Ki67 deficiency impedes chromatin accessibility and BCR gene rearrangement.

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.

Log D7.4 and plasma protein binding of synthetic cannabinoid receptor agonists and a comparison of experimental and predicted lipophilicity.

The emergence of new synthetic cannabinoid receptor agonists (SCRAs) onto the illicit drugs market continues to cause harm, and the overall availability of physicochemical and pharmacokinetic data for new psychoactive substances is lacking. The lipophilicity of 23 SCRAs and the plasma protein binding (PPB) of 11 SCRAs was determined. Lipophilicity was determined using a validated chromatographic hydrophobicity index (CHI) log D method; tested SCRAs showed moderate to high lipophilicity, with experimental log D7.4 ranging from 2.48 (AB-FUBINACA) to 4.95 (4F-ABUTINACA). These results were also compared to in silico predictions generated using seven commercially available software packages and online tools (Canvas; ChemDraw; Gastroplus; MoKa; PreADMET; SwissADME; and XlogP). Licenced, dedicated software packages provided more accurate lipophilicity predictions than those which were free or had prediction as a secondary function; however, the latter still provided competitive estimates in most cases. PPB of tested SCRAs, as determined by equilibrium dialysis, was in the upper range of the lipophilicity scale, ranging from 90.8% (ADB-BUTINACA) to 99.9% (BZO-HEXOXIZID). The high PPB of these drugs may contribute to reduced rate of clearance and extended durations of pharmacological effects compared to lesser-bound SCRAs. The presented data improve understanding of the behaviour of these drugs in the body. Ultimately, similar data and predictions may be used in the prediction of the structure and properties of drugs yet to emerge on the illicit market.

Detection in seized samples, analytical characterization, and in vitro metabolism of the newly emerged 5-bromo-indazole-3-carboxamide synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after ß-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge.

In vitro cannabinoid activity profiling of generic ban-evading brominated synthetic cannabinoid receptor agonists and their analogs.

Following the enactment of a generic ban in China in 2021, the synthetic cannabinoid market has been evolving, now encompassing even wider structural diversity. Compounds carrying a brominated core such as ADB-5'Br-BUTINACA (ADMB-B-5Br-INACA) and tail-less analogs, such as ADB-5'Br-INACA (ADMB-5Br-INACA), MDMB-5'Br-INACA, and ADB-INACA (ADMB-INACA), have been detected since late 2021. This study investigated the cannabinoid receptor (CB) activation potential of synthesized (S)-enantiomers of these substances, as well as of two predicted analogs MDMB-5'Br-BUTINACA (MDMB-B-5Br-INACA) and ADB-5'F-BUTINACA (ADMB-B-5F-INACA), using CB1 and CB2 ß-arrestin 2 recruitment assays and a CB1 intracellular calcium release assay. Surprisingly, the tail-less (S)-ADB-5'Br-INACA and (S)-MDMB-5'Br-INACA retained CB activity, albeit with a decreased potency compared to their tailed counterparts (S)-ADB-5'Br-BUTINACA and (S)-MDMB-5'Br-BUTINACA, respectively, which were potent and efficacious CB1 agonists. Also, at CB2 , tail-less analogs showed a lower potency but increased efficacy. Removing the bromine substitution ((S)-ADB-INACA) resulted in a reduced activity at CB1 ; however, this effect was less prominent at CB2 . Looking at tailed analogs, replacing the bromine with a fluorine substitution ((S)-ADB-5'F-BUTINACA) resulted in an increased potency and efficacy at both receptors. Furthermore, as ADB-5'Br-INACA and MDMB-5'Br-INACA have been frequently detected together in Scottish prisons, this study also evaluated the CB1 receptor activation potential of different mixtures of their respective reference standards, showing no unexpected cannabimimetic effect of combining both substances. Lastly, two powders seized by Belgian Customs and confirmed to contain ADB-5'Br-INACA and MDMB-5'Br-INACA, respectively, were assessed for CB activity. Based on the comparison with their reference standards, varying degrees of purity were suspected.

The metabolic profile of the synthetic cannabinoid receptor agonist ADB-HEXINACA using human hepatocytes, LC-QTOF-MS and synthesized reference standards.

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography-quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

Changing trends in novel benzodiazepine use within Scottish prisons: detection, quantitation, prevalence, and modes of use.

Drug use within prisons is increasingly complex and unpredictable. Benzodiazepines are currently one of the most common drugs detected in individuals leaving Scottish prisons; however, understanding illicit benzodiazepine use within prisons and assessing the potential harm to individuals is challenging due to the lack of available analytical data on the substances circulating. Increasingly, materials, such as paper and clothing, infused with novel benzodiazepines have been identified as a smuggling route into Scottish prisons. Methods were developed for the qualitative and quantitative analysis of benzodiazepines using gas chromatography-mass spectrometry (GC-MS) and applied to 495 seized samples from 11 Scottish prisons, including papers, cards, blotters, powders, tablets, and clothing. Evolution in the benzodiazepines being detected was demonstrated, with etizolam being the most prevalent throughout 2020/2021 following which flubromazepam and bromazolam detections increased. Additionally, significant changes in the smuggling methods and drug formats detected occurred over time following policy changes within prisons. These data represent the first reported widescale etizolam quantitation data and demonstrate high levels of variability across all sample types, most notably within tablets (0.34-2.33 mg per tablet). Additionally, concentration mapping of a whole seized card sample revealed the total concentration of drug present (312.5 mg) and demonstrated variability across the surface of the card (1.16-1.87 mg/cm2 ). These data highlight the challenges of consistent dosing for individuals and the high risks of unintentional overdose. Increased understanding of the challenge of such drug smuggling and benzodiazepine use will aid in the development of strategies to reduce supply and mitigate harm.

Evaluation of fentanyl immunoassay test strips for rapid in-situ detection of fentanyl and fentanyl analogs in seized samples and alternative matrices.

BACKGROUND: Ion mobility spectrometry is used for the rapid detection of drugs at points of security but are unable to differentiate some drugs leading to the instrument alarming for a drug not present in the sample. This can be particularly problematic for samples that alarm for fentanyl. In this study, fentanyl immunoassay strips were evaluated for use as a secondary test for fentanyl, including for the testing of alternative matrices, such as powders, e-liquids, and infused papers and textiles. METHODS: The limit of detection of fentanyl immunoassay strips was examined along with their selectivity to 18 fentanyl analogsand 72 other drugs and cutting agents. The effectiveness of the test strips at the detection of fentanyl in the presence of other drugs was examined by testing a series of concentrations of fentanyl in solution in combination with other drugs. The testing of alternative matrices was explored with laboratory prepared samples through sampling with cotton buds and extraction in water. RESULTS: The fentanyl immunoassay strips detected fentanyl at concentrations of 45 ng/mL and reacted with 16 of 18 tested fentanyl analogs with carfentanil and norfentanyl being the only analogs to not react. There was no reactivity with other drugs or cutting agents. The effectiveness of the fentanyl test strips was not reduced when fentanyl was mixed with other drugs. Fentanyl was successfully detected with high sensitivity in all alternative matrices. CONCLUSION: The fentanyl immunoassay strips were found to be an effective secondary test for fentanyl and at least 16 fentanyl analogs in seized drug samples, including when mixed with other drugs. The effectiveness of the sampling methods for alternative matrices should be further evaluated using fentanyl and fentanyl analog casework samples. The use of this method by law enforcement and other agencies should be examined to assess its effectiveness and ease of use in operational settings.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Systematic In Vitro Metabolic Profiling of the OXIZID Synthetic Cannabinoids BZO-4en-POXIZID, BZO-POXIZID, 5F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.

In vitro characterization of the pyrazole-carrying synthetic cannabinoid receptor agonist 5F-3,5-AB-PFUPPYCA and its structural analogs.

The synthetic cannabinoid receptor agonist (SCRA) market is undergoing important changes since the enactment of the 2021 class-wide generic SCRA ban in China, one of the most important source countries for new psychoactive substances (NPS). Recently, various compounds with new structural features, synthesized to bypass this legislation, have entered the recreational drug market. Certain monocyclic pyrazole-carrying "FUPPYCA" SCRAs have been sporadically detected since 2015 without gaining further popularity. However, as evidenced by their recent detection in Scottish prisons, 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA have re-emerged, potentially triggered by the new legislative ban. The aim of this study was to characterize the in vitro intrinsic CB1 and CB2 receptor activation potential of 5F-3,5-AB-PFUPPYCA and 3,5-ADB-4en-PFUPPYCA, as well as 4 analogs (5F-3,5-ADB-PFUPPYCA, 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA) using live cell ß-arrestin 2 recruitment assays. Most analogs were essentially inactive at either CB1 or CB2, with only 3,5-AB-CHMFUPPYCA, 5,3-AB-CHMFUPPYCA and 5,3-ADB-4en-PFUPPYCA showing a limited activation potential at CB1. Furthermore, the importance of the position of the tail structure was demonstrated, with 5,3 regioisomers being more active than their 3,5 analogs. Moreover, all compounds exhibited antagonistic behavior at both receptors, which may be associated with their structural resemblance to cannabinoid antagonists and inverse agonists. Although the 3,5 regioisomers of these "FUPPYCA" SCRAs circumvent the Chinese ban, it is unlikely that these SCRAs will pose a major threat to public health, given the lack of pronounced CB receptor activity.

RNA sequencing of single allergen-specific memory B cells after grass pollen immunotherapy: Two unique cell fates and CD29 as a biomarker for treatment effect.

BACKGROUND: Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgG4 . IgG4 competitively inhibits functional IgE on the surface of effector cells, such as mast cells and basophils, from binding to allergens. To further understand the important role memory B-cell (Bmem) responses play in mediating the beneficial effects of SLIT, we assessed changes in allergen-specific Bmem subsets induced by SLIT for grass pollen allergy. METHODS: Blood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort. RESULTS: Four months of SLIT increased RGP-specific IgE and IgG4 in serum and induced two Lol p 1-specific Bmem subsets with unique transcriptional profiles. Both subsets had upregulated expression of beta 1 integrin ITGB1 (CD29), whereas IGHE (IgE), IGHG4 (IgG4 ), FCER2 (CD23), and IL13RA1 were upregulated in one subset. There was an increase in the proportion of Lol p 1+ Bmem expressing surface IgG4 , CD23, and CD29 after SLIT. CONCLUSIONS: A clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

Is it a judgment of representativeness? Re-examining the birth sequence problem.

Although all birth orders in the "birth sequence problem" are equiprobable, most participants judge the less representative order as less likely than the more representative order. But this well-known problem confounds representativeness with the direction in which birth orders are compared. We hypothesized and corroborated in three experiments (total N = 1,136) that participants pragmatically infer the birth orders' relative prevalence from the direction of comparison. Experiment 1 found that participants judged the less representative sequence as more common when we reversed the comparison. Experiment 2 reproduced these results despite removing representativeness as a cue. In Experiment 3, participants preferred to place the relatively common sequence as the referent in an inverted "speaker" problem. Our results turn the iconic problem's interpretation on its head: Rather than indicating flawed human cognition, the birth sequence problem illustrates people's ability to adaptively extract subtle linguistic meaning beyond the literal content.

Incomplete preferences and rational framing effects.

The normative principle of description invariance presupposes that rational preferences must be complete. The completeness axiom is normatively dubious, however, and its rejection opens the door to rational framing effects. In this commentary, we suggest that Bermúdez's insightful challenge to the standard normative view of framing can be clarified and extended by situating it within a broader critique of completeness.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation.

The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.

The concentration and biomagnification of PCBs and PBDEs across four trophic levels in a marine food web.

Contracting Parties to the OSPAR Convention for the Protection of the Maine Environment of the North-East Atlantic are required to undertake monitoring and assessment of both inorganic and organic contaminants. There is a requirement to assess contaminants across different trophic levels on an ecosystem-specific basis. However, this is currently constrained by the availability of relevant samples to cover the full range of trophic levels. This study investigates the variability (inter- and intra-species variation) of the concentrations and distributions of thirty-two polychlorinated biphenyl (PCB) congeners and nine polybrominated diphenyl ether (PBDE) congeners in twenty-six species covering four trophic levels from different geographic locations around Scotland. Trophic magnification factors (TMFs) were calculated using a traditional method and a balanced method for both the ICES-7 PCBs and BDE47, to refine and improve the application of TMFs to assess and predict biomagnification risk to biota in the marine environment. There were clear differences in congener percentage distribution between sample categories and species, with differences influenced by physiological processes and eco-biological parameters. Trophic magnification was found to occur for the ICES-7 PCBs and BDE47 using the traditional method, with the highest degree of trophic magnification reported for CB52. An unbalanced dataset was found to influence the calculated TMF and in some cases, the overall conclusion of the trophic transfer of PCB and PBDE congeners. The balanced method is highly recommended for calculating TMFs to ensure that the TMF is a true indication of the biomagnification potential, particularly when conducting regional comparisons for which sampling requirements are difficult to achieve.

The metabolism of the synthetic cannabinoids ADB-BUTINACA and ADB-4en-PINACA and their detection in forensic toxicology casework and infused papers seized in prisons.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50 , 11.5 nM and ADB-4en-PINACA (EC50 , 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50 , 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.

The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21.

Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.

The concentration and biomagnification of trace metals and metalloids across four trophic levels in a marine food web.

To be able to assess progress towards "Good Environmental Status" adopted across European Member States, and by the United Kingdom through its 3-stage Marine Strategy, contaminant concentrations and their biological effects need to be assessed in environmental samples by comparison to assessment criteria. This study examines the variability of concentrations (inter- and intra- species variation) of three priority heavy metals (Hg, Cd and Pb) and six additional trace metals and metalloids (As, Ni, Se, Zn, Cu and Cr) in twenty-three species across four trophic levels from different locations around Scotland. Trophic magnification factors (TMFs) were calculated using two methods for metals/metalloids possessing a significant trophic relationship (Hg, Cd, Cu, Ni and Zn) to refine and improve the application of TMFs to assess and predict biomagnification risk of metals/metalloids to biota in the environment. It was concluded that a reasonable balance in sample numbers of lower- versus higher-trophic level organisms is highly recommended when calculating TMFs and appropriate species selection is vital to ensure TMFs accurately represent the selected ecosystem.

Advances in allergen-specific immune cell measurements for improved detection of allergic sensitization and immunotherapy responses.

Over the past two decades, precision medicine has advanced diagnostics and treatment of allergic diseases. Component-resolved analysis of allergen sensitization facilitates stratification of patients. Furthermore, new formulations of allergen immunotherapy (AIT) products can more effectively deliver the relevant components. Molecular insights from the identification of allergen component sensitization and clinical outcomes of treatment with new AIT formulations can now be utilized for a deeper understanding of the nature of the pathogenic immune response in allergy and how this can be corrected by AIT. Fundamental in these processes are the allergen-specific B and T cells. Within the large B- and T-cell compartments, only those that specifically recognize the allergen with their immunoglobulin (Ig) or T-cell receptor (TCR), respectively, are of clinical relevance. With peripheral blood allergen-specific B- and T-cell frequencies below 1%, bulk cell analysis is typically insufficiently sensitive. We here review the latest technologies to detect allergen-specific B and T cells, as well as new developments in utilizing these tools for diagnostics and therapy monitoring to advance precision medicine for allergic diseases.