From soap bubbles to multicellular organisms: Unraveling the role of cell adhesion and physical constraints in tile pattern formation and tissue morphogenesis.

Tile patterns, in which numerous cells are arranged in a regular pattern, are found in a variety of multicellular organisms and play important functional roles. Such regular arrangements of cells are regulated by various cell adhesion molecules. On the other hand, cell shape is also known to be regulated by physical constraints similar to those of soap bubbles. In particular, circumference minimization plays an important role, and cell adhesion negatively affects this process, thereby regulating tissue morphogenesis based on physical properties. Here, we focus on the Drosophila compound eye and the mouse auditory epithelium, and summarize the mechanisms of tile pattern formation by cell adhesion molecules such as cadherins, Irre Cell Recognition Modules (IRMs), and nectins. Phenomena that cannot be explained by physical stability based on cortical tension alone have been reported in the tile pattern formation in the compound eye, suggesting that previously unexplored forces such as cellular concentric expansion force may play an important role. We would like to summarize perspectives for future research on the mechanisms of tissue morphogenesis.

An FDA-Validated, Self-Cleaning Liquid Chromatography-Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium.

As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

National Institute of Health and Care Excellence Guidelines for Displaced Intracapsular Hip Fractures: Examining Satisfaction With the Guidelines and Effects on Outcomes.

BACKGROUND: The objectives of the study were to: (1) evaluate satisfaction with the new 2023 National Institute of Health and Care Excellence (NICE) criteria for selecting total hip arthroplasty (THA) over hemiarthroplasty and surgical recommendations for treatment of displaced intracapsular hip fractures; (2) describe why THA is performed when NICE criteria are not met; and (3) determine whether satisfaction with these guidelines is associated with improved outcomes. METHODS: A retrospective chart review of patients who had a displaced intracapsular hip fracture treated with THA at a single tertiary academic center between 2010 and 2022 was performed. Preoperative patient characteristics were reviewed to determine if the indication for THA met NICE criteria. Operative details, perioperative complications, reoperation, and revision arthroplasty within 12 months of surgery were recorded. RESULTS: Data from 196 patients (63% women; age 67 ± 10 years) were used. There were 161 THAs (82.1%) that satisfied NICE criteria. The 2 most common reasons for performing a THA when NICE criteria were not met (n = 35) included preoperative radiographic osteoarthritis (Tönnis grade ≥ 2; 48.6%) and decreased patient age (< 65 years; 31.5%). Satisfaction with the NICE criteria was associated with fewer perioperative complications (0.6 versus 37.1%; P < .001), reoperations (0.6 versus 31.4%; P < .001), and revisions (0.6 versus 28.6%; P < .001). The most common reason for revision was periprosthetic fracture, possibly secondary to the use of uncemented femoral stems (171 of 196, 87.2%). CONCLUSIONS: Satisfaction with the new NICE criteria is associated with improved perioperative outcomes. Further studies are necessary to determine if preexisting hip osteoarthritis and younger age merit consideration in patient selection.

Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids.

For studying stem cell-derived islet organoids (SC-islets) in an organ-on-chip (OoC) platform, we have developed a reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method allowing for simultaneous determination of insulin, somatostatin-14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl-C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin-14, and 0.05 µg/L for glucagon. The here-developed RPLC-MS/MS method showed that the SC-islets have an insulin response dependent on glucose concentration, and the SC-islets produce and release somatostatin-14 and glucagon. The RPLC-MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC-islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC-islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well-plate. Taken together, RPLC-MS/MS method is well suited for multi-hormone measurements of SC-islets on an OoC platform.

Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications.

Ultralow flow LC employs ultra-narrow bore columns and mid-range pL/min to low nL/min flow rates (i.e., ≤20 nL/min). The separation columns that are used under these conditions are typically 2-30 µm in inner diameter. Ultralow flow LC systems allow for exceptionally high sensitivity and frequently high resolution. There has been an increasing interest in the analysis of scarce biological samples, for example, circulating tumor cells, extracellular vesicles, organelles, and single cells, and ultralow flow LC was efficiently applied to such samples. Hence, advances towards dedicated ultralow flow LC instrumentation, technical approaches, and higher throughput (e.g., tens-to-hundreds of single cells analyzed per day) were recently made. Here, we review the types of ultralow flow LC technology, followed by a discussion of selected representative ultralow flow LC applications, focusing on the progress made in bioanalysis of amount-limited samples during the last 10 years. We also discuss several recently reported high-sensitivity applications utilizing flow rates up to 100 nL/min, which are below commonly used nanoLC flow rates. Finally, we discuss the path forward for future developments of ultralow flow LC.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

"Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry.

Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

Marine Microbial-Derived Resource Exploration: Uncovering the Hidden Potential of Marine Carotenoids.

Microbes in marine ecosystems are known to produce secondary metabolites. One of which are carotenoids, which have numerous industrial applications, hence their demand will continue to grow. This review highlights the recent research on natural carotenoids produced by marine microorganisms. We discuss the most recent screening approaches for discovering carotenoids, using in vitro methods such as culture-dependent and culture-independent screening, as well as in silico methods, using secondary metabolite Biosynthetic Gene Clusters (smBGCs), which involves the use of various rule-based and machine-learning-based bioinformatics tools. Following that, various carotenoids are addressed, along with their biological activities and metabolic processes involved in carotenoids biosynthesis. Finally, we cover the application of carotenoids in health and pharmaceutical industries, current carotenoids production system, and potential use of synthetic biology in carotenoids production.

Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1-0.4 and 2-10%, respectively, for a wide polarity range of metabolites (log P -4.4 to 8.8). The method's performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.

Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies.

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

Pancreas-on-a-Chip Technology for Transplantation Applications.

PURPOSE OF REVIEW: Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. RECENT FINDINGS: PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions.

Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells.

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

Nano liquid chromatography columns.

Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 µm, can provide enhanced sensitivity and enable analysis of limited samples. NanoLC has become an established tool in omics research, and is gaining ground in other applications as well. There are several variants and formats of nanoLC columns, including packed columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while e.g. the monolith and open tubular columns are still less established as routine tools. The pillar array format is a new variant with excellent resolution and low backpressure, and has recently been commercialized and used for bio-applications. In this minireview, we summarize and discuss recent research on nanoLC column development and uses, focusing on literature between 2016 and medio 2019.

Distinguishing Between Cyclopropylfentanyl and Crotonylfentanyl by Methods Commonly Available in the Forensic Laboratory.

BACKGROUND: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity, and legal status, and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. METHODS: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by ultra-high-performance liquid chromatography was investigated using 3 different stationary phases (high strength silica T3, ethylsiloxane/silica hybrid C18, and Kinetex biphenyl) using gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by tandem mass spectrometry. In addition, the major metabolites of the 2 compounds formed on incubation with human liver microsomes were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. RESULTS: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the ethylsiloxane/silica hybrid C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the 2 analogues formed by human liver microsomes differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the 2 approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in 2 authentic postmortem blood samples. CONCLUSIONS: In this study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.

Addressing the Fentanyl Analogue Epidemic by Multiplex UHPLC-MS/MS Analysis of Whole Blood.

BACKGROUND: Fentanyl and fentanyl analogues (fentanyls) are very potent opioids posing a serious threat to the public health. Thousands of overdose deaths across the world are caused by fentanyls, and the numbers are increasing. Rapid mapping of current trends in opioid abuse is necessary to accelerate preventive measures. To ensure this, there is a need for sensitive targeted multiplex MS/MS methods to pinpoint drugs of abuse. We present a fully validated UHPLC-MS/MS method for the determination of 26 fentanyls, including several structural isomers, and the opioid antagonist naloxone in human whole blood. METHODS: Blood samples were prepared by liquid-liquid extraction with ethyl acetate and heptane. The fentanyls were separated with UHPLC, using a Kinetex biphenyl column (2.1 × 100 mm, 1.7 µm; Phenomenex, Verløse, Denmark) with an acidic mobile phase. Quantification was performed by MS/MS. The method was validated according to SWGTOX guidelines. RESULTS: The developed method could successfully separate all 27 analytes, including 7 isomers, and was validated according to SWGTOX guidelines with very low limits of quantification (4-20 pg/mL). The applicability of the method was demonstrated by determination of fentanyls in postmortem blood samples from 2 cases. CONCLUSIONS: A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.

Multichannel Open Tubular Enzyme Reactor Online Coupled with Mass Spectrometry for Detecting Ricin.

For counterterrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys-C enzyme-immobilized multichannel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed online digestion of proteins (5 min reaction time, instead of 4-16 h in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial online coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.

Timing of Permanent Ventricular Shunt Placement Following External Ventricular Drain Placement in Primary Intracerebral Hemorrhage.

BACKGROUND: Intraventricular hemorrhage requiring ventriculostomy placement is a frequent complication of spontaneous intracerebral hemorrhage. Although a subset of patients will require permanent ventricular shunt placement, little is known about contemporary practices regarding the timing of ventriculostomy and ventricular shunt placement after intracerebral hemorrhage. METHODS: Using the 2010-2012 National Inpatient Sample, we identified patients with International Classification of Diseases, Ninth Revision codes for intracerebral hemorrhage, excluded secondary causes, and examined procedure dates. RESULTS: Of 35,899 patients with primary intracerebral hemorrhage, 2443 (6.8%) received ventriculostomy, 93% within the first 3 days of admission and 66% within the first day. Permanent shunt placement occurred in 173 (7.1%) patients following ventriculostomy at a median interval of 15 days (interquartile range: 11-20). Among those remaining alive and in hospital at 14, 21, and 28 days, 5%, 11%, and 15%, respectively, underwent shunt placement following ventriculostomy, and 24% of those with multiple ventriculostomy insertions required permanent shunt by 4 weeks of hospitalization. Multiple ventriculostomies, tracheostomy, and black race were associated with longer time to permanent shunt. CONCLUSIONS: A wide variation in delay to permanent shunt placement is present, with substantial and increasing prevalence with time in hospital. Better understanding of the risk factors associated with persistent hydrocephalus will help optimize patient selection and timing of treatment.

Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.

Metabolites in vertebrate Hedgehog signaling.

The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field.