Mining Biosynthetic Gene Clusters in Carnobacterium maltaromaticum by Interference Competition Network and Genome Analysis.

Carnobacterium maltaromaticum is a non-starter lactic acid bacterium (LAB) of interest in the dairy industry for biopreservation. This study investigated the interference competition network and the specialized metabolites biosynthetic gene clusters (BGCs) content in this LAB in order to explore the relationship between the antimicrobial properties and the genome content. Network analysis revealed that the potency of inhibition tended to increase when the inhibition spectrum broadened, but also that several strains exhibited a high potency and narrow spectrum of inhibition. The C. maltaromaticum strains with potent anti-L. monocytogenes were characterized by high potency and a wide intraspecific spectrum. Genome mining of 29 strains revealed the presence of 12 bacteriocin BGCs: four of class I and eight of class II, among which seven belong to class IIa and one to class IIc. Overall, eight bacteriocins and one nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) BGCs were newly described. The comparison of the antimicrobial properties resulting from the analysis of the network and the BGC genome content allowed us to delineate candidate BGCs responsible for anti-L. monocytogenes and anti-C. maltaromaticum activity. However, it also highlighted that genome analysis is not suitable in the current state of the databases for the prediction of genes involved in the antimicrobial activity of strains with a narrow anti-C. maltaromaticum activity.

Pharmacokinetics/pharmacodynamics by race: Analysis of a peginterferon ß-1a phase 1 study.

BACKGROUND: Black/African American participants are underrepresented in clinical trials for multiple sclerosis but can experience a greater burden of disease than other racial groups in the United States. A phase 1, open-label, crossover study that demonstrated bioequivalence of subcutaneous and intramuscular injection of peginterferon ß-1a in healthy volunteers enrolled similar proportions of Black and White participants, enabling a post hoc subgroup analysis comparing these groups. METHODS: Peginterferon ß-1a (125 µg) was administered by subcutaneous or intramuscular injection, followed by a washout period before a second injection using the alternative method. The primary pharmacokinetic and pharmacodynamic endpoints were maximum observed concentration (Cmax) and area under the concentration-time curve from hour 0 to infinity (AUCinf) of study drug and serum concentration of neop-terin, respectively. Safety and tolerability were included as secondary endpoints. FINDINGS: This analysis included 70 (51.5%) Black and 59 (43.3%) White participants. Peginterferon ß-1a Cmax was 29.8% higher in Black than in White participants following subcutaneous administration but was similar following intramuscular administration. Mean AUCinf was 31.0% and 11.8% greater in Black than in White participants with subcutaneous and intramuscular administration, respectively. Pharmacodynamics and safety signals were similar between groups, although Black participants reported numerically fewer adverse events. CONCLUSIONS: No clinically meaningful differences were identified between Black and White participants related to peginterferon ß-1a administration, supporting the approved dose of 125 µg/mL peginterferon ß-1a. Future clinical studies should include sufficiently diverse populations to ensure accurate assessments of treatment response. FUNDING: Funding for medical writing support was provided by Biogen (Cambridge, MA, USA).

Peginterferon beta-1a concentrations in breast milk of lactating multiple sclerosis patients.

BACKGROUND: Peginterferon beta-1a is an interferon beta-1a formulation that has been pegylated, resulting in a longer half-life than other interferon beta formulations. We examined concentrations of peginterferon beta-1a in breast milk of lactating patients with multiple sclerosis (MS) receiving peginterferon beta-1a as their postpartum disease-modifying therapy. METHODS: After completion of titration to a full dose of peginterferon beta-1a and following a single full dose peginterferon beta-1a injection (125 µg), breast milk samples (≥10 mL) were collected by 5 women on days 1-14 post injection. Peginterferon beta-1a concentrations in breast milk samples were measured by a qualified enzyme-linked immunosorbent assay (detection threshold: 15 pg/mL). Mean and median daily concentrations and median maximum concentration (Cmax), time of Cmax (Tmax), time of last measurable concentration (Tlast), area under the concentration-time curve (AUClast), and relative infant dose (RID) were determined. RESULTS: After receiving a single full dose peginterferon beta-1a injection, the maximum breast milk concentration recorded in an individual patient was 126.2 pg/mL (0.00013 µg/mL) on day 6. The remaining patients all had maximum breast milk concentrations <72 pg/mL. The geometric mean of Cmax was 48.9 pg/mL and the median Tmax and Tlast were 4 and 7 days, respectively. The median AUClast was 210.9 day*pg/mL. Among the 5 study patients, the mean breast milk concentration across all study days was 35.95 pg/mL, with an estimated RID of 0.0054% of the maternal dose. CONCLUSION: Minimal concentrations of peginterferon beta-1a were detected in the breast milk samples. These findings may be useful for clinicians considering postpartum MS treatment options.

Identification of Potential Citrate Metabolism Pathways in Carnobacterium maltaromaticum.

In the present study, we describe the identification of potential citrate metabolism pathways for the lactic acid bacterium (LAB) Carnobacterium maltaromaticum. A phenotypic assay indicated that four of six C. maltaromaticum strains showed weak (Cm 6-1 and ATCC 35586) or even delayed (Cm 3-1 and Cm 5-1) citrate utilization activity. The remaining two strains, Cm 4-1 and Cm 1-2 gave negative results. Additional analysis showed no or very limited utilization of citrate in media containing 1% glucose and 22 or 30 mM citrate and inoculated with Cm 6-1 or ATCC 35586. Two potential pathways of citrate metabolism were identified by bioinformatics analyses in C. maltaromaticum including either oxaloacetate (pathway 1) or tricarboxylic compounds such as isocitrate and α-ketoglutarate (pathway 2) as intermediates. Genes encoding pathway 1 were present in two out of six strains while pathway 2 included genes present in all six strains. The two potential citrate metabolism pathways in C. maltaromaticum may potentially affect the sensory profiles of milk and soft cheeses subjected to growth with this species.

Bioequivalence of intramuscular and subcutaneous peginterferon beta-1a: results of a phase I, open-label crossover study in healthy volunteers.

BACKGROUND: Peginterferon beta-1a administered every 2 weeks via subcutaneous (SC) injection is approved to treat adult patients with relapsing-remitting multiple sclerosis (RRMS) and relapsing forms of multiple sclerosis (RMS). However, associated injection site reactions (ISRs) can lead to treatment discontinuation. Prior studies with interferon beta-1a reported a lower frequency of ISRs with intramuscular (IM) administration than with SC administration. IM administration of peginterferon beta-1a may therefore represent a useful alternative treatment option. METHODS: A phase I, open-label, two-period crossover study randomized healthy volunteers to receive a single dose of peginterferon beta-1a 125 mcg administered IM followed by a single 125 mcg dose administered SC after a 28-day washout or vice versa. Blood samples were collected up to 504 h post dose to determine pharmacokinetic (PK) and pharmacodynamic (PD) profiles. The primary endpoint was assessment of bioequivalence based on maximum serum concentration (Cmax) and area under the curve from time zero extrapolated to infinity (AUCinf). Other PK parameters, as well as PD (serum neopterin) and safety profiles, were also evaluated. RESULTS: The study enrolled 136 participants. Bioequivalence of IM and SC peginterferon beta-1a was established for both Cmax ([least squares (LS)] mean IM/SC ratio: 1.083 [90% confidence interval (CI), 0.975-1.203]) and AUCinf (LS mean IM/SC ratio: 1.089 [90% CI, 1.020-1.162]). Other PK and PD parameters were similar between administration routes, although moderate to high inter-subject variability was observed for IM and SC. Safety profiles were generally balanced between IM and SC administration. ISRs occurred at a lower frequency with IM [14.4% (95% CI, 8.89-21.56%)] than with SC [32.1% (95% CI, 24.29-40.70%)] administration (p = 0.0005). CONCLUSIONS: These results demonstrate bioequivalence between peginterferon beta-1a IM and SC and support the consideration of IM injection of peginterferon beta-1a as a viable treatment option in patients with RRMS and RMS.

Nested structure of intraspecific competition network in Carnobacterium maltaromaticum.

While competition targeting food-borne pathogens is being widely documented, few studies have focused on competition among non-pathogenic food bacteria. Carnobacterium maltaromaticum is a genetically diverse lactic acid bacterium known for comprising several bacteriocinogenic strains with bioprotective potentialities against the food-borne pathogen Listeria monocytogenes. The aim of our study is to examine the network properties of competition among a collection of 73 strains of C. maltaromaticum and to characterize their individual interaction potential. The performed high-throughput competition assays, investigating 5 329 pairwise interactions, showed that intraspecific competition was major in C. maltaromaticum with approximately 56% of the sender strains antagonizing at least one receiver strain. A high diversity of inhibitory and sensitivity spectra was identified along with a majority of narrow inhibitory as well as sensitivity spectra. Through network analysis approach, we determined the highly nested architecture of C. maltaromaticum competition network, thus showing that competition in this species is determined by both the spectrum width of the inhibitory activity of sender strains and the spectrum width of the sensitivity of receiver strains. This study provides knowledge of the competition network in C. maltaromaticum that could be used in rational assembly of compatible microbial strains for the design of mixed starter cultures.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes.

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow's milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR-Cas system.

CRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Essential structural and functional roles of the Cmr4 subunit in RNA cleavage by the Cmr CRISPR-Cas complex.

The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes.

Staphylococcus epidermidis Csm1 is a 3'-5' exonuclease.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) offer an adaptive immune system that protects bacteria and archaea from nucleic acid invaders through an RNA-mediated nucleic acid cleavage mechanism. Our knowledge of nucleic acid cleavage mechanisms is limited to three examples of widely different ribonucleoprotein particles that target either DNA or RNA. Staphylococcus epidermidis belongs to the Type III-A CRISPR system and has been shown to interfere with invading DNA in vivo. The Type III-A CRISPR system is characterized by the presence of Csm1, a member of Cas10 family of proteins, that has a permuted histidine-aspartate domain and a nucleotidyl cyclase-like domain, both of which contain sequence features characteristic of nucleases. In this work, we show in vitro that a recombinant S. epidermidis Csm1 cleaves single-stranded DNA and RNA exonucleolytically in the 3'-5' direction. We further showed that both cleavage activities are divalent-metal-dependent and reside in the GGDD motif of the cyclase-like domain. Our data suggest that Csm1 may work in the context of an effector complex to degrade invading DNA and participate in CRISPR RNA maturation.

Structure of an RNA silencing complex of the CRISPR-Cas immune system.

Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs (crRNAs) containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multicomponent Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here, we report cryoelectron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ~12 Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits: Cmr2 and Cmr3 at the conserved 5' crRNA tag sequence and Cmr1 and Cmr6 near the 3' end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.

Structure of the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex.

The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here, we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution. The structure revealed a dual ferredoxin fold and glycine-rich loops characteristic of previously known repeat-associated mysterious proteins and two unique insertion elements in Cmr3 that mediate its interaction with Cmr2. Surprisingly, while mutation of both insertion elements significantly weakened Cmr3-Cmr2 interaction, they exhibit differential effects on Cmr-mediated RNA cleavage by the Cmr complex, suggesting stabilization of Cmr2-Cmr3 interactions by other subunits. Further mutational analysis of the two conserved (but non-Cmr2-binding) glycine-rich loops of Cmr3 identified a region that is likely involved in assembly or the RNA cleavage function of the Cmr complex.

Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex.

Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3Å crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two α-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

TNF-alpha reduces the Na+/K+ ATPase activity in LLC-PK1 cells by activating caspases and JNK and inhibiting NF-kappaB.

TNF-alpha has recently been implicated in diabetic nephropathy, which is usually accompanied by higher sodium retention. The kidneys play a major role in sodium homeostasis by regulating tubular sodium reabsorption, a process geared by the sodium gradient established by the Na(+)/K(+) ATPase. The aim of this work was to investigate the effect of TNF on the ATPase, and consequently its implication in kidney malfunction, using LLC-PK1 cells. The cytokine reduced the Na(+)/K(+)ATPase activity significantly. In an attempt to elucidate the signalling pathway involved, PDTC (pyrrolidinedithiocarbamate), SP600125 and FK009 respectively inhibitors of NF-kappaB, c-JNK and caspases, were added to the cells in the presence and absence of TNF, and changes in the activities of JNK and PDTC were determined. The activity of the pump was assayed by measuring the ouabain-inhibitable release of inorganic phosphate. The effect of the cytokine was abrogated completely when JNK and caspases were inhibited but was unaffected by NF-kappaB inhibition. The role of each mediator in the signalling cascade was studied further by applying different combinations of the inhibitors. TNF-alpha was found to act at 1 h by activating caspases, which in turn activate JNK; the latter exerts an inhibitory effect on NF-kappaB, a transcription factor that stimulates the Na(+)/K(+) ATPase when active. It was concluded that TNF-alpha exerts opposite effects on the Na(+)/K(+)ATPase at different times, though the effects are always mediated via cJNK, NF-kappaB and caspases.