Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices.

Untargeted lipidomics, with its ability to take a snapshot of the lipidome landscape, is an important tool to highlight lipid changes in pathology or drug treatment models. One of the shortcomings of most untargeted lipidomics based on UHPLC-HRMS is the low throughput, which is not compatible with large-scale screening. In this contribution, we evaluate the application of a sub-5-min high-throughput four-dimensional trapped ion mobility mass spectrometry (HT-4D-TIMS) platform for the fast profiling of multiple complex biological matrices. Human AC-16 cells and mouse brain, liver, sclera, and feces were used as samples. By using a fast 4-min RP gradient, the implementation of TIMS allows us to differentiate coeluting isomeric and isobaric lipids, with correct precursor ion isolation, avoiding co-fragmentation and chimeric MS/MS spectra. Globally, the HT-4D-TIMS allowed us to annotate 1910 different lipid species, 1308 at the molecular level and 602 at the sum composition level, covering 58 lipid subclasses, together with quantitation capability covering more than three orders of magnitude. Notably, TIMS values were highly comparable with respect to longer LC gradients (CV% = 0.39%). These results highlight how HT-4D-TIMS-based untargeted lipidomics possess high coverage and accuracy, halving the analysis time with respect to conventional UHPLC methods, and can be used for fast and accurate untargeted analysis of complex matrices to rapidly evaluate changes of lipid metabolism in disease models or drug discovery campaigns.

A High Copy Number from a Pharyngeal Swab Is Not Associated with Different Presenting Features in 100 Children with Acute Adenovirus Infection from a Cluster in Italy.

Human mastadenoviruses, frequently denominated adenoviruses (HAdVs), may cause respiratory tract, gastrointestinal or, less frequently, other involvements. Epidemics of HAdV infections occur globally, in communities, and in closed or crowded settings. In our institution, a cluster of infants and children admitted for HAdV infection was recently observed. The aim of this study was to describe the pattern of their presenting features and investigate the possible correlation between the HAdV copy number and the clinical picture. Two main patterns of clinical presentation were observed: 68 patients had mainly respiratory symptoms (pharyngitis n = 67, cough n = 44; tonsillar exudate n = 17; other respiratory signs n = 4) while 26 patients showed prevalent gastrointestinal involvement (diarrhea n = 26, vomiting n = 8). Patients with respiratory symptoms had a significantly higher count of WBC, PMN, and platelets, while CRP level approached statistical significance (p = 0.07) for higher values in the patients with diarrhea. In order to explore the impact of selected presenting features, the possible association between the level of CRP and the presence of pharyngeal exudate, cough, vomiting, diarrhea, duration of fever, number of neutrophils, and administration of antibiotics was analyzed. Patients falling in the tertile with more elevated CRP values had tonsillar exudate and diarrhea significantly more often, while those in the lower tertile had a 4.4-day duration fever vs. ≥5.0 days in the remaining patients. Antibiotic therapy was administered more frequently to patients with higher values of CRP (p = 0.006). The duration of hospitalization was not associated with the CRP level. The median time from the receipt of a positive HAdV PCR test result to patient discharge was 1 day in 73% of cases. The number of copies of HAdV detected via PCR ranged between 47 million and 15/µL. Falling in the highest tertile of copy number was significantly associated with pharyngitis. The 24 patients with evidence of viral coinfection had no difference in the demographics or presenting features, with the only exception being a significantly higher leukocyte count. The rapid turn-around of the results of the molecular testing of the HAdV genome on a pharyngeal swab allowed us to rapidly diagnose HAdV infection, allowing us to stop antibiotic therapy and immediately discharge the patients, with reduced discomfort for the families and more appropriate use of hospital beds. A high copy number of HAdV from a pharyngeal swab should not be taken as an indicator of worse prognosis, thus allowing for the preferential use of qualitative rather than quantitative assay.

Enhanced selective capture of phosphomonoester lipids enabling highly sensitive detection of sphingosine 1-phosphate.

Sphingolipids play crucial roles in cellular membranes, myelin stability, and signalling responses to physiological cues and stress. Among them, sphingosine 1-phosphate (S1P) has been recognized as a relevant biomarker for neurodegenerative diseases, and its analogue FTY-720 has been approved by the FDA for the treatment of relapsing-remitting multiple sclerosis. Focusing on these targets, we here report three novel polymeric capture phases for the selective extraction of the natural biomarker and its analogue drug. To enhance analytical performance, we employed different synthetic approaches using a cationic monomer and a hydrophobic copolymer of styrene-DVB. Results have demonstrated high affinity of the sorbents towards S1P and fingolimod phosphate (FTY-720-P, FP). This evidence proved that lipids containing phosphate diester moiety in their structures did not constitute obstacles for the interaction of phosphate monoester lipids when loaded into an SPE cartridge. Our suggested approach offers a valuable tool for developing efficient analytical procedures.

Optimization of microwave-assisted extraction of antioxidant compounds from spring onion leaves using Box-Behnken design.

Many studies have explored the extraction of bioactive compounds from different onion solid wastes, such as bulb, skin, and peel. However, onion leaves have received limited attention despite their potential as a valuable source of nutraceutical compounds. This study aimed to valorise, for the first time, the agricultural waste in the form of spring onion leaves (CN, Cipollotto Nocerino) to obtain antioxidant-rich polyphenolic extracts. A Box-Behnken design (BBD) was used to assess the impact of microwave-assisted extraction (MAE) variables (temperature, time, extraction volume, and ethanol concentration) on total polyphenol content (TPC) measured by Folin-Ciocalteu method and the antioxidant power determined by FRAP assay. Response surface methodology (RSM) was applied, and regression equations, analysis of variance, and 3D response curves were developed. Our results highlighted that the TPC values range from 0.76 to 1.43 mg GAE g-1 dw, while the FRAP values range from 8.25 to 14.80 mmol Fe(II)E g-1 dw. The optimal extraction conditions predicted by the model were 60 °C, 22 min, ethanol concentration 51% (v/v), and solvent volume 11 mL. These conditions resulted in TPC and FRAP values of 1.35 mg GAE g-1 dw and 14.02 mmol Fe(II)E g-1 dw, respectively. Furthermore, the extract obtained under optimized conditions was characterized by UHPLC-ESI-Orbitrap-MS analysis. LC/MS-MS platform allowed us to tentatively identify various compounds belonging to the class of flavonoids, saponins, fatty acids, and lipids. Finally, the ability of CN optimal extract to inhibit the intracellular reactive oxygen species (ROS) release in a hepatocarcinoma cell line using an H2O2-induced oxidative stress model, was evaluated. The results highlighted the potential of CN extract as a valuable source of polyphenols with significant antioxidant properties, suitable for various applications in the food and pharmaceutical industries.

Integrating ZooMS and zooarchaeology: New data from the Uluzzian levels of Uluzzo C Rock Shelter, Roccia San Sebastiano cave and Riparo del Broion.

In this study we explore the potential of combining traditional zooarchaeological determination and proteomic identification of morphologically non-diagnostic bone fragments (ZooMS) collected from the Uluzzian levels of three Italian sites: Uluzzo C Rock Shelter, Roccia San Sebastiano cave, and Riparo del Broion. Moreover, we obtained glutamine deamidation ratios for all the contexts analysed during routine ZooMS screening of faunal samples, giving information on collagen preservation. We designed a selection protocol that maximizes the efficiency of the proteomics analyses by excluding particularly compromised fragments (e.g. from taphonomic processes), and that aims to identify new human fragments by favouring bones showing morphological traits more similar to Homo. ZooMS consistently provided taxonomic information in agreement with the faunal spectra outlined by traditional zooarchaeology. Our approach allows us to delineate and appreciate differences between the analysed contexts, particularly between the northern and southern sites, related to faunal, environmental, and climate composition, although no human remains were identified. We reconstructed the faunal assemblage of the different sites, giving voice to morphologically undiagnostic bone fragments. Thus, the combination of these analyses provides a more complete picture of the faunal assemblage and of the paleoenvironment during the Middle-Upper Palaeolithic transition in Italy.

Catalytic Enantioselective Access to Dihydroquinoxalinones via Formal α-Halo Acyl Halide Synthon in One Pot.

An enantioselective one-pot catalytic strategy to dihydroquinoxalinones, featuring novel 1-phenylsulfonyl-1-cyano enantioenriched epoxides as masked α-halo acyl halide synthons, followed by a domino ring-opening cyclization (DROC), is documented. A popular quinine-derived urea served as the catalyst in two out of the three steps performed in the same solvent using commercially available aldehydes, (phenylsulfonyl)acetonitrile, cumyl hydroperoxide and 1,2-phenylendiamines. Medicinally relevant 3-aryl/alkyl-substituted heterocycles are isolated in generally good to high overall yield and high enantioselectivity (up to 99 % ee). A rare example of excellent reusability of an organocatalyst at higher scale, subjected to oxidative conditions, is demonstrated. Mechanistically, labile α-ketosulfone has been detected as the intermediate involved in the DROC process. Theoretical calculations on the key epoxidation step rationalize the observed stereocontrol, highlighting the important role played by the sulfone group.

Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine.

Short peptide sequences represent emerging analytes in a variety of fields, including biomarker discovery, but also a well-known analytical challenge in complex matrices, due to the low abundance, extensive suppression during MS analysis, and lack of workflows, as they cannot be identified by ordinary peptidomics strategies and coverage is extremely limited by metabolomics as well. In this context, in this work, a solid phase extraction method was developed for the cleanup and enrichment of dipeptides, tripeptides, and tetrapeptides in urine using graphitized carbon black Carbograph 4 as the sorbent. The method was first developed on analytical standards spiked in urine, with recoveries in the range of 60-100%. Then the method was applied to urine samples from healthy volunteers. The enriched urine samples were analyzed by ultrahigh performance liquid chromatography (UHPLC) using an orthogonal strategy in which both a reversed phase (RP) C18 column and a zwitterionic hydrophilic interaction liquid chromatography (HILIC) column were used, for better coverage of peptide polarity and improved detection of peptides. High-resolution mass spectra were acquired in data-dependent mode using a suspect screening strategy with inclusion list; peptides were identified by a semiautomated workflow for feature extraction, candidate mass filtering, and MS/MS spectra comparison with in silico mass spectra. The complementarity of the orthogonal separation strategy was confirmed by peptide identification, resulting in 101 peptides identified from the RP runs and 111 peptides from the HILIC runs, with 60 common identifications. The method is applicable to both hydrophobic and hydrophilic peptides. Peptides were stable over 2 h after collection and protease inhibitors were not necessary, as no formation of artifacts was observed.

Sensitive untargeted identification of short hydrophilic peptides by high performance liquid chromatography on porous graphitic carbon coupled to high resolution mass spectrometry.

The combination of an efficient chromatographic separation with post-column addition of a supercharging agent was evaluated for the determination of small peptides. The procedure takes advantage of porous graphitic carbon (PGC) ability in retaining very polar and ionic molecules to overstep the poor retention of small peptides on conventional reversed phase (RP) columns. The method was developed specifically for the most hydrophilic di-, tri- and tetrapeptides, which are not identified in ordinary peptidomics experiments. In addition to retention mechanisms acting on conventional RP, the method exploited the charge induced interactions generated by the charges on the peptides with the polarizable surface of PGC. This results in efficient retention of very short and highly polar peptides using classical RP mobile phases. The effects of varying mobile phase composition (organic solvent and ion-pairing additives) as well as column temperature have been thoroughly investigated using short peptide standards. Under optimized conditions (water and acetonitrile/tetrahydrofuran 99:1 (v/v), both with 0.15% trifluoroacetic acid, as phase A and B, respectively, 0.5 mL min-1 flowrate at 50 °C) the effect of post-column addition of 3-nitrobenzylic alcohol was also investigated allowing effective coupling of the chromatographic system with high resolution mass spectrometry. Finally, an untargeted approach for peptide identification was pursued, based on precursor identification in database with all possible combinations of the 20 natural amino acids and fragmentation spectra matching to in silico generated spectra. The method was then applied to investigation of the short endogenous peptides in human serum from healthy individuals resulting in the identification of 30 short peptides.

Chemicals from textiles to skin: an in vitro permeation study of benzothiazole.

Despite the possible impact on human health, few studies have been conducted to assess the penetration and accumulation of contaminants in the skin after a prolonged contact with textile materials. In previous studies, we have shown that benzothiazole and its derivatives, as well as other potentially hazardous chemicals, often are present as textile contaminants in clothes available on the retail market. Since benzothiazole is a common contaminant in clothes, these can be a possible route for human chemical exposure, both systemic and onto the skin. To investigate this potential exposure, Franz-type and flow-through cells were used for the permeation studies together with a Strat-M® artificial membranes. Experiments were performed using solutions of benzothiazole, as well as contaminated textile samples in the donor chamber. Benzothiazole was demonstrated to penetrate through, as well as being accumulated in the membrane mimicking the skin. After 24 h, up to 62% of benzothiazole was found in the acceptor cell, while up to 37% was found absorbed in the skin mimicking membrane. It also was shown that there was release and permeation from contaminated fabrics. The results indicate that benzothiazole can be released from textile materials, penetrate through the skin, and further enter the human body. This will possibly also apply to other chemical contaminants in textiles, and the results of this study indicate that the presence of these textile contaminants entails potential health risks. A rough risk assessment was made for clothing textiles according to Environmental Protection Agency (EPA) and European regulations for carcinogenic and non-carcinogenic compounds, using literature data for benzothiazole.

Determination of aniline and quinoline compounds in textiles.

A simple method for simultaneous determination of twenty-one analytes, belonging to two classes of compounds, aromatic amines and quinolines, is presented. Several of the analytes considered in this study frequently occur in textiles goods on the open market and have been related to allergic contact dermatitis and/or are proven or suspected carcinogens. The method includes an efficient clean-up step using graphitized carbon black (GCB) that simplifies and improves the robustness of the subsequent GC-MS analysis. Briefly, after solvent extraction of the textile sample, the extract is passed through a GCB SPE cartridge that selectively retain dyes and other interfering compounds present in the matrix, producing a clean extract, suitable for GC-MS analysis, is obtained. The method was evaluated by spiking blank textiles with the selected analytes. Method quantification limits (MQL) ranged from 5 to 720ng/g depending on the analyte. The linear range of the calibration curves ranged over two order magnitude with coefficients of determination (R2) higher than 0.99. Recoveries ranged from 70 to 92% with RSDs 1.7-14%. The effectiveness of the method was tested on a variety of textile materials samples from different origin. In a pilot explorative survey, 2,6-dichloro-4-nitroaniline was detected in all the analysed clothing samples in concentrations ranging from 1.0 to 576µg/g. 2,4-dinitroaniline was detected in four of the seven samples with a highest concentration of 305µg/g. Quinoline was detected in all samples in concentrations ranging from 0.06 to 6.2µg/g.

Application of graphitic sorbent for online microextraction of drugs in human plasma samples.

In the present work a new sorbent based on graphitized carbon (CarbonX(®) COA) was evaluated in microextraction by packed sorbent (MEPS) for extraction of lidocaine and ropivacaine from human plasma samples. The new graphitic sorbent showed high recoveries of lidocaine and ropivacaine compared to C18 sorbent. In the present study the G-MEPS (syringe packed with graphitic sorbent) was connect online with liquid chromatography tandem mass spectrometry (LC-MS/MS). In order to obtain a fast and reliable method different factors affecting MEPS performance were investigated. The extraction efficiency of the graphitic sorbent was compared with silica-based sorbents used in MEPS. The G-MEPS was also evaluated for reuse (50-100 times). The recoveries of lidocaine and ropivacaine from plasma samples were 79% and 82%; respectively. The method was validated according to FDA (Food and Drug Administration) guideline for bioanalytical method validation. Linearity was assessed in the range 5-2000nmol/L, with coefficient of determination r(2)>0,995 (n=3) for lidocaine and r(2)>0.997 (n=3) for ropivacaine. The lower limit of quantification (LLOQ) was 5nmol/L and the limit of detection (LOD) was 1nmol/L for studied analytes in plasma samples. For both analytes considered in this study the accuracy values in plasma samples were ranged from 86% to 113%. The Inter-day precisions, expressed as relative standard deviation (%RSD), at three different concentrations (QC-samples) ranged from 8% to 9% for lidocaine, and from 4% to 11% for ropivacaine.

Fast determination of underivatized gentamicin C components and impurities by LC-MS using a porous graphitic carbon stationary phase.

Gentamicin C antibiotics are important because they are active against many multidrug-resistant Gram-negative bacilli. Unfortunately, their clinical usefulness is limited by their toxicity. Because of the difficulty involved in separating its different components, the US and European pharmacopeias both specify that the composition of gentamicin C should be determined by liquid chromatography with pulsed electrochemical detection. Here, we assess the usefulness of a porous graphitic carbon (PGC) HPLC column for separating the components of gentamicin C, and report chromatographic conditions that enable its direct characterization by PGC chromatography directly coupled to electrospray mass spectrometry. Native major components of gentamicin and impurities in commercial formulations were retained and separated on the PGC column without any need for derivatization, using mobile phases basified with ammonium hydroxide. When coupled with detection by conventional electrospray ion trap mass spectrometry (ESI-IT-MS), several previously reported impurities were detected easily, including the most polar gentamicin impurity, garamine. When operating in full-scan mode, it was possible to identify and quantitate gentamicin-related compounds using injected samples of only a few picograms. Under the described conditions, all analytes were eluted in less than 10 min and the LC-MS analyses exhibited excellent stability and linearity. The method's effectiveness was evaluated by analyzing commercial gentamicin batches and in-house formulations. When the PGC chromatographic system was coupled to an evaporative light-scattering detector, detection limits of 40-70 ng were achieved for various major gentamicin components. The chromatographic method was applied on a semi-preparative scale to purify the five major components.

Micro-solid-phase extraction coupled to desorption electrospray ionization-high-resolution mass spectrometry for the analysis of explosives in soil.

Home-made micro-solid-phase extraction (SPE) cartridges using different adsorbent materials were tested for the desorption electrospray ionization-high-resolution mass spectrometry (DESI-HRMS) determination of explosives like 2,4,6-trinitrotoluene, cyclotrimethylene-trinitramine, cyclotetramethylene-tetranitramine, pentaerythritol tetranitrate, and trinitrophenylmethylnitramine in soil samples. Quantitation limits in the low nanogram per kilogram range proved the reliability of the method for the detection of explosives at ultra-trace levels. The reduced sample preparation allowed for low costs and high-throughput analyses. Finally, the superior extraction capability of the method was proved by obtaining DESI-HRMS responses at least five times higher than those achieved by performing DESI-HRMS analyses of solid-liquid extracts spotted onto commercial polytetrafluoroethylene slides.

Comparison of extraction methods for the identification and quantification of polyphenols in virgin olive oil by ultra-HPLC-QToF mass spectrometry.

In this work, liquid-liquid extraction (LLE) and solid phase extraction (SPE), of polyphenols from a VOO sample were optimised by a Plackett-Burman experimental design; then the two extraction techniques capabilities were compared. By using HPLC-DAD, the extraction ability of SPE with the diol phase and LLE were similar. The two methods were further evaluated with ultra HPLC-ESI QToF in negative ion mode by recoveries of standards and matched comparison of the peak area of 40 identified and 27 unidentified compounds. Conclusions indicate that LLE gives better recoveries for highly polar, non-polar, and some polyphenols suspected to contain a nitrogen atom, while for the others the two methods seem to be equally suitable. The presence of nitrogen-containing polyphenols was confirmed in positive ionisation mode in LLE extract, whereas in the SPE extract they were not present. One of them was tentatively identified as a compound containing tyrosine and methyl-decarboxymetyl-eleanoic acid moieties.

Gentamicin and leucine inhalable powder: what about antipseudomonal activity and permeation through cystic fibrosis mucus?

The aim of this study was to evaluate the permeation properties of gentamicin (G) in a novel dry powder form for inhalation through an artificial mucus model. Moreover, since respiratory infections sustained by Pseudomonas are a major cause of sickness and death in CF patients, the susceptibility of P. aeruginosa to engineered G powders was investigated. Micronized G and G/leucine (85:15) formulations were produced by co-spray-drying, using process parameters and conditions previously set. Powders were characterized in terms of yield, drug content and aerodynamic profiles, analyzed by Andersen Cascade Impactor. Different mucus models were prepared, showing composition and viscosity similar to those of the native CF mucus. To investigate the impact on drug permeation, Franz-type vertical diffusion cells were used; the powders were applied directly on a synthetic membrane with or without the interposition of the artificial mucus layer. In buffer, gentamicin showed a diffusion controlled release; the presence of leucine reduced powder wettability and, consequently, the permeation rate. Otherwise, mucus delayed drug permeation from both G and G/leucine formulations, with a faint influence of the aminoacid. Antimicrobial tests revealed that G/leu engineered particles are able to preserve the antipseudomonal activity, even in presence of the mucus.

Development and validation of a method for the determination of (E)-resveratrol and related phenolic compounds in beverages using molecularly imprinted solid phase extraction.

A molecularly imprinted polymer was prepared using (E)-resveratrol as template and was evaluated for multicomponent multiclass analysis of polyphenolic compounds in complex matrices such as natural and alcoholic beverages. Chromatographic evaluation of the polymer exhibited high selectivity for (E)-resveratrol and its structural analogues, quercetin, and other flavonoids. An analytical procedure based on molecularly imprinted solid phase extraction (MISPE) and high-performance liquid chromatography coupled to UV detector was developed and validated for determination of (E)-resveratrol and quercetin in wine and fruit juice samples. The specific binding capacity of the MIP was estimated as 80 µg g(-1) polymer by the cartridge test. MISPE sample pretreatment allows an excellent sample cleanup, enormously decreasing the number of coextracted potentially interfering compounds. Under the described conditions, by extracting 2 mL samples a clean extract is obtained and (E)-resveratrol and quercetin could be easily identified at concentration levels of, respectively, 1.5 and 7.0 µg L(-1).

Surgical wound infection by Streptococcus pneumoniae after a cat-scratch disease.

Streptococcus pneumoniae (pneumococcus) is mostly known as an agent of meningitis and pneumonia. We present what is believed to be a previously unreported case of pneumococcal disease, involving an inguinal wound. A 10-year-old male patient underwent surgical removal of a cat-scratch disease-related enlarged groin lymph node; infection of the wound was soon observed, with multidrug-resistant S pneumoniae growing from the lesion discharge. This communication expands the spectrum of skin ulcer-infecting pathogens; in fact, although pneumococcus has been known to mostly affect central nervous system and airways, we observed a surgical wound infection by this organism.

Extending the applicability of pressurized hot water extraction to compounds exhibiting limited water solubility by pH control: curcumin from the turmeric rhizome.

Pressurized hot water extraction (PHWE, also known as subcritical water extraction) is commonly considered to be an environmentally friendly extraction technique that could potentially replace traditional methods that use organic solvents. Unfortunately, the applicability of this technique is often limited by the very low water solubility of the target compounds, even at high temperatures. In this paper, the scope for broadening the applicability of PHWE by adjusting the pH of the water used in the extraction is demonstrated in the extraction of curcumin (which exhibits very limited water solubility) from untreated turmeric (Curcuma longa L.) rhizomes. Although poor extraction yields were obtained, even at high temperatures when using degassed water or neutral phosphate buffer as the extraction medium, yields exceeding those obtained by Soxhlet extraction were achieved using highly acidic pH buffers due to curcumin protonation. The influence of the temperature, pH, and buffer concentration on the extraction yield were investigated in detail by means of a series of designed experiments. Optimized conditions for the extraction of curcumin from turmeric by PHWE were estimated at 197 °C using 62 g/L buffer concentration at pH 1.6. The relationships between these variables were subjected to statistical analysis using response surface methodology.

Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface.

The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids.