Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Seven-year outcomes of venetoclax-ibrutinib therapy in mantle cell lymphoma: durable responses and treatment-free remissions.

In the phase-2 clinical trial (AIM) of venetoclax-ibrutinib, 24 patients with mantle cell lymphoma (MCL; 23 with relapsed/refractory [R/R] disease) received ibrutinib 560mg and venetoclax 400mg both once daily. High complete remission (CR) and measurable residual disease negative (MRD-negative) CR rates were previously reported. With median survivor follow-up now exceeding 7 years, we report long-term results. Treatment was initially continuous, with elective treatment interruption (ETI) allowed after protocol amendment for patients in MRD-negative CR. For R/R MCL, the estimated 7-year progression-free survival (PFS) was 30% [95%CI: 14-49] (median 28 months [95%CI: 13-82]) and overall survival was 43% [95%CI: 23-62] (median 32 months [95%CI: 15-NE]). Eight patients in MRD-negative CR entered ETI for a median of 58 months (95%CI, 37-79), with four experiencing disease recurrence. Two of 3 re-attained CR on retreatment. Time-to-treatment-failure (TTF), which excluded progression in ETI for those reattaining response, was 39% overall and 68% at 7-years for responders. Beyond 56 weeks Grade 3 and serious adverse events were uncommon. Newly emergent or increasing cardiovascular toxicity were not observed beyond 56 weeks. We demonstrate long-term durable responses and acceptable toxicity profile of venetoclax-ibrutinib in R/R MCL and show feasibility of treatment interruption while maintaining ongoing disease control. (NCT02471391).

Acquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Sorafenib plus intensive chemotherapy in newly diagnosed FLT3-ITD AML: a randomized, placebo-controlled study by the ALLG.

Sorafenib maintenance improves outcomes after hematopoietic cell transplant (HCT) for patients with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) acute myeloid leukemia (AML). Although promising outcomes have been reported for sorafenib plus intensive chemotherapy, randomized data are limited. This placebo-controlled, phase 2 study (ACTRN12611001112954) randomized 102 patients (aged 18-65 years) 2:1 to sorafenib vs placebo (days 4-10) combined with intensive induction: idarubicin 12 mg/m2 on days 1 to 3 plus either cytarabine 1.5 g/m2 twice daily on days 1, 3, 5, and 7 (18-55 years) or 100 mg/m2 on days 1 to 7 (56-65 years), followed by consolidation and maintenance therapy for 12 months (post-HCT excluded) in newly diagnosed patients with FLT3-ITD AML. Four patients were excluded in a modified intention-to-treat final analysis (3 not commencing therapy and 1 was FLT3-ITD negative). Rates of complete remission (CR)/CR with incomplete hematologic recovery were high in both arms (sorafenib, 78%/9%; placebo, 70%/24%). With 49.1-months median follow-up, the primary end point of event-free survival (EFS) was not improved by sorafenib (2-year EFS 47.9% vs 45.4%; hazard ratio [HR], 0.87; 95% confidence interval [CI], 0.51-1.51; P = .61). Two-year overall survival (OS) was 67% in the sorafenib arm and 58% in the placebo arm (HR, 0.76; 95% CI, 0.42-1.39). For patients who received HCT in first remission, the 2-year OS rates were 84% and 67% in the sorafenib and placebo arms, respectively (HR, 0.45; 95% CI, 0.18-1.12; P = .08). In exploratory analyses, FLT3-ITD measurable residual disease (MRD) negative status (<0.001%) after induction was associated with improved 2-year OS (83% vs 60%; HR, 0.4; 95% CI, 0.17-0.93; P = .028). In conclusion, routine use of pretransplant sorafenib plus chemotherapy in unselected patients with FLT3-ITD AML is not supported by this study.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

Clonal hematopoiesis, myeloid disorders and BAX-mutated myelopoiesis in patients receiving venetoclax for CLL.

The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy.

Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.

Chemoselective, Oxidation-Induced Macrocyclization of Tyrosine-Containing Peptides.

Inspired by nature's wide range of oxidation-induced modifications to install cross-links and cycles at tyrosine (Tyr) and other phenol-containing residue side chains, we report a Tyr-selective strategy for the preparation of Tyr-linked cyclic peptides. This approach leverages N4-substituted 1,2,4-triazoline-3,5-diones (TADs) as azo electrophiles that react chemoselectively with the phenolic side chain of Tyr residues to form stable C-N1-linked cyclic peptides. In the developed method, a precursor 1,2,4-triazolidine-3,5-dione moiety, also known as urazole, is readily constructed at any free amine revealed on a solid-supported peptide. Once prepared, the N4-substituted urazole peptide is selectively oxidized using mild, peptide-compatible conditions to generate an electrophilic N4-substituted TAD peptide intermediate that reacts selectively under aqueous conditions with internal and terminal Tyr residues to furnish Tyr-linked cyclic peptides. The approach demonstrates good tolerance of native residue side chains and enables access to cyclic peptides ranging from 3- to 11-residues in size (16- to 38-atom-containing cycles). The identity of the installed Tyr-linkage, a stable covalent C-N1 bond, was characterized using NMR spectroscopy. Finally, we applied the developed method to prepare biologically active Tyr-linked cyclic peptides bearing the integrin-binding RGDf epitope.

BCL2 and MCL1 inhibitors for hematologic malignancies.

BCL2 and MCL1 are commonly expressed prosurvival (antiapoptotic) proteins in hematologic cancers and play important roles in their biology either through dysregulation or by virtue of intrinsic importance to the cell-of-origin of the malignancy. A new class of small-molecule anticancer drugs, BH3 mimetics, now enable specific targeting of these proteins in patients. BH3 mimetics act by inhibiting the prosurvival BCL2 proteins to enable the activation of BAX and BAK, apoptosis effectors that permeabilize the outer mitochondrial membrane, triggering apoptosis directly in many cells and sensitizing others to cell death when combined with other antineoplastic drugs. Venetoclax, a specific inhibitor of BCL2, is the first approved in class, demonstrating striking single agent activity in chronic lymphocytic leukemia and in other lymphoid neoplasms, as well as activity against acute myeloid leukemia (AML), especially when used in combination. Key insights from the venetoclax experience include that responses occur rapidly, with major activity as monotherapy proving to be the best indicator for success in combination regimens. This emphasizes the importance of adequate single-agent studies for drugs in this class. Furthermore, secondary resistance is common with long-term exposure and often mediated by genetic or adaptive changes in the apoptotic pathway, suggesting that BH3 mimetics are better suited to limited duration, rather than continuous, therapy. The success of venetoclax has inspired development of BH3 mimetics targeting MCL1. Despite promising preclinical activity against MYC-driven lymphomas, myeloma, and AML, their success may particularly depend on their tolerability profile given physiological roles for MCL1 in several nonhematologic tissues.

Efficacy of venetoclax plus rituximab for relapsed CLL: 5-year follow-up of continuous or limited- duration therapy.

We report long-term follow-up of the phase 1b study of venetoclax and rituximab (VenR) in patients with relapsed chronic lymphocytic leukemia (CLL), including outcomes with continuous or limited-duration therapy. Patients received venetoclax daily (200-600 mg) and rituximab over 6 months and then received venetoclax monotherapy. Patients achieving complete response (CR), CR with incomplete marrow recovery (CRi), or undetectable minimal residual disease (uMRD) assessed by flow cytometry (<10-4 cutoff) were allowed, but not required, to discontinue therapy, while remaining in the study and could be retreated with VenR upon progression. Median follow-up for all patients (N = 49) was 5.3 years. Five-year rates (95% CI) for overall survival, progression-free survival, and duration of response were 86% (72-94), 56% (40-70), and 58% (40-73), respectively. Of the 33 deep responders (CR/CRi or uMRD), 14 remained on venetoclax monotherapy (continuous therapy), and 19 stopped venetoclax therapy (limited-duration therapy) after a median of 1.4 years. Five-year estimates of ongoing response were similar between continuous (71%; 95% CI, 39-88) or limited-duration therapy (79% [49-93]). Six of 19 patients in the latter group had subsequent disease progression, all >2 years off venetoclax (range, 2.1-6.4). Four patients were retreated with VenR, with partial responses observed in the 3 evaluable to date. VenR induced deep responses that were highly durable with either continuous or limited-duration therapy. Retreatment with VenR induced responses in patients with CLL progression after discontinuing therapy. Continuous exposure to venetoclax in deep responders does not appear to provide incremental benefit.

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

GEnZ explorer: a tool for visualizing agroclimate to inform research and regulatory risk assessment.

Confined field trials (CFT) of genetically engineered (GE) crops are used to generate data to inform environmental risk assessments (ERA). ERAs are required by regulatory authorities before novel GE crops can be released for cultivation. The transportability of CFT data to inform risk assessment in countries other than those where the CFT was conducted has been discussed previously in an analysis showing that the primary difference between CFT locations potentially impacting trial outcomes is the physical environment, particularly the agroclimate. This means that data from trials carried out in similar agroclimates could be considered relevant and sufficient to satisfy regulatory requirements for CFT data, irrespective of the country where the CFTs are conducted. This paper describes the development of an open-source tool to assist in determining the transportability of CFT data. This tool provides agroclimate together with overall crop production information to assist regulators and applicants in making informed choices on whether data from previous CFTs can inform an environmental risk assessment in a new country, as well as help developers determine optimal locations for planning future CFTs. The GEnZ Explorer is a freely available, thoroughly documented, and open-source tool that allows users to identify the agroclimate zones that are relevant for the production of 21 major crops and crop categories or to determine the agroclimatic zone at a specific location. This tool will help provide additional scientific justification for CFT data transportability, along with spatial visualization, to help ensure regulatory transparency.

Healthcare utilisation and costing for decompensated chronic liver disease hospitalisations at a Victorian network.

BACKGROUND: The economic burden of decompensated chronic liver disease (CLD) on Australian healthcare services is poorly characterised. AIMS: To evaluate the in-patient healthcare utilisation costs associated with decompensated CLD at Monash Health, an Australian tertiary healthcare service. METHODS: The current retrospective cost analysis examined patients with decompensated CLD admitted between 1 January 2012 and 31 December 2018. Hospitalisations were identified using CLD-specific International Classification of Diseases, Tenth Revision, codes. Cost measures were estimated using the Victorian Weighted Inlier Equivalent Separation funding data based on the Australian Refined Diagnosis Related Groups cost weights. RESULTS: There were 707 hospitalisations in 435 adult patients. The mean age was 56.7 ± 11.7 years and the mean length of stay was 10.28 ± 11.2 days. Median survival was 31 months (interquartile range, 2-94 months) and 177 (40.8%) patients died within 1 year of admission. The cost of admission varied according to decompensation: hepatorenal syndrome ($20 162 AUD), variceal bleed ($16 630 AUD), spontaneous bacterial peritonitis ($12 664 AUD), hepatic encephalopathy ($9973 AUD) and ascites ($9001 AUD). There was no significant difference in the admissions or 30-day readmission rate from 2012 to 2018 financial year (FY). The total adjusted cost of cirrhotic admissions per year increased by 78% from FY2012 to FY2018. CONCLUSION: Hospital admission and readmission for decompensated CLD is common and associated with 40.8% 1-year mortality and high costs. Clearer delineation of goals of care and alternative ambulatory care models for decompensated CLD are urgently required to reduce the high costs and burden on health services.

Gluteal muscle fatty infiltration, fall risk, and mobility limitation in older women with urinary incontinence: a pilot study.

OBJECTIVE: Gluteal muscle quality influences risk of falling and mobility limitation. We sought (1) to compare gluteal muscle fatty infiltration (FI) between groups of older women with urinary incontinence (UI) at risk for falling (at-risk group) and not at risk for falling (not-at-risk group), and (2) to determine correlation of gluteal FI with Timed Up and Go (TUG) and Short Physical Performance Battery (SPPB) performance. MATERIALS AND METHODS: Prospective pilot study of gluteal FI on pelvis MRI for 19 women with UI, aged ≥ 70 years. A musculoskeletal radiologist selected axial T1-weighted MR images; then, two blinded medical student research assistants analyzed gluteal FI by quantitative fuzzy C-means segmentation. TUG and SPPB tests were performed. TUG ≥ 12 s defined participants as at risk for falling. Descriptive, correlation, and reliability analyses were performed. RESULTS: Mean age, 76.3 ± 4.8 years; no difference for age or body mass index (BMI) between the at-risk (n = 5) versus not-at-risk (n = 14) groups. SPPB score (p = 0.013) was lower for the at-risk group (6.4 ± 3.1) than for the not-at-risk group (10.2 ± 1.9). Fuzzy C-means FI-%-estimate differed between the at-risk group and the not-at-risk group for bilateral gluteus medius/minimus (33.2% ± 15.6% versus 19.5% ± 4.1%, p = 0.037) and bilateral gluteus maximus (33.6% ± 15.6% versus 19.7% ± 6.9%, p = 0.047). Fuzzy C-means FI-%-estimate for bilateral gluteus maximus had significant (p < 0.050) moderate correlation with age (rho = - 0.64), BMI (rho = 0.65), and TUG performance (rho = 0.52). Fuzzy C-means FI-%-estimates showed excellent inter-observer and intra-observer reliability (intraclass correlation coefficient, ≥ 0.892). CONCLUSION: Older women with UI at risk for falling have greater levels of gluteal FI and mobility limitation as compared to those not at risk for falling.

Notoamide R: A Prominent Diketopiperazine Fermentation Metabolite amongst Others of Aspergillus ochraceus in the Absence of Ochratoxins.

Ochratoxin A is historically the most notable secondary metabolite of Aspergillus ochraceus on account of its toxicity to animals and fish. Currently, over 150 compounds of diverse structure and biosynthesis is a challenge to predict the array for any particular isolate. A brief focus 30 years ago on the failure to produce ochratoxins in foods in Europe and the USA revealed consistent failures to produce ochratoxin A by isolates from some USA beans. Analysis for familiar or novel metabolites particularly focused on a compound for which mass and NMR analyses were inconclusive. Resort to 14C-labelled biosynthetic precursors, particularly phenylalanine, to search for any close alternative to ochratoxins, was combined with conventional shredded-wheat/shaken-flask fermentation. This yielded, for an extract, an autoradiograph of a preparative silica gel chromatogram, which was subsequently analysed for an excised fraction using spectroscopic methodologies. Circumstances then delayed progress for many years until the present collaboration revealed notoamide R. Meanwhile, pharmaceutical discovery around the turn of the millennium revealed stephacidins and notoamides, biosynthetically combining indole, isoprenyl and diketopiperazine components. Later, in Japan, notoamide R was added as a metabolite of an Aspergillus sp. isolated from a marine mussel, and the compound was recovered from 1800 Petri dish fermentations. Renewed attention to our former studies in England has since shown for the first time that notoamide R can be a prominent metabolite of A. ochraceus, sourced from a single shredded wheat flask culture with its structure confirmed by spectroscopic data, and in the absence of ochratoxins. Renewed attention to the archived autoradiographed chromatogram allowed further exploration, but in particular has stimulated a fundamental biosynthetic approach to considering influences redirecting intermediary metabolism to secondary metabolite accumulation.

Identification and characterization of magnetotactic Gammaproteobacteria from a salt evaporation pool, Bohai Bay, China.

Magnetotactic bacteria (MTB) are phylogenetically diverse prokaryotes that can produce intracellular chain-assembled nanocrystals of magnetite (Fe3 O4 ) or greigite (Fe3 S4 ). Compared with their wide distribution in the Alpha-, Eta- and Delta-proteobacteria classes, few MTB strains have been identified in the Gammaproteobacteria class, resulting in limited knowledge of bacterial diversity and magnetosome biomineralization within this phylogenetic branch. Here, we identify two magnetotactic Gammaproteobacteria strains (tentatively named FZSR-1 and FZSR-2 respectively) from a salt evaporation pool in Bohai Bay, at the Fuzhou saltern, Dalian City, eastern China. Phylogenetic analysis indicates that strain FZSR-2 is the same species as strains SHHR-1 and SS-5, which were discovered previously from brackish and hypersaline environments respectively. Strain FZSR-1 represents a novel species. Compared with strains FZSR-2, SHHR-1 and SS-5 in which magnetite particles are assembled into a single chain, FZSR-1 cells form relatively narrower magnetite nanoparticles that are often organized into double chains. We find a good relationship between magnetite morphology within strains FZSR-2, SHHR-1 and SS-5 and the salinity of the environment in which they live. This study expands the bacterial diversity of magnetotactic Gammaproteobacteria and provides new insights into magnetosome biomineralization within magnetotactic Gammaproteobacteria.

Identification of sulfate-reducing magnetotactic bacteria via a group-specific 16S rDNA primer and correlative fluorescence and electron microscopy: Strategy for culture-independent study.

Magnetotactic bacteria (MTB) biomineralize intracellular magnetic nanocrystals and swim along geomagnetic field lines. While few axenic MTB cultures exist, living cells can be separated magnetically from natural environments for analysis. The bacterial universal 27F/1492R primer pair has been used widely to amplify nearly full-length 16S rRNA genes and to provide phylogenetic portraits of MTB communities. However, incomplete coverage and amplification biases inevitably prevent detection of some phylogenetically specific or non-abundant MTB. Here, we propose a new formulation of the upstream 390F primer that we combined with the downstream 1492R primer to specifically amplify 1100-bp 16S rRNA gene sequences of sulfate-reducing MTB in freshwater sediments from Lake Weiyanghu, Xi'an, northwestern China. With correlative fluorescence in situ hybridization and scanning/transmission electron microscopy, three novel MTB strains (WYHR-2, WYHR-3 and WYHR-4) from the Desulfobacterota phylum were identified phylogenetically and structurally at the single-cell level. Strain WYHR-2 produces bullet-shaped magnetosome magnetite, while the other two strains produce both cubic/prismatic greigite and bullet-shaped magnetite. Our results expand knowledge of bacterial diversity and magnetosome biomineralization of sulfate-reducing MTB. We also propose a general strategy for identifying and characterizing uncultured MTB from natural environments.

Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells.

In this study we demonstrate a new form of immunoregulation: engagement on CD4(+) T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T(H)1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4(+) T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4(+) T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-gamma (IFN-gamma). Finally, gammadelta T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) gammadelta and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.