A Decline in p38 MAPK Signaling Underlies Immunosenescence in Caenorhabditis elegans

The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans

Targeting the Replication Initiator of the Second Vibrio Chromosome: Towards Generation of Vibrionaceae-Specific Antimicrobial Agents

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms

Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function

Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNAPro peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748–A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNAPro against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel

Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs

Brentuximab vedotin in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone as frontline treatment for patients with CD30-positive B-cell lymphomas

We conducted a phase I/II multicenter trial using 6 cycles of brentuximab vedotin (BV) in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHP) for treatment of patients with CD30-positive (+) B-cell lymphomas. Thirty-one patients were evaluable for toxicity and 29 for efficacy including 22 with primary mediastinal B-cell lymphoma (PMBCL), 5 with diffuse large B-cell lymphoma (DLBCL), and 2 with gray zone lymphoma (GZL). There were no treatment-related deaths; 32% of patients had non-hematological grade 3/4 toxicities. The overall response rate was 100% (95% CI: 88-100) with 86% (95% CI: 68-96) of patients achieving complete response at the end of systemic treatment. Consolidative radiation following end of treatment response assessment was permissible and used in 52% of all patients including 59% of patients with PMBCL. With a median follow-up of 30 months, the 2-year progression-free survival (PFS) and overall survival (OS) were 85% (95% CI: 66-94) and 100%, respectively. In the PMBCL cohort, 2-year PFS was 86% (95% CI: 62-95). In summary, BV-R-CHP with or without consolidative radiation is a feasible and active frontline regimen for CD30+ B-cell lymphomas (NCT01994850)

The PP2A/4/6 subfamily of phosphoprotein phosphatases regulates DAF-16 and confers resistance to environmental stress in postreproductive adult C. elegans.

Insulin and insulin-like growth factors are longevity determinants that negatively regulate Forkhead box class O (FoxO) transcription factors. In C. elegans mutations that constitutively activate DAF-16, the ortholog of mammalian FoxO3a, extend lifespan by two-fold. While environmental insults induce DAF-16 activity in younger animals, it also becomes activated in an age-dependent manner in the absence of stress, modulating gene expression well into late adulthood. The mechanism by which DAF-16 activity is regulated during aging has not been defined. Since phosphorylation of DAF-16 generally leads to its inhibition, we asked whether phosphatases might be necessary for its increased transcriptional activity in adult C. elegans. We focused on the PP2A/4/6 subfamily of phosphoprotein phosphatases, members of which had been implicated to regulate DAF-16 under low insulin signaling conditions but had not been investigated during aging in wildtype animals. Using reverse genetics, we functionally characterized all C. elegans orthologs of human catalytic, regulatory, and scaffolding subunits of PP2A/4/6 holoenzymes in postreproductive adults. We found that PP2A complex constituents PAA-1 and PPTR-1 regulate DAF-16 transcriptional activity during aging and that they cooperate with the catalytic subunit LET-92 to protect adult animals from ultraviolet radiation. PP4 complex members PPH-4.1/4.2, and SMK-1 also appear to regulate DAF-16 in an age-dependent manner, and together with PPFR-2 they contribute to innate immunity. Interestingly, SUR-6 but no other subunit of the PP2A complex was necessary for the survival of pathogen-infected animals. Finally, we found that PP6 complex constituents PPH-6 and SAPS-1 contribute to host defense during aging, apparently without affecting DAF-16 transcriptional activity. Our studies indicate that a set of PP2A/4/6 complexes protect adult C. elegans from environmental stress, thus preserving healthspan. Therefore, along with their functions in cell division and development, the PP2A/4/6 phosphatases also appear to play critical roles later in life

Efficacy and Limitations of Chemically Diverse Small-Molecule Enzyme-Inhibitors against the Synergistic Coagulotoxic Activities of <i>Bitis</i> Viper Venoms

Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have begun to focus on the use of small-molecule inhibitors as potential candidates for the neutralisation of key snake venom toxins and as potential field therapies. Bitis vipers represent some of the most medically important as well as frequently encountered snake species in Africa, with a number of species possessing anticoagulant phospholipase A2 (PLA2) toxins that prevent the prothrombinase complex from inducing clot formation. Additionally, species within the genus are known to exert pseudo-procoagulant activity, whereby kallikrein enzymatic toxins cleave fibrinogen to form a weak fibrin clot that rapidly degrades, thereby depleting fibrinogen levels and contributing to the net anticoagulant state. Utilising well-validated coagulation assays measuring time until clot formation, this study addresses the in vitro efficacy of three small molecule enzyme inhibitors (marimastat, prinomastat and varespladib) in neutralising these aforementioned activities. The PLA2 inhibitor varespladib showed the greatest efficacy for the neutralisation of PLA2-driven anticoagulant venom activity, with the metalloproteinase inhibitors prinomastat and marimastat both showing low and highly variable degrees of cross-neutralisation with PLA2 anticoagulant toxicity. However, none of the inhibitors showed efficacy in neutralising the pseudo-procoagulant venom activity exerted by the venom of B. caudalis. Our results highlight the complex nature of snake venoms, for which single-compound treatments will not be universally effective, but combinations might prove highly effective. Despite the limitations of these inhibitors with regards to in vitro kallikrein enzyme pseudo-procoagulant venom activity, our results further support the growing body of literature indicating the potential use of small molecule inhibitors to enhance first-aid treatment of snakebite envenoming, particularly in cases where hospital and thus antivenom treatment is either unavailable or far away

DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie age-dependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegans DAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16-mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life

Differential Antivenom and Small-Molecule Inhibition of Novel Coagulotoxic Variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American Viperid Snake Venoms

Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for Atropoides picadoi, Cerrophidiontzotzilorum, Metlapilcoatlus mexicanus, M. nummifer, M. occiduus, M. olmec, and Porthidium porrasi. In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with C. godmani and C. wilsoni being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but Cerrophidion godmani and C. wilsoni were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. C. godmani and C. wilsoni were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, Porthidium dunni potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, Porthidium volcanicum was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the Oxyuranus/Pseudonaja elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of P. volcanicum, neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies