(SO(3))-Gly-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains an additional Gly(5) residue compared with X. laevis caerulein and caerulein-B2 (pGlu-Asp-Tyr(SO(3))-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains a Gln(2) deletion. X. amieti caerulein was identical to the X. laevis peptide. In addition, xenopsin, identical to the peptide from X. laevis, together with xenopsin-AM2 (pGlu-Gly-Arg-Arg-Pro-Trp-Ile- Leu) that contains the substitution Lys(3)→Arg were isolated from X. amieti secretions. X. borealis caerulein-B1, and X. amieti xenopsin and xenopsin-AM2 produced significant (P<0.05) and concentration-dependent stimulations of insulin release from the rat BRIN-BD11 clonal ß cell line at concentrations ⩾30nM. The peptides did not stimulate the release of lactate dehydrogenase at concentrations up to 3µM demonstrating that the integrity of the plasma membrane had been preserved. While their precise biological role is unclear, the caerulein- and xenopsin-related peptides may constitute a component of the animal's chemical defenses against predators.

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity.

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal ß cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30nM, with a maximum response (236% of basal rate, P<0.001) at a concentration of 3µM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3µM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P<0.001 at 3µM) but the peptide was cytotoxic at this concentration.

Investigation of the pyrolysis products of methionine-enkephalin-Arg-Gly-Leu using liquid chromatography-tandem mass spectrometry.

Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC-UV/Vis-MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides.

Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae).

The Volcano clawed frog Xenopus amieti Kobel, du Pasquier, Fischberg, and Gloor, 1980, with a chromosome number of 2n=72, is believed to have undergone two rounds of genome duplication since evolving from a diploid ancestor. Nine peptides with differential antimicrobial activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of X. amieti that showed structural similarity to peptides previously isolated from the tetraploid frog X. laevis (2n=36) and the diploid frog Silurana (formerly Xenopus) tropicalis (2n=20). Two peptides (magainin-AM1 and -AM2) are othologous to the magainins, two peptides (PGLa-AM1 and -AM2) orthologous to peptide glycine-leucine-amide, four peptides (CPF-AM1, -AM2, -AM3, -AM4) orthologous to caerulein-precursor fragments, and one peptide (XPF-AM1) structurally similar to xenopsin-precursor fragments were characterized. CFP-AM1 (GLGSVLGKALKIGANLL.NH(2)) was the most potent peptide present in the secretions and magainin-AM2 (GVSKILHSAGKFGKAFLGEIMKS) was the most abundant. The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage. However, the very low antimicrobial activity of the magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).

Antimicrobial peptides from the skin secretions of the New World frogs Lithobates capito and Lithobates warszewitschii (Ranidae).

Taxonomic revisions within the anuran family Ranidae have established the genus Lithobates that currently comprises 49 species of frogs from the New World. Peptidomic analysis, using reversed-phase HPLC with on-line detection by electrospray mass spectrometry, has led to the identification of multiple antimicrobial peptides in norepinephrine-stimulated skin secretions of the North American frog Lithobates capito and the Central American frog Lithobates warszewitschii. Structural characterization of the peptides demonstrated that the L. capito secretions contained brevinin-1 (1), esculentin-1 (1), esculentin-2 (1), ranatuerin-2 (3), and temporin (2) peptides. L. warszewitschii secretions contained brevinin-1 (1), esculentin-2 (1), ranatuerin-2 (2), and temporin (1) peptides. Values in parentheses indicate number of peptides in each family. Temporin-CPa from L. capito, with the atypical structure IPPFIKKVLTTVF.NH(2), also showed atypical growth-inhibitory activity having greater potency against Escherichia coli (MIC=25 microM) and Candida albicans (MIC=25 microM) than against Staphylococcus aureus (MIC=50 microM). Phylogenetic analysis based upon the amino acid sequences of 37 ranatuerin-2 peptides from 17 species belonging to the genus Lithobates provides support for currently accepted taxonomic relationships. L. capito is sister-group to Lithobates sevosus in a clade that also contains Lithobates areolatus, and Lithobates palustris. L. warszewitschii is most closely related to the Central American species Lithobates tarahumarae and Lithobates vaillanti.

Desorption electrospray ionization mass spectrometry of intact bacteria.

Desorption electrospray ionization (DESI) mass spectrometry (MS) was used to differentiate seven bacteria species on the basis of their measured DESI-mass spectral profile. Both gram-positive and gram-negative bacteria were tested and included Escherichia coli, Staphyloccocus aureus, Enterococcus sp., Bordetella bronchiseptica, Bacillus thuringiensis, Bacillus subtilis and Salmonella typhimurium. Distinct DESI-mass spectra, in the mass range of 50-500 u, were obtained from whole bacteria in either positive or negative ion modes in less than 2 mins analysis time. Positive ion DESI-mass spectral fingerprints were compared using principal components analysis (PCA) to investigate reproducibility for the intraday and the day-to-day measurements and the method selectivity to differentiate the bacteria studied. Detailed study of variances in the assay revealed that a large contribution to the DESI-mass spectral fingerprint variation was the growth media preparation procedure. Specifically, experiments conducted with the growth media prepared using the same batch yielded highly reproducible DESI-mass spectra, both in intraday and in day-to-day analyses (i.e. one batch of growth media used over a 3-day period versus a new batch every day over the same 3-day period). Conclusions are drawn from our findings in terms of strategies for rapid biodetection with DESI-MS.