Cannabidiol (CBD) use has grown exponentially more popular in the last two decades, particularly among older adults (>55 yr), though very little is known about the effects of CBD use during age-associated metabolic dysfunction. In addition, synthetic analogues of CBD have generated great interest because they can offer a chemically pure product, which is free of plant-associated contaminants. To assess the effects of a synthetic analogue of CBD (H4CBD) on advanced metabolic dysfunction, a cohort of 41-wk-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 200 mg H4CBD/kg by oral gavage for 4 wk. Animals were fed ad libitum and monitored alongside vehicle-treated OLETF and Long-Evans Tokushima Otsuka (LETO) rats, the lean-strain controls. An oral glucose-tolerance test (oGTT) was performed after 4 wk of treatment. When compared with vehicle-treated, OLETF rats, H4CBD decreased body mass (BM) by 15%, which was attributed to a significant loss in abdominal fat. H4CBD reduced glucose response (AUCglucose) by 29% (P < 0.001) and insulin resistance index (IRI) by 25% (P < 0.05) compared with OLETF rats. However, H4CBD did not statically reduce fasting blood glucose or plasma insulin, despite compensatory increases in skeletal muscle native insulin receptor (IR) protein expression (54%; P < 0.05). H4CBD reduced circulating adiponectin (40%; P < 0.05) and leptin (47%; P < 0.05) and increased ghrelin (75%; P < 0.01) compared with OLETF. Taken together, a chronic, high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of the profound loss of visceral adiposity.NEW & NOTEWORTHY Cannabis product use has grown in the last two decades despite the lack of research on Cannabidiol (CBD)-mediated effects on metabolism. Here, we provide seminal data on CBD effects during age-associated metabolic dysfunction. We gave 41-wk-old OLETF rats 200 mg H4CBD/kg by mouth for 4 wk and noted a high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of loss of visceral adiposity.

Introduction:While laboratory-developed tests (LDTs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely employed to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is often overlooked. This paper reports on the important role of LC-MS/MS LDTs in demonstrating improved performance of the Roche FEN2 fentanyl immunoassay compared with the Thermo DRI fentanyl immunoassay. Methods:The FEN2 assay was implemented according to the manufacturer's instructions and its performance was compared to the existing DRI assay using LC-MS/MS as a reference. Clinical sensitivity and specificity were determined using 250 consecutive random patient specimens. Spiking experiments were conducted to determine cross-reactivity with 31 fentanyl analogs. Select DRI false-positive samples were analyzed by the FEN2 assay via time-of-flight mass spectrometry method (LC-QTOF). Results:The FEN2 assay showed improved clinical sensitivity compared to the DRI (98% vs 61%) in 250 consecutive patient samples due to its ability to detect norfentanyl. It also showed better clinical specificity by correctly classifying select DRI false-positive results. Upon implementation in clinical practice, the FEN2 resulted in a higher screening positivity rate than the DRI (17.3% vs 13.3%) and a greater LC-MS/MS confirmation rate of immunoassay-positive samples (96.8% vs 88.8%, respectively). Conclusion:The use of LC-MS/MS LDTs demonstrated that the FEN2 assay has greater clinical sensitivity and is less prone to false-positives than the DRI assay. These findings support the use of FEN2 in routine clinical practice and emphasize the role of mass spectrometry-based LDTs in clinical toxicology testing.

Background:The VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites. Methods:Precision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation. Results:The precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations. Conclusions:We demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

Methaqualone, known previously under the brand name Quaalude, is a Schedule I sedative hypnotic drug that may cause neurotoxicity in overdose, characterized by somnolence, hyperreflexia and muscular hyperactivity. We present a case of a 21-year-old male who reportedly ingested methaqualone in addition to insufflation of street cocaine. He subsequently developed hypoxia, hyperreflexia, myoclonus and altered mental status. His laboratory results were notable for the presence of methemoglobinemia, which was most likely due to a cocaine contaminant. Laboratory analysis of the alleged methaqualone pills identified the substance as SL-164, a dichlorinated methaqualone analog. Urine toxicology results were positive for SL-164 (and presumed metabolites) as well as for cocaine and tetrahydrocannabinol metabolites. The patient was treated with supplemental oxygen and a benzodiazepine (lorazepam) and observed in the Emergency Department until his symptoms resolved. This case highlights current community access to methaqualone analogs. The case also focuses on laboratory techniques used to identify the methaqualone analog.

Objective Antibiotic resistance by beta lactamase expression is a serious and growing threat. We aimed to determine whether beta-lactamase activity is detectable in urine specimens to enable faster identification of resistance. Methods Urine specimens from patients with extended spectrum beta lactamase (ESBL)-expressing urinary infections were incubated with beta lactam antibiotics. Beta lactam hydrolysis was determined by mass spectrometry methods. Results Ceftriaxone hydrolysis was observed in 45 of 45 ESBL-containing specimens from patients not treated with a beta lactamase inhibitor before specimen collection. Ceftriaxone hydrolysis was not observed in 108 of 108 non-ESBL-containing specimens. Spiking studies show that beta lactam hydrolysis can be observed within 30 minutes. Beta lactam hydrolysis is evidenced by mass spectrometry preceded by either liquid chromatography or matrix-assisted laser desorption ionization specimen processing methods. Conclusion Clinically significant beta lactamase activity is detectable directly from urine specimens. The described methods would enable the detection of beta lactam resistance 24 to 48 hours sooner than culture based methods.

Background. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. Methods. The ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. Results. The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284). Conclusions. The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming methods of MSC, interferon-gamma (IFNγ) and hypoxia (HYP), could modify their EV miRNA content. Human bone marrow MSCs (BM-MSCs) from five healthy donors were cultured with IFNγ or in HYP or in control (CONT) conditions. The conditioned media were collected after 48 h in serum-free condition and EV were isolated by ultracentrifugation. Total RNA was isolated, pools of CONT, IFN, and HYP cDNA were prepared, and a miRNA profiling was performed using RT-qPCR. Then, miRNAs were selected based on their detectability and measured on each individual EV sample. Priming had no effect on EV amount or size distribution. A set of 81 miRNAs was detected in at least one of the pools of EVs. They were measured on each individual sample; 41 miRNAs were detected in all samples. The principal component analysis (PCA) failed to discriminate the groups. HYP induced a significant decrease in EV hsa-miR-34a-3p content and IFN induced a significant increase in five miRNAs (hsa-miR-25-3p, hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-451a, and hsa-miR-665). Taken together, we found only limited alterations in the miRNA landscape of MSC EV with a high inter-individual variability.

Septic patients often die in a context of multiple organ dysfunction syndrome (MODS), despite the macro-hemodynamic parameters being normalized and after the onset of antibiotic therapy. Microcirculation injury during sepsis affects capillary permeability and leukocyte-endothelium interactions and is thought to be instrumental in organ injury. Several studies have demonstrated a beneficial effect of mesenchymal stromal cells (MSCs) injection on survival and organ dysfunctions in sepsis models. In vivo activity of MSCs also appears to be very much dependent on the information provided before injection. Indeed preconditioning by interferon gamma (IFN gamma; MSC-IFN gamma) increases immunosuppressive capacity of MSCs in vitro and in vivo. Therefore, the objective was to evaluate the effect of MSC naive or IFN gamma preconditioned on leukocyte-endothelium interactions in a polymicrobial sepsis model by intraperitoneal feces injection. Six hours (H6) after this induction, we used intravital microscopy in mice cremaster muscle venules to study the flow behavior of leukocytes. Plasmas were harvested to evaluate inflammation level and endothelial activation. We showed that MSC-IFN gamma have a beneficial effect on microcirculation, by increasing the flow of white blood cells (WBCs) and the percentage of venules containing flowing WBCs, by significantly reducing the adhesion of WBCs and by increasing the average red blood cell velocity (V-RBC). In conclusion, our results suggest that intravenous injection of preconditioned MSC-IFN gamma improves microvascular hemodynamics in early phases of sepsis.

Sepsis is a complex disease characterized by an inflammatory host response to infectious stimulus. Patients often die within a context of multiple organ dysfunction syndrome (MODS), while the macro-hemodynamic parameters have been corrected and that early antibiotic therapy was administered. For several years, many studies have shown that microcirculation plays a key role in the development of MODS. Microcirculation injury affects vascular autoregulatory mechanisms, capillary permeability and leukocyte-endothelium interactions. Even if leukocytes activation and recruitment are essential to an effective host defense against infection, these interactions have been implicated in inflammation mediated vascular injury, which leads to end-organ injury.

Mesenchymal stromal cells are multipotent cells found in a large number of adult tissues. Their ability to participate in the repair of these damaged tissues is the origin of the enthusiasm that they elicit in the field of cell therapy. It gradually became apparent that their ability to change a pathological environment is more related to their ability to modulate the behavior of other cell types than their capacity of differentiation. Recent years have expanded the scope of our knowledge about their way of communication with their environment but also the amount of information that they receive from this environment. In this brief review, we will present some of the mechanisms by which sMSCs can communicate remotely with other cell types and how it currently appears possible to direct the secretion pattern of these cells

Mesenchymal stromal cells are multipotent cells found in a large number of adult tissues. Their ability to participate in the repair of these damaged tissues is the origin of the enthusiasm that they elicit in the field of cell therapy. It gradually became apparent that their ability to change a pathological environment is more related to their ability to modulate the behavior of other cell types than their capacity of diferentiation. Recent years have expanded the scope of our knowledge about their way of communication with their environment but also the amount of information that they receive from this environment. In this brief review, we will present some of the mechanisms by which MSCs can communicate remotely with other cell types and how it currently appears possible to direct the secretion pattern of these cells.

Introduction: During the course of systemic inflammation, most of the immune cell types get activated to a certain degree as part of, or contributing to, the cascade of physiopathological events.Whether for some cells, classically phagocytes of the innate immune system, it is clear that direct sensing of pathogen-associated molecular patterns leads to activation initiating systemic inflammation, the picture is not so clear for natural killer (NK) cells.While NK cells have been shown to express toll-like receptors (TLR), the role of these receptors on NKs during systemic inflammation has not been directly addressed.Methods: To directly assess the role of TLR expression on NK cells we used an adoptive transfer model in which NKs purified from the spleens of WT, TLR4KO and TLR2/4DKO mice were transferred intravenously to RAG2 -/-γc -/-(devoid of T, B and NK cells).Five days after reconstitution the mice were challenged intraperitoneally with conventional or TLR-grade lipopolysaccharide (LPS).Immune cell activation and production of IFNγ by NK cells was determined after 6 hours by FACS analysis.Results: We observed no differences in reconstitution of the recipient mice with NK cells from different backgrounds suggesting no difference in trafficking and survival of the transferred cells.At 6 hours after LPS challenge, WT, TLR4KO or TLR2/4DKO NK cells recovered from the spleen and lungs of RAG2 -/-γc -/-mice showed comparable levels of CD69 activation marker expression.Intracellular labeling for IFNγ in NK cells also revealed no significant differences.Conclusion: Whether there is a role for direct TLR signaling on NK cells remains the objective of further investigations; however, our data show that in the course of a systemic inflammatory process, like endotoxinemia, the expression of TLR2 and TLR4 by NK cells makes no difference in terms of their activation and secretion of IFNγ P2 Role of 6-hour, 12-hour, and 24-hour lactate clearance in mortality of severe sepsis and septic shock patients.

Since the secretory pathway is essential for Candida albicans to transition from a commensal organism to a pathogen, an understanding of how this pathway functions may be beneficial for identifying novel drug targets to prevent candidiasis. We have cloned the C. albicans KAR2 gene, which performs many roles during the translocation of proteins into the endoplasmic reticulum (ER) during the first committed step of the secretory pathway in many eukaryotes. Our results show that C. albicans KAR2 is essential, and that the encoded protein rescues a temperature-sensitive growth defect found in a Saccharomyces cerevisiae strain harboring a mutant form of the Kar2 protein. Additionally, S. cerevisiae containing CaKAR2 as the sole copy of this essential gene are viable, and ER microsomes prepared from this strain exhibit wild-type levels of post-translational translocation during in vitro translocation assays. Finally, ER microsomes isolated from a C. albicans strain expressing reduced amounts of KAR2 mRNA are defective for in vitro translocation of a secreted substrate protein, establishing a new method to study ER translocation in this organism. Together, these results suggest that C. albicans Kar2p functions during the translocation of proteins into the ER during the first step of the secretory pathway.

A synthetic molecular nanorobot dubbed a "spider" composed of a streptavidin protein "body" bound to three biotinylated 8-17 deoxyribozyme-bearing "legs" completes the programmed task of traversing a predetermined DNA origami track. The track is assembled by folding and holding in place a long single-stranded DNA molecule into a two-dimensional landscape by using hundreds of short oligonucleotides ("staples") and hybridizing chimeric DNA-RNA substrates to specific staples to realize a pseudo-one-dimensional track. Spiders are designed to undergo a biased random walk as their legs repeatedly cleave oligonucleotide substrates on a track, causing the legs to more readily dissociate from the cleaved product and progress towards the uncleaved substrate. We image Cy3-labeled spiders and Cy5-labeled origami using total internal reflection fluorescence microscopy (TIRFM). By fitting Gaussian functions to the imaged point spread functions, we are able to monitor with nanometer spatial precision the real-time motion of the spiders along DNA origami tracks.

The probing and characterization of the behavior of individual nucleic acid based molecular robots is presented, using real-time single-particle tracking with super-resolution total internal reflection fluorescence microscopy (TIRFM). Nucleic acid based molecular assemblies, called "spiders", implemented as robots with multiple deoxyribozyme sensor-actuator legs traverse and cleave two-dimensional landscapes of surface bound oligonucleotide substrates. We analyze the movement of spiders to test the hypothesis that they walk by biased diffusion. The experimental approach demonstrated here should allow for control over the cybernetic properties of spiders, resulting in the integration and synthesis of complex robotic behaviors at the nanoscale based on DNA and RNA nanotechnology.

Programming the behaviour of molecules in time and space is a big challenge in nanotechnology. Traditional robots can be given a memory in which to store internal representations of their goals and environment, and coordinate their response. Individual molecules are limited in their ability to store information and programs, but robotic behaviour can still be realized by exploiting the interaction of simple single-molecule robots with a precisely defined environment. This has now been demonstrated with spider-shaped DNA 'walkers', which sense and modify tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. The robots carry out actions such as 'start', 'follow', 'turn', and 'stop' programmed into the DNA landscape, with more complex robotic behaviour expected once secondary levels of control can be introduced. Traditional robots need to store internal representations of their goals and environment, and to coordinate sensing and the movement of components required in response. Individual molecules are limited in their ability to store complex information, but robotic behaviour can still be realized — as has now been shown with DNA walkers, which can carry out a sequence of actions such as 'start', 'follow', 'turn' and 'stop' that are programmed into the DNA landscape on which the walkers move. Traditional robots1 rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment2,3,4. A first step in this direction was the development of DNA walkers5, which have developed from being non-autonomous6,7 to being capable of directed but brief motion on one-dimensional tracks8,9,10,11. Here we demonstrate that previously developed random walkers12—so-called molecular spiders that comprise a streptavidin molecule as an inert ‘body’ and three deoxyribozymes as catalytic ‘legs’—show elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape13. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as ‘start’, ‘follow’, ‘turn’ and ‘stop’. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour14,15; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour2,16,17.

We present the development of a new graphical user interface driven molecular modeling, editing and visualization tool called Tiamat. Tiamat addresses the challenge of how to efficiently model large and complex DNA nanostructures. We describe the three major components of our system. First, we discuss design guidelines and data structures that form the basis of flexible and large-scale editing. Second, we explain a semi-automatic sequence generator that combines user input with random sequence generation to efficiently label the molecules in the DNA structure. Third, we outline the visualization techniques including a simplification algorithm that are used to render large designs. The results demonstrate how Tiamat was used to generate large and complex designs.

Hochdichte Peptidanordnungen, die eine Vielzahl unterschiedlicher Aminosäuresequenzen an genau definierten und addressierbaren Stellen derselben DNA-Nanostruktur präsentieren können, wurden erzeugt. Dabei wurde die genetische Information genutzt, die im Nucleinsäureteil eines DNA-markierten Peptids verschlüsselt ist, um die Aminosäuresequenz an der vorbestimmten Stelle zu positionieren. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2007/z603919_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Here we report a modular design of self-assembly of DNA nanostructures in a combinatorial approach; a square with approximately 25 nm cavity dimension, a chair with approximately 80 nm in height and a line with approximately 100 nm in length are formed through combinations of four cross-shaped DNA tiles which are kept constant and six variable linker tiles.