Preparation of Simple Bicyclic Carboxylate-Rich Alicyclic Molecules for the Investigation of Dissolved Organic Matter.

Dissolved organic matter (DOM) is a vast and complex chemical mixture that plays a key role in the mediation of the global carbon cycle. Fundamental understanding of the source and fate of oceanic organic matter is obscured due to poor definition of the key molecular contributors to DOM, which limits accurate sample analysis and prediction of the Earth's carbon cycle. Previous work has attempted to define the components of the DOM through a variety of chromatographic and spectral techniques. However, modern preparative and analytical methods have not isolated or unambiguously identified molecules from DOM. Therefore, previously proposed structures are based solely on the mixture's aggregate properties and do not accurately describe any true individual molecular component. In addition to this, there is a lack of appropriate analogues of the individual chemical classes within DOM, limiting the scope of experiments that probe the physical, chemical, and biological contributions from each class. To address these problems, we synthesized a series of analogues of carboxylate-rich alicyclic molecules (CRAM), a molecular class hypothesized to exist as a major contributor to DOM. Key analytical features of the synthetic CRAMs were consistent with marine DOM, supporting their suitability as chemical substitutes for CRAM. This new approach provides access to a molecular toolkit that will enable previously inaccessible experiments to test many unproven hypotheses surrounding the ever-enigmatic DOM.

Characterization of a Newly Available Coastal Marine Dissolved Organic Matter Reference Material (TRM-0522).

Recent methodological advances have greatly increased our ability to characterize aquatic dissolved organic matter (DOM) using high-resolution instrumentation, including nuclear magnetic resonance (NMR) and mass spectrometry (HRMS). Reliable DOM reference materials are required for further method development and data set alignment but do not currently exist for the marine environment. This presents a major limitation for marine biogeochemistry and related fields, including natural product discovery. To fill this resource gap, we have prepared a coastal marine DOM reference material (TRM-0522) from 45 m deep seawater obtained ∼1 km offshore of Sweden's west coast. Over 3000 molecular formulas were assigned by direct infusion HRMS, confirming sample diversity, and the distribution of formulas in van Krevelen space was typical for a marine sample, with the majority of formulas in the region H/C 1-1.5 and O/C 0.3-0.7. The extracted DOM pool was more nitrogen (N)- and sulfur (S)-rich than a typical terrestrial reference material (SRFA). MZmine3 processing of ultrahigh-performance liquid chromatography (UPLC)-HRMS/MS data revealed 494 resolvable features (233 in negative mode; 261 in positive mode) over a wide range of retention times and masses. NMR data indicated low contributions from aromatic protons and, generally speaking, low lignin, humic, and fulvic substances associated with terrestrial samples. Instead, carboxylic-rich aliphatic molecules were the most abundant components, followed by carbohydrates and aliphatic functionalities. This is consistent with a very low specific UV absorbance SUVA254 value of 1.52 L mg C-1 m-1. When combined with comparisons with existing terrestrial reference materials (Suwannee River fulvic acid and Pony Lake fulvic acid), these results suggest that TRM-0522 is a useful and otherwise unavailable reference material for use in marine DOM biogeochemistry.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Does the Reaction of Cyclopropyl Acid Chlorides and Imines To Form 1,3-Oxazin-4-enone Heterocycles Proceed via a Ketene or an N-Acyl-iminium Mechanism?

The mechanism of the reaction between cyclopropyl acid chlorides and imines to form 1,3-oxazin-4-enones was probed through physical and computational experiments. The data gathered strongly support the reaction proceeding through an N-acyl iminium intermediate mechanism rather than a ketene intermediate mechanism.

Sphincterotomy for biliary sphincter of Oddi dysfunction.

The review is withdrawn as it has not been maintained since its first publication in 2001 (searches date back to the year 2000). Since then, new trials have been published that may or may not change the conclusions of the review. A new team of authors overtook the review on 26.10.2020, and the new review is expected to be published by the beginning of 2022. The review will be prepared based on most recent Cochrane methods. Readers may still find the outdated review on the CDSR (the Cochrane Library).

Siloxymethylamines as Aminomethylation Reagents for Amines Leading to Labile Diaminomethanes That can be Trapped as Their [Mo(CO)4 ] Complexes.

Compound Et3 SiOCH2 NMe2 transfers Me2 NCH2 to R2 NH (R2 =Et2 , PhMe, [Cr(η(6) -C6 H5 )(CO)3 ]Me, PhH) to form previously unknown diaminomethanes, Me2 NCH2 NR2 and, in the case of R2 =PhH, the triamine Me2 NCH2 N(Ph)CH2 NMe2 . The diaminomethanes exhibit an unreported disproportionation to a mixture of (R2 N)2 CH2 , (Me2 N)2 CH2 , and Me2 NCH2 NR2 , which can be trapped as their [Mo(CO)4 (diamine)] complexes. Whereas PhMeNCH2 NMe2 is a labile material, the metal-substituted ([(η(6) -C6 H5 )Cr(CO)3 ]MeNCH2 NMe2 is a stable material. The triamine Me2 NCH2 N(Ph)CH2 NMe2 is unstable with respect to transformation to 1,3,5-triphenyltriazine, but is readily trapped as the bidentate-triamineMo(CO)4 . All metal complexes were characterized by single-crystal X-ray diffraction.

Utility of PD-1, PD-L1, and IDO-1 Stains in Ocular Extranodal Marginal Zone Lymphoma (MZL) and Diffuse Large B-cell Lymphoma (DLBCL).

Extranodal marginal zone lymphoma (EMZL) is the most common subtype of ocular lymphomas. Diffuse large B-cell lymphoma (DLBCL) and EMZL with large-cell transformation present diagnostic challenges. Radiotherapy is the standard treatment for ocular lymphomas, but complications and relapse are common. Diagnostic utility in challenging cases, as well as treatment options using immune checkpoint inhibitors, are unclear in ocular lymphomas. We herein investigated the PD-1, PD-L1, and IDO1 staining patterns in 20 cases of ocular lymphomas, including EMZL (n=14), EMZL with increased large cells (n=2), and DLBCL (n=4). PD-1, PD-L1, and IDO1 staining was not detected in lymphoma cells in any cases but was observed within the tumor microenvironment in all cases. Positivity for PD-1, PD-L1, and IDO1 in inflammatory cells was seen either intratumorally or peritumorally. In all 6 cases with significantly more large B cells, the density of PD-1, PD-L1, and IDO1 expression in the tumor microenvironment was higher than that of the remaining 14 cases without large B cells (P-value<0.0001), whereas other clinicopathologic features showed no statistical correlation. Increased expression of PD-1, PD-L1, and IDO1 in the inflammatory milieu in cases with large cells may provide diagnostic utility in small biopsies as well as therapeutic potential.

ALK+ ALCL in the setting of adalimumab-related hidradenitis suppurativa.

This ALK-positive anaplastic large cell lymphoma (ALCL) case highlights the importance of considering ALCL in the setting of iatrogenic immunosuppression in hidradenitis suppurativa patients. Diagnosis of ALCL can be challenging in situations with very low viability/cellularity, negative CD3 and/or other T-cell markers, and immunosuppression.

Antimicrobial Peptides Incorporating Halogenated Marine-Derived Amino Acid Substituents.

Small synthetic mimics of cationic antimicrobial peptides represent a promising class of compounds with leads in clinical development for the treatment of persistent microbial infections. The activity and selectivity of these compounds rely on a balance between hydrophobic and cationic components, and here, we explore the activity of 19 linear cationic tripeptides against five different pathogenic bacteria and fungi, including clinical isolates. The compounds incorporated modified hydrophobic amino acids inspired by motifs often found in bioactive marine secondary metabolites in combination with different cationic residues to probe the possibility of generating active compounds with improved safety profiles. Several of the compounds displayed high activity (low µM concentrations), comparable with the positive controls AMC-109, amoxicillin, and amphotericin B. A higher activity was observed against the fungal strains, and a low in vitro off-target toxicity was observed against erythrocytes and HeLa cells, thereby illustrating effective means for tuning the activity and selectivity of short antimicrobial peptides.

A phase 1/2 study of lenalidomide and obinutuzumab with CHOP for newly diagnosed DLBCL.

Diffuse large B-cell lymphoma (DLBCL) can be cured with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP); however, one-third of patients experience refractory or relapsed disease. Studies comparing R-CHOP with modified regimens replacing R with obinutuzumab (O) or adding lenalidomide (L) did not result in improved outcomes; however, L and O together may enhance natural killer-cell mediated antibody-dependent cellular toxicity when paired with CHOP. Here, we report on a phase 1b/2 study of 53 patients with newly diagnosed DLBCL who received 6 cycles of LO-CHOP. The end of treatment overall and complete response rates of the 50 evaluable patients were 98% and 90%, respectively. After a median follow-up of 4.5 years, the 4-year progression free and overall survival rates were 87.4% and 91.3%, respectively. Grade 3 to 4 adverse events were experienced by 70% of patients, including neutropenia (38%), thrombocytopenia (17%), fatigue (13%), and neutropenic fever (13%). Of the 33 patients profiled with circulating tumor DNA (ctDNA) sequencing, 31 (94%) had detectable pretreatment ctDNA with cancer personalized profiling by deep sequencing, 24 (73%) were classifiable by the LymphGen classifier, and 15/20 (75%) and 12/17 (71%) patients achieved early and major molecular responses after 1 and 2 cycles, respectively. Using phased variant enrichment and detection sequencing, 16/18 evaluable patients (89%) showed no detectable ctDNA after at least 5 cycles of LO-CHOP. LO-CHOP demonstrates high efficacy and tolerability in newly diagnosed DLBCL, leading to a high rate of undetectable minimal residual disease by ctDNA. This trial has been registered at www.clinicaltrials.gov as NCT02529852.

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

Baseline physical performance, health, and functioning of participants in the Frequent Hemodialysis Network (FHN) trial.

BACKGROUND: Self-reported physical health and functioning and direct measures of physical performance are decreased in hemodialysis patients and are associated with mortality and hospitalization. STUDY DESIGN: We determined baseline cross-sectional associations of physical performance, health, and functioning with demographics, clinical characteristics, nutritional indexes, laboratory benchmarks, and measures of body composition in participants in the Frequent Hemodialysis Network (FHN) trial. SETTING & PARTICIPANTS: 375 persons enrolled in the FHN with data for physical performance, health, and functioning. PREDICTORS: Explanatory variables were categorized into fixed factors of age, race, comorbid conditions (diabetes mellitus, heart failure, and peripheral arterial disease) and potentially modifiable factors of dialysis dose, phosphorus level, hemoglobin level, equilibrated normalized protein catabolic rate (enPCR), body composition, body mass index, phase angle, and ratio of intracellular water volume to body weight (calculated from bioelectrical impedance). OUTCOMES: Scores on tests of physical performance, health, and functioning. MEASUREMENTS: Physical performance measured using the Short Physical Performance Battery, self-reported physical health and functioning using the 36-Item Short Form Health Survey (SF-36). Body composition (body mass index and bioimpedance analysis) and laboratory data were obtained from affiliated dialysis providers. RESULTS: Relative to population norms, scores for all 3 physicality metrics were low. Poorer scores on all 3 metrics were associated with diabetes mellitus and peripheral arterial disease. Poorer scores on the SF-36 Physical Functioning subscale and Short Physical Performance Battery also were associated with age, lower ratio of intracellular water volume to body weight, and lower enPCR. Black race was associated with poorer scores on the Short Physical Performance Battery. LIMITATIONS: This was a cross-sectional study of individuals agreeing to participate in the FHN study and may not be generalizable to the general dialysis population. CONCLUSIONS: Hemodialysis patients show markedly impaired physical performance, health, and functioning relative to population norms. Although some factors associated with these impairments are not modifiable, others may change with improvement in nutritional status or body composition.

A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling.

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Short Diagnosis-to-Treatment Interval Is Associated With Higher Circulating Tumor DNA Levels in Diffuse Large B-Cell Lymphoma.

PURPOSE: Patients with Diffuse Large B-cell Lymphoma (DLBCL) in need of immediate therapy are largely under-represented in clinical trials. The diagnosis-to-treatment interval (DTI) has recently been described as a metric to quantify such patient selection bias, with short DTI being associated with adverse risk factors and inferior outcomes. Here, we characterized the relationships between DTI, circulating tumor DNA (ctDNA), conventional risk factors, and clinical outcomes, with the goal of defining objective disease metrics contributing to selection bias. PATIENTS AND METHODS: We evaluated pretreatment ctDNA levels in 267 patients with DLBCL treated across multiple centers in Europe and the United States using Cancer Personalized Profiling by Deep Sequencing. Pretreatment ctDNA levels were correlated with DTI, total metabolic tumor volumes (TMTVs), the International Prognostic Index (IPI), and outcome. RESULTS: Short DTI was associated with advanced-stage disease (P < .001) and higher IPI (P < .001). We also found an inverse correlation between DTI and TMTV (RS = -0.37; P < .001). Similarly, pretreatment ctDNA levels were significantly associated with stage, IPI, and TMTV (all P < .001), demonstrating that both DTI and ctDNA reflect disease burden. Notably, patients with shorter DTI had higher pretreatment ctDNA levels (P < .001). Pretreatment ctDNA levels predicted short DTI independent of the IPI (P < .001). Although each risk factor was significantly associated with event-free survival in univariable analysis, ctDNA level was prognostic of event-free survival independent of DTI and IPI in multivariable Cox regression (ctDNA: hazard ratio, 1.5; 95% CI [1.2 to 2.0]; IPI: 1.1 [0.9 to 1.3]; -DTI: 1.1 [1.0 to 1.2]). CONCLUSION: Short DTI largely reflects baseline tumor burden, which can be objectively measured using pretreatment ctDNA levels. Pretreatment ctDNA levels therefore have utility for quantifying and guarding against selection biases in prospective DLBCL clinical trials.

Enhanced detection of minimal residual disease by targeted sequencing of phased variants in circulating tumor DNA.

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.

Gonadotropin-releasing hormone and protein kinase C signaling to ERK: spatiotemporal regulation of ERK by docking domains and dual-specificity phosphatases.

Activated ERK translocates to the nucleus to regulate transcription. Spatiotemporal aspects of this response dictate biological consequences and are influenced by dual-specificity phosphatases (DUSPs) that can scaffold and dephosphorylate ERK. In HeLa cells, GnRH causes transient and protein kinase C (PKC)-dependent ERK activation, but termination mechanisms are unknown. We now explore DUSP roles using short inhibitory RNA to knock down endogenous ERK, adenoviruses to express GnRH receptors and add-back ERK2-GFP, and automated microscopy to monitor ERK location and activation. GnRH caused rapid and transient increases in dual phosphorylated ERK2 (ppERK2) and nuclear to cytoplasmic ERK2-green fluorescent protein (GFP) ratio, whereas responses to a PKC-activating phorbol ester were more sustained. In cells expressing D319N ERK2-GFP (D319N mutation impairs docking-domain-dependent binding to DUSPs), GnRH caused more sustained increases in ppERK2 and nuclear to cytoplasmic ERK2-GFP ratio and also had more pronounced effects on Egr-1 luciferase (a transcriptional reporter for ERK activation). Cycloheximide caused more sustained effects of GnRH and phorbol ester on ppERK, suggesting termination by nuclear-inducible DUSPs. GnRH also increased expression of nuclear-inducible DUSP1 and -4, but their knockdown did not alter GnRH-mediated ERK signaling. Screening a short inhibitory RNA library targeting 16 DUSPs (nuclear-inducible DUSPs, cytoplasmic ERK MAPK phosphatases, c-Jun N-terminal kinase/p38 MAPK phosphatases, and atypical DUSPs) revealed GnRH effects to be influenced by DUSPs 5, 9, 10, 16, and 3 (i.e. by each DUSP class). Thus, GnRH-mediated ERK responses (like PKC-mediated ERK responses) are dependent on protein neosynthesis and docking-domain-dependent binding, but for GnRH activation (unlike PKC activation), this does not reflect dependence on nuclear-inducible DUSPs. Termination of these GnRH effects is apparently dependent upon a preexisting rapid turnover protein.

Liquid biopsy in lymphoma: Molecular methods and clinical applications.

In this article, we broadly review the application of cfDNA analysis to the diagnosis and management of lymphoma. We introduce the advantages of cfDNA measurement over conventional tissue biopsy and describe how cfDNA may be utilized for both genotyping and detection of minimal residual disease. First, we discuss genotyping, beginning with differences in identifying mutations from the blood plasma vs. from circulating cells. We review the technical distinctions between PCR- and NGS-based assays and describe two important applications of NGS-based cfDNA tests, namely the identification of resistance mutations and classification of disease subtype. We discuss difficulties in genotyping diseases with low burden of tumor cells and the application of cfDNA assays in these contexts. Second, we describe the utility of ctDNA measurement in assessing MRD. We cover recent advances in the assessment of pre-treatment disease burden as a prognostic biomarker, detection of molecular response to therapy, and early detection of relapsing disease. Third, we explore select emerging areas of research in ctDNA technologies that show promise in boosting the performance of existing ctDNA-based assays. These include cell-free DNA fragment structure analysis or 'fragmentomics', epigenetic modifications, and novel circulating analytes such as tumor-educated platelets and extracellular vesicular DNA. We also discuss alternative analytes to blood plasma for tumor detection, such as urine, saliva, and stool. Finally, we present a case that highlights potential applications of ctDNA approaches to the management of patients with lymphoma, while also defining important prerequisite advances before this can be fully realized. We close with a look to the future of cfDNA applications, outlining one potential timeline and path forward towards routine clinical application.

Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein.

BACKGROUND: Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. METHODS: The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. RESULTS: The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. CONCLUSION: The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function.

How Should Physicians Care for Dying Patients with Amyotrophic Lateral Sclerosis?

We discuss physician aid in dying, euthanasia, and other dimensions of palliative care decision making and define relevant terms raised by this case of a dying patient with amyotrophic lateral sclerosis in Washington State who is unable to self-administer a lethal prescription. We then present a concrete framework that clinicians can directly apply when faced with difficult cases such as this one. We outline how exploring motivations, obtaining informed consent, defining goals, and examining alternatives can help guide physicians like the one in this case. We conclude by summarizing one way in which physicians might balance these issues while still remaining within the constraints of the law.