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N-Heterocyclic carbene (NHC)-derived selenoureas comprise a fundamentally important class of NHC derivatives, with key applications in coordination chemistry and the determination of NHC electronic properties. Considering the broad reactivity of chalcogen-containing compounds, it is surprising to note that the use of NHC-derived selenoureas as organic synthons remains essentially unexplored. The present contribution introduces a novel, straightforward transformation leading to azines bearing a guanidine moiety, through the reaction of a wide range of NHC-derived selenoureas with commercially available diazo compounds, in the presence of triphenylphosphine. This transformation offers a new approach to such products, having biological, materials chemistry, and organic synthesis applications. The guanidine-bearing azines are obtained in excellent yields, with all manipulations taking place in air. A reaction mechanism is proposed, based on both experimental mechanistic findings and density functional theory (DFT) calculations. A one-pot, multicomponent transesterification reaction between selenoureas, α-diazoesters, alcohols, and triphenylphosphine was also developed, providing highly functionalized azines., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

The melt analysis feature in most real-time polymerase chain reaction (PCR) instruments is a simple method for determining if expected or unexpected products are present. High-resolution melt (HRM) analysis seeks to improve the precision of melt temperature measurements for better PCR product sequence characterization. In the area of tuberculosis (TB) drug susceptibility screening, sequencing has shown that a single base change can be sufficient to make a first-line TB drug ineffective. In this study, a reagent-based calibration strategy based on synthetic left-handed (L)-DNA, designated LHRM, was developed to confirm validation of a PCR product with single base resolution. To test this approach, a constant amount of a double-stranded L-DNA melt comparator was added to each sample and used as a within-sample melt standard. The performance of LHRM and standard HRM was used to classify PCR products as drug-susceptible or not drug-susceptible with a test bed of nine synthetic katG variants, each containing single or multiple base mutations that are known to confer resistance to the first-line TB drug isoniazid (INH). LHRM achieved comparable classification to standard HRM relying only on within-sample melt differences between L-DNA and the unknown PCR product. Using a state-of-the-art calibrated instrument and multiple sample classification analysis, standard HRM was performed at 66.7% sensitivity and 98.8% specificity. Single sample analysis incorporating L-DNA for reagent-based calibration into every sample maintained high performance at 77.8% sensitivity and 98.7% specificity. LHRM shows promise as a high-resolution single sample method for validating PCR products in applications where the expected sequence is known.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm ( N = 6) or preset cycling conditions ( N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

Cancer is a complex disease and the second leading cause of death globally, and breast cancer is still a leading cause of cancer death in women. Tamoxifen is the most commonly used drug for breast cancer (ER-positive) treatment and chemoprevention, saving the lives of millions of patients every year. In addition, the tamoxifen template has been explored extensively for the development of selective estrogen receptor modulators (SERMs) applicable in breast cancer, osteoporosis, and postmenopausal symptom treatment. Numerous anticancer drugs, including tamoxifen, are in use, but the complexity and heterogeneous nature of cancer complicate the effect of conventional targeted drugs, leading to adverse reactions and resistance. One of the significant approaches to overcome these shortcomings is drug hybrids, generated by covalently linking two or more active pharmacophores. These drug hybrids are remarkably effective in acting on multiple drug targets with higher selectivity and specificity. In recent years, several tamoxifen hybrids have been discovered as potential candidates for cancer treatment. The review highlights the recent progress in developing anticancer hybrids, including organometallic, fluorescent, photocaged, and novel ligand-based tamoxifen hybrids. It also demonstrates the significance of merging various pharmacophores with tamoxifen to produce more potent, precise, and effective anticancer agents. The study offers valuable knowledge to researchers working on cancer research with the hope of enhancing drug potency and reducing drug toxicity to improve cancer patients' lives., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na + /H + exchange, but apical proton uptake is undefined. We found high expression of the Cl - /H + exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl - dependency of H + transport in osteoblast membranes, we queried whether ClC3 Cl - /H + exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo . In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H + uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs., Competing Interests: All of the authors declare no financial and personal relationships with people or organizations that might inappropriately influence the work. No author is under investigation for responsible conduct in research, animal welfare, human subjects, or laboratory safety compliance at the time of submission., (© 2024 The Authors. Published by Elsevier Inc.)

This research introduces a novel fluorescence sensor 'on-off-on' employing nitrogen-doped carbon dots (N-CDs) with an 'on-off-on' mechanism for the selective and sensitive detection of Hg(II) and L-cysteine (L-Cys). N-CDs was synthesized using citric acid as the carbon precursor and urea as the nitrogen source in dimethylformamide (DMF) solvent, resulting in red emissive characteristics under UV light. Comprehensive spectroscopic analyses, including UV-Vis, fluorescence, FT-IR, XRD, XPS, Raman, and Zeta potential techniques, validated the structural and optical characteristics of the synthesized N-CDs. The maximum excitation and emission of N-CDs were observed at 548 and 622 nm, respectively. The quantum yield of N-CDs was calculated to be 16.1%. The fluorescence of N-CDs effectively quenches upon the addition of Hg(II) due to the strong coordination between Hg(II) and the surface functionalities of N-CDs. Conversely, upon the subsequent addition of L-Cys, the fluorescence of N-CDs was restored. This restoration can be attributed to the stronger affinity of the -SH group in L-Cys towards Hg(II) relative to the surface functionalities of N-CDs. This dual-mode response enabled the detection of Hg(II) and L-Cys with impressive detection limits of 15.1 nM and 8.0 nM, respectively. This sensor methodology effectively detects Hg(II) in lake water samples and L-Cys levels in human urine, with a recovery range between 99 and 101%. Furthermore, the N-CDs demonstrated excellent stability, high sensitivity, and selectivity, making them a promising fluorescence on-off-on probe for both environmental monitoring of Hg(II) and clinical diagnostics of L-Cys., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bilirubin plays a significant role in human health management, particularly in the case of jaundice. Because of the need for the monitoring of bilirubin levels in jaundice patients, the development of a robust sensitive method becomes essential. Here, we describe the development of a highly sensitive and selective turn-off fluorometric detection method for bilirubin in blood serum samples using nitrogen-doped carbon dots (N-CDs). N-CDs was synthesized by the pyrolysis process, using citric acid and L-asparagine as the carbon and nitrogen sources, respectively. The prepared N-CDs solution showed highly intense blue emission with good stability. The HR-TEM image of N-CDs revealed spherical dot-like structures with an average size calculated to be 7.16 nm. Further, the surface functional groups of N-CDs were analyzed by FT-IR, Raman, XRD, and XPS techniques. Fluorescence spectra showed the maximum emission intensity at 443 nm (λ ex ). The linear range of addition was performed from 1 to 150 µM, and the limit of detection (LOD) was determined to be 1.97 nM. The emission of N-CDs was quenched by Förster Resonance Energy Transfer (FRET) by adding bilirubin. These N-CDs showed extraordinary sensitivity and selectivity in the detection of bilirubin. Hence, this fluorescent probe has been proven successful in detecting the concentration of free bilirubin in human serum samples., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ-carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs., (© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

Palladium salts and complexes were tested separately and in the presence of added ligands as potential sources of aryl radicals in ground-state coupling reactions of aryl halide with arenes under basic conditions (KO t Bu). Our recently developed assay for aryl radicals was employed to test for aryl radicals. In this assay, aryl radicals derived from the test substrate, 1-iodo-2,6-dimethylbenzene 7 , undergo base-promoted homolytic aromatic substitution (BHAS) with benzene to produce 2,6-dimethylbiphenyl 8 and biphenyl 9 in an approximately 1:4 ratio as well as m -xylene 10. The biphenyl arises from a diagnostic radical transfer reaction with the solvent benzene. Using substrate 7 with a range of Pd sources as potential initiators led to formation of 8 , 9 , and 10 in varying amounts. However, when any one of a range of diphosphinoferrocenes (e.g., dppf or dippf) or BINAP or the monophosphine, diphenylphosphinoferrocene, was added as a ligand to Pd(OAc) 2 , the ratio of [2,6-dimethylbiphenyl 8 : biphenyl 9 ] moved decisively to that expected from the BHAS (radical) pathway. Further studies were conducted with dppf. When dppf was added to each of the other Pd sources, the ratio of coupled products was also diverted to that expected for radical BHAS chemistry. Deuterium isotope studies and radical trap experiments provide strong additional support for the involvement of aryl radicals. Accordingly, under these ground-state conditions, palladium sources, in the presence of defined ligands, convert aryl iodides to aryl radicals. A rationale is proposed for these observations.

Exhaled biologic material is the source for the spread of many respiratory tract infections. To avoid the high-level of biosafety required to manage dangerous pathogens, we developed a safer framework using the endogenous surrogate targets RNase P and Streptococcus mitis as a means to sample exhaled biologics. Our exhalation collection scheme uses nanoscale fibrous poly(vinyl alcohol) substrates as facemask inserts. After a period of breathing or speaking, the inserts are removed and dissolved. RNase P RNA and S. mitis DNA are extracted for quantification by multiplexed RT-qPCR. Both surrogate biomarkers were detected in all samples obtained during breathing for at least five minutes or speaking for one minute. Phrases repeated 30 times had the most copies with 375 ± 247 of S. mitis and 54 ± 33 of RNase P. When the phrases were repeated just 5 times, the S. mitis copies collected were still detectable but at a significantly lower level of 11 ± 5 for S. mitis and 12 ± 9 for RNase P. These results demonstrate a collection and quantification framework that can be readily adapted to further characterize the exhalation of nanoscale biologic materials from healthy individuals, explore new collection designs safely, and serve as a method to incorporate sample controls for future pathogen exhalation studies.

Monodentate phosphine-ligated nickel compounds, e.g. , [Ni(PPh 3 ) 4 ] are relevant as active catalysts across a broad range of reactions. This report expands upon the coordination chemistry of this family, offering the reactivity of allyl- and vinyl-substituted diphenylphosphine (PPh 2 R) with [Ni(COD) 2 ] (COD = 1,5-cyclooctadiene). These reactions provide three-coordinate dinickelacycles that are intermolecularly tethered through adjacent {Ni}-olefin interactions. The ring conformation of such cycles has been studied in the solid-state and using theoretical calculations. Here, a difference in reaction outcome is linked to the presence of an allyl vs vinyl group, where the former is observed to undergo rearrangement, bringing about challenges in clean product isolation., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Coaching, mentorship, and leadership are all paramount for the creation of a championship-winning football team. Looking back and studying the great coaches of professional football provides valuable insight into the qualities and the characteristics they possessed and how that impacted their leadership. Many of the great coaches from this game have instilled team standards and a culture that led to unprecedented success and sprouted into many other great coaches and leaders. Leadership at all levels of an organization is essential to consistently achieve a championship-caliber team., Competing Interests: Disclosure The authors have nothing to disclose about this work. Other disclosures: Dr M.T. Provencher receives royalties from Arthrex, Inc. and Elsevier, Inc., consulting fees from Arthrex, Inc., Joint Restoration Foundation, and SLACK, Inc., and is an honorarium for Arthrosurface. He is currently a Board or Committee member of the following: AAOS: Board or Committee member; AANA: Board or Committee member; AOSSM: Board or Committee member; ASES: Board or Committee member; Arthroscopy: Editorial or governing board; ISAKOS: Board or Committee member; Knee: Editorial or governing board; Orthopedics: Editorial or governing board; San Diego Shoulder Institute: Board or Committee member; SLACK Inc: Editorial or governing board; Society of Military Orthopaedic Surgeons: Board or Committee member., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The importance of the immune microenvironment in triple negative and HER2-amplified breast cancer (BC) is well-established; less is known about the immune environment in luminal breast cancers. We aimed to assess for the impact of immune biomarkers on BC outcome in a group of luminal B patients with archived tissue and annotated clinical information. Patients with early breast cancer (EBC) treated in a single institution over a 14-year period, with prospectively collected data were included. Luminal B EBC patients were identified and defined into three cohorts: A: grade 2 or 3, ER & PR positive, HER2-negative; B: Any grade, ER positive, PR and HER2-negative (Ki67 ≥ 14% in cohorts A & B); and C: Any grade, ER or PR positive, HER2-positive. Within each cohort, patients with a relapsed BC event (R) were compared on a 1:1 basis with a control patient (C) who remained disease-free, balanced for key characteristics in an effort to balance the contribution of each clinical group to outcome. Archival breast, involved and uninvolved axillary nodes were assessed by immunohistochemistry for biomarkers identifying effector and suppressor immune cells, and compared between R and C. In total, 120 patients were included (80, 22, and 18 patients in cohorts A, B, and C, respectively). R were 1.5 years older (p = 0.016), with all other characteristics being balanced. Overall, there were no statistically significant differences in immune biomarkers in breast or nodal tissue of R and C. However, there was a trend toward higher levels of TILs in breast tumors of C, while GAL-9 was consistently expressed on lymphocytes and tumor cells in all breast and nodes of C and was absent from all tissues of R. These trends in checkpoint molecule expression deserve further research., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing., Methods: A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance., Results: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 10 4 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples., Conclusions: The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance., (© 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken., Competing Interests: Author IL was employed by Becton Dickinson Pty Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Graham, Nowak, Cornwall, Larma and Nelson.)

Human and canine sarcomas are difficult to treat soft tissue malignancies with an urgent need for new improved therapeutic options. Local recurrence rates for humans are between 10%-30%, and 30%-40% develop metastases. Outcomes for dogs with sarcoma vary with grade but can be similar. Pet dogs share the human environment and represent human cancer with genetic variation in hosts and tumours. We asked if our murine studies using genetically identical mice and cloned tumour cells were translatable to larger, genetically diverse domestic dogs with naturally occurring tumours, to (i) develop a canine cancer therapeutic, and (ii) to use as a translational pathway to humans. Our murine studies showed that intra-tumoral delivery of interleukin-2 (IL-2) plus an agonist anti-CD40 antibody (Ab) induces long-term curative responses ranging from 30% to 100%, depending on tumour type. We developed an agonist anti-canine-CD40 Ab and conducted a phase I dose finding/toxicology 3 + 3 clinical trial in dogs (n = 27) with soft tissue sarcomas on account of suitability for intratumoral injection and straightforward monitoring. Dogs were treated with IL-2 plus anti-CD40 antibody for 2 weeks. Three dose levels induced tumour regression with minimal side effects, measured by monitoring, haematological and biochemical assays. Importantly, our mouse and canine studies provide encouraging fundamental proof-of-concept data upon which we can develop veterinary and human immunotherapeutic strategies., (© 2022 Selvax Pty Ltd. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Increased cancer incidence occurs with the emergence of immunosenescence, highlighting the indispensability of the immune system in preventing cancer and its dysregulation with aging. Tumor-associated macrophages (TAMs) are often present in high numbers and are associated with poor clinical outcomes in solid cancers, including mesothelioma. Monocytes and macrophages from the bone marrow and spleen can respond to tumor-derived factors, such as CSF-1, and initiation of the CSF-1R signaling cascade results in their proliferation, differentiation, and migration to the tumor. Age-related changes occur in monocytes and macrophages in terms of numbers and function, which in turn can impact tumor initiation and progression. Whether this is due to changes in CSF-1R expression with aging is currently unknown and was investigated in this study. We examined monocytes and macrophages in the bone marrow and spleen during healthy aging in young (3-4 months) and elderly (20-24 months) female C57BL/6J mice. Additionally, changes to these tissues and in TAMs were examined during AE17 mesothelioma tumor growth. Healthy aging resulted in an expansion of Ly6C high monocytes and macrophages in the bone marrow and spleen. CSF-1R expression levels were reduced in elderly splenic macrophages only, suggesting differences in CSF-1R signaling between both cell type and tissue site. In tumor-bearing mice, Ly6C high monocytes increased with tumor growth in the spleen in the elderly and increased intracellular CSF-1R expression occurred in bone marrow Ly6C high monocytes in elderly mice bearing large tumors. Age-related changes to bone marrow and splenic Ly6C high monocytes were reflected in the tumor, where we observed increased Ly6C high TAMs earlier and expansion of Ly6C low TAMs later during AE17 tumor growth in the elderly compared to young mice. F4/80 high TAMs increased with tumor growth in both young and elderly mice and were the largest subset of TAMs in the tumor. Together, this suggests there may be a faster transition of Ly6C high towards F4/80 high TAMs with aging. Amongst TAM subsets, expression of CSF-1R was lowest in F4/80 high TAMs, however Ly6C low TAMs had higher intracellular CSF-1R expression. This suggests downstream CSF-1R signaling may vary between macrophage subsets, which can have implications towards CSF-1R blockade therapies targeting macrophages in cancer., Competing Interests: DN acts as a non-salaried Chief Scientific Officer for Selvax. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duong, Pixley, Nelson and Jackaman.)

We have investigated the reactions of chalcogenoureas derived from N -heterocyclic carbenes, referred to here as [E(NHC)], with halogens. Depending on the structure of the chalcogenourea and the identity of the halogen, a diverse range of reactivity was observed and a corresponding range of structures was obtained. Cyclic voltammetry was carried out to characterise the oxidation and reduction potentials of these [E(NHC)] species; selenoureas were found to be easier to oxidise than the corresponding thioureas. In some cases, a correlation was found between the oxidation potential of these compounds and the electronic properties of the corresponding NHC. The reactivity of these chalcogenoureas with different halogenating reagents (Br 2 , SO 2 Cl 2 , I 2 ) was then investigated, and products were characterised using NMR spectroscopy and single-crystal X-ray diffraction. X-ray analyses elucidated the solid-state coordination types of the obtained products, showing that a variety of possible adducts can be obtained. In some cases, we were able to extrapolate a structure/activity correlation to explain the observed trends in reactivity and oxidation potentials.

HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactions─reverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT → OLA by 8.06 cycles (∼250-fold) compared to direct pipette processing and detected between 10 3 and 10 4 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level., (© 2022. The Author(s).)

Introduction: In COVID-19 pneumonia, there is a massive increase in fatty acid levels and lipid mediators with a predominance of cyclooxygenase metabolites, notably TxB 2 ≫ PGE 2 > PGD 2 in the lungs, and 11-dehydro-TxB 2 , a TxA 2 metabolite, in the systemic circulation. While TxA 2 stimulates thromboxane prostanoid (TP) receptors, 11-dehydro-TxB 2 is a full agonist of DP2 (formerly known as the CRTh2) receptors for PGD 2 . Anecdotal experience of using ramatroban, a dual receptor antagonist of the TxA 2 /TP and PGD 2 /DP2 receptors, demonstrated rapid symptomatic relief from acute respiratory distress and hypoxemia while avoiding hospitalization., Areas Covered: Evidence supporting the role of TxA 2 /TP receptors and PGD 2 /DP2 receptors in causing rapidly progressive lung injury associated with hypoxemia, a maladaptive immune response and thromboinflammation is discussed. An innovative perspective on the dual antagonism of TxA 2 /TP and PGD 2 /DP2 receptor signaling as a therapeutic approach in COVID-19 is presented. This paper examines ramatroban an anti-platelet, immunomodulator, and antifibrotic agent for acute and long-haul COVID-19., Expert Opinion: Ramatroban, a dual blocker of TP and DP2 receptors, has demonstrated efficacy in animal models of respiratory dysfunction, atherosclerosis, thrombosis, and sepsis, as well as preliminary evidence for rapid relief from dyspnea and hypoxemia in COVID-19 pneumonia. Ramatroban merits investigation as a promising antithrombotic and immunomodulatory agent for chemoprophylaxis and treatment.

Transmetalation is a key method for the construction of element-element bonds. Here, we disclose the reactivity of [Ni II (Ar)(I)(diphosphine)] compounds with arylgold(I) transmetalating agents, which is directly relevant to cross-coupling catalysis. Both aryl-for-iodide and unexpected aryl-for-aryl transmetalation are witnessed. Despite the strong driving force expected for Au-I bond formation, aryl scrambling can occur during transmetalation and may complicate the outcomes of attempted catalytic cross-coupling reactions.

Acidification in intracellular organelles is tightly linked to the influx of Cl - counteracting proton translocation by the electrogenic V-ATPase. We quantified the dynamics of Cl - transfer accompanying cargo incorporation into single phagosomes in alveolar macrophages (AMs). Phagosomal Cl - concentration and acidification magnitude were followed in real time with maximal acidification achieved at levels of approximately 200 mM. Live cell confocal microscopy verified that phagosomal Cl - influx utilized predominantly the Cl - channel CFTR. Relative levels of elemental chlorine (Cl) in hard X-ray fluorescence microprobe (XFM) analysis within single phagosomes validated the increase in Cl - content. XFM revealed the complex interplay between elemental K content inside the phagosome and changes in Cl - during phagosomal particle uptake. Cl - -dependent changes in phagosomal membrane potential were obtained using second harmonic generation (SHG) microscopy. These studies provide a mechanistic insight for screening studies in drug development targeting pulmonary inflammatory disease., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) x 10 6 pores per cm 2 . Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm 2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oxidative addition represents a critical elementary step in myriad catalytic transformations. Here, the importance of thoughtful ligand design cannot be overstated. In this work, we report the intermolecular activation of iodobenzene (PhI) at a coordinatively saturated 18-electron [Ni 0 (diphosphine) 2 ] complex bearing a Lewis acidic secondary coordination sphere. Whereas alkyl-substituted diphosphine complexes of Group 10 are known to be unreactive in such reactions, we show that [Ni 0 (P 2 B Cy 4 ) 2 ] (P 2 B Cy 4 =1,2-bis(di(3-dicyclohexylboraneyl)-propylphosphino)ethane) is competent for room-temperature PhI cleavage to give [Ni II (P 2 B Cy 4 )(Ph)(I)]. This difference in oxidative addition reactivity has been scrutinized computationally - an outcome that is borne out in ring-opening to provide the reactive precursor - for [Ni 0 (P 2 B Cy 4 ) 2 ], a "boron-trapped" 16-electron κ 1 -diphosphine Ni(0) complex. Moreover, formation of [Ni II (P 2 B Cy 4 )(Ph)(I)] is inherent to the P 2 B Cy 4 secondary coordination sphere: treatment of the Lewis adduct, [Ni 0 (P 2 B Cy 4 ) 2 (DMAP) 8 ] with PhI provides [Ni II (P 2 B Cy 4 ) 2 (DMAP) 8 (I)]I via iodine-atom abstraction and not a [Ni II (Ph)(I)(diphosphine)] compound - an unusual secondary sphere effect. Finally, the reactivity of [Ni 0 (P 2 B Cy 4 ) 2 ] with 4-iodopyridine was surveyed, which resulted in a pyridyl-borane linked oligomer. The implications of these outcomes are discussed in the context of designing strongly donating, and yet labile diphosphine ligands for use in a critical bond activation step relevant to catalysis., (© 2021 Wiley-VCH GmbH.)