Relationship between Facial Color Changes and Psychological Problems Associated with Lower Back Pain.

Background and Objectives: The aim of this study was to determine whether a non-contact sensor that detects complexion changes can be used to assess the psychological state of patients with chronic lower back pain (LBP). Materials and Methods: Twenty-six patients with LBP (LBP group; mean age = 68.0 ± 13.9 years) and 18 control subjects without LBP (control group; mean age = 60.8 ± 16.1 years) were included in the study. All the subjects in the two groups wore headphones when asked LBP-related and LBP-unrelated questions. During questioning, the facial image of the subjects was captured using a video camera, and the complexion of the subjects was converted into red, green, and blue (RGB) values. RGB correlation coefficients (RGBCCs; range: 0-1) represent the difference in complexion between LBP-related and LBP-unrelated questions. A high RGBCC indicates that the brain is more activated by LBP-related questions than by LBP-unrelated questions. We also noted the scores of subjects on the Numerical Rating Scale (NRS), Japanese Orthopedic Association Back Pain Evaluation Questionnaire (JOABPEQ), Pain Catastrophizing Scale (PCS), and Hospital Anxiety and Depression Scale (HADS). Results: There were no significant differences in RGBCC between the control and LBP groups (0.64 versus 0.56, p = 0.08). In the LBP group, no correlation was observed between RGBCC and each examination item of NRS, JOABPEQ, and HADS. In contrast, a correlation was observed between RGBCC and the rumination subscale of PCS in the LBP group (Spearman's rank correlation coefficient = 0.40, p = 0.04). Conclusions: The complexion of patients with catastrophic thinking changes when the patients are asked LBP-related questions.

Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

Analysis of the Valgamicin Biosynthetic Pathway Reveals a General Mechanism for Cyclopropanol Formation across Diverse Natural Product Scaffolds.

Cyclopropanol rings are highly reactive and may function as molecular "warheads" that affect natural product bioactivity. Yet, knowledge on their biosynthesis is limited. Using gene cluster analyses, isotope labeling, and in vitro enzyme assays, we shed first light on the biosynthesis of the cyclopropanol-substituted amino acid cleonine, a residue in the antimicrobial depsipeptide valgamicin C and the cytotoxic glycopeptide cleomycin A2. We decipher the biosynthetic origin of valgamicin C and show that the cleonine cyclopropanol ring is derived from dimethylsulfoniopropionate (DMSP). Furthermore, we demonstrate that part of the biosynthesis is analogous to the formation of malleicyprol polyketides in pathogenic bacteria. By genome mining and metabolic profiling, we identify the potential to produce cyclopropanol rings in other bacterial species. Our results reveal a general mechanism for cyclopropyl alcohol biosynthesis across diverse natural products that may be harnessed for bioengineering and drug discovery.

Amycolamicin: a novel broad-spectrum antibiotic inhibiting bacterial topoisomerase.

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Tripropeptin C blocks the lipid cycle of cell wall biosynthesis by complex formation with undecaprenyl pyrophosphate.

Tripropeptin C (TPPC) is a naturally occurring cyclic lipodepsipeptide antibiotic produced by a Lysobacter sp. TPPC exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. This antibiotic also inhibits the incorporation of N-acetylglucosamine into the peptidoglycan of S. aureus at a 50% inhibitory concentration (IC(50)) of 0.7 µM, which is proportional to its MIC (0.87 µM; equivalent to 1.0 µg/ml). Treatment of exponential-phase S. aureus cells with TPPC resulted in accumulation of UDP-MurNAc-pentapeptide in the cytoplasm. The antimicrobial activity of TPPC was weakened by the addition of prenyl pyrophosphates but not by prenyl phosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. The direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin-layer chromatography analysis, indicating that TPPC can potentially inhibit C(55)-PP phosphatase activity, which plays a crucial role in the lipid cycle of peptidoglycan synthesis. As expected, TPPC inhibits this enzymatic reaction at an IC(50) of 0.03 to 0.1 µM in vitro, as does bacitracin. From the analysis of accumulation of lipid carrier-related compounds, TPPC was found to cause the accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-containing lipid intermediate. This suggested that the TPPC/C(55)-PP complex also inhibits the transglycosylation step or flippase activity, adding to the inhibition of C(55)-PP dephosphorylation. This mode of action is different from that of currently available drugs such as vancomycin, daptomycin, and bacitracin.

Genome Sequence of Lysobacter sp. Strain BMK333-48F3, the Producer Strain of Potent Lipopeptide Antibiotics of the Tripropeptin Family.

Lysobacter sp. strain BMK333-48F3 is known primarily for its production of the antibiotically active tripropeptins. Here, we report its draft genome sequence, which will give insight into the biosynthesis of tripropeptins and enable genome mining for further secondary metabolites.

New chloptosins B and C from an Embleya strain exhibit synergistic activity against methicillin-resistant Staphylococcus aureus when combined with co-producing compound L-156,602.

Two new dimeric cyclohexapeptides, chloptosins B and C, were discovered from the culture broth of Embleya sp. MM621-AF10 together with the known compounds chloptosin and L-156,602. The structures of the new chloptosins were determined by spectroscopic studies and advanced Marfey's methods. The stereo structure of the constituent isoleucine was determined by C3 Marfey's analysis. Chloptosins demonstrated potent antimicrobial activity against Gram-positive bacteria including drug-resistant strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci with MICs of 0.5-2 µg ml-1. The antimicrobial activities of chloptosins were enhanced by addition of co-producing compound L-156,602, as shown by checkerboard analysis.

Pain Evaluation During Colonoscopy by the Erythema Index of the Facial Image.

BACKGROUND: Endoscopy of the digestive tract is useful but is associated with significant pain to the patient. Its safety and tolerability could be improved by an immediate and objective method to evaluate the pain level and give feedback to the examiner. However, under the current circumstances, it is difficult to measure and assess the pain level objectively. METHODS: We previously developed a discomfort assessment device that measures the changes in brain activity caused by changes in the pain level by extracting the changes in the erythema index from facial color data. In this study, to evaluate the usefulness of this discomfort assessment device, the association between the changes in the erythema index of facial images during colonoscopy and the subjective pain level during the examination were evaluated. For the recording of the subjective pain level during the examination, a subjective pain level recording device that we developed to measure grip strength over time was used. The subjective pain level, facial image, and percutaneous venous oxygen saturation during the examination were recorded in 30 patients who underwent colonoscopy at our hospital. RESULTS: The duration of colonoscopy was divided into the insertion section and the removal section. The subjective pain level was found to be significantly greater during the insertion section than during the removal section, and the changes in the erythema index of the facial images were significantly different between the two groups. CONCLUSION: These findings indicate that the erythema index changes on facial images determined by the discomfort assessment device may facilitate objective evaluation of the pain level during colonoscopy.

A new type of tripropeptin with anteiso-branched chain fatty acid from Lysobacter sp. BMK333-48F3.

Branched chain amino acids are often utilized as the precursors of many lipid-containing bacterial secondary metabolites. The effect of isoleucine on the composition of the mixture of cyclic lipopeptide antibiotics, tripropeptins from Lysobacter sp. BMK333-48F3 was evaluated. As expected, a novel tripropeptin analog with an anteiso-branched fatty acid was produced. The new compound, TPPaiC shows potent antibacterial activity against Gram-positive bacteria including MRSA and VRE. On the other hand, no increase was observed in the production of other tripropeptins by the addition of isoleucine.

Pargamicin A, a novel cyclic peptide antibiotic from Amycolatopsis sp.

A novel cyclic peptide antibiotic, pargamicin A was isolated from the culture broth of an actinomycete strain. The producing organism, designated ML1-hF4, was identified as a member of the genus Amycolatopsis. Pargamicin A was identified as a novel cyclic hexapeptide antibiotic containing piperazic acid by various spectroscopic analyses. Pargamicin A showed potent antibacterial activity against Staphylococcus aureus strains including MRSA and Enterococcus faecalis/faecium strains including VRE.

In vivo efficacy of ß-lactam/tripropeptin C in a mouse septicemia model and the mechanism of reverse ß-lactam resistance in methicillin-resistant Staphylococcus aureus mediated by tripropeptin C.

Natural lipopeptide antibiotic tripropeptin C (TPPC) revitalizes and synergistically potentiates the activities of the class of ß-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) but not against methicillin-sensitive S. aureus in vitro; however, the mode of action remains unclear. In the course of the study to reveal its mode of action, we found that TPPC inhibited the ß-lactamase production induced by cefotiam. This prompted us to focus on the ß-lactam-inducible ß-lactam-resistant genes blaZ (ß-lactamase) and mecA (foreign penicillin-binding protein), as they are mutually regulated by the blaZ/I/R1 and mecA/I/R1 systems. Quantitative reverse-transcription polymerase chain reaction analysis revealed that TPPC reversed ß-lactam resistance by reducing the expression of the genes blaZ and mecA, when treated alone or in combination with ß-lactam antibiotics. In a mouse/MRSA septicemia model, subcutaneous injection of a combination of TPPC and ceftizoxime demonstrated synergistic therapeutic efficacy compared with each drug alone. These observations strongly suggested that reverse ß-lactam resistance by TPPC may be a potentially effective new therapeutic strategy to overcome refractory MRSA infections.The Journal of Antibiotics advance online publication, 26 July 2017; doi:10.1038/ja.2017.88.

Valgamicin C, a novel cyclic depsipeptide containing the unusual amino acid cleonine, and related valgamicins A, T and V produced by Amycolatopsis sp. ML1-hF4.

In the course of optimizing pargamicin A production in Amycolatopsis sp. ML1-hF4, we discovered novel cyclic depsipeptide compounds in the broth and designated them valgamicins A, C, T and V. The structures of these molecules were determined by spectroscopic studies, advanced Marfey's method and X-ray crystal structural analysis. Valgamicin C contains the extremely rare amino acid cleonine. To our knowledge, this is the first report of a cleonine-containing metabolite from a naturally isolated microorganism without any breeding or mutation treatment. None of the valgamicins showed potent antibacterial activity against either Gram-positive or -negative bacteria. Valgamicins A, C and T exhibited moderate cytotoxicity against human tumor cell lines.The Journal of Antibiotics advance online publication, 15 November 2017; doi:10.1038/ja.2017.135.

Natural lipopeptide antibiotic tripropeptin C revitalizes and synergistically potentiates the activity of beta-lactams against methicillin-resistant Staphylococcus aureus.

Tripropeptin C (TPPC) is a natural calcium-ion-dependent lipopeptide antibiotic that inhibits peptidoglycan biosynthesis by binding to prenyl pyrophosphate. It displays very potent antimicrobial activity both in vitro and in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) septicemia. The combination of TPPC with all classes of beta-lactams tested (including penam, carbapenem, cephem and oxacephem) showed highly synergistic (SYN) effects against MRSA strains, but not against methicillin-sensitive S. aureus strains. These SYN effects were observed with both a checkerboard methodology and a time-kill analysis. The TPPC analog, bis-methyl ester-TPPC, which has neither antimicrobial activity nor the ability to bind prenyl pyrophosphate, also potentiated the activity of beta-lactams. This result indicates that the mechanism of the SYN activity of TPPC is independent of its binding to prenyl pyrophosphate. Therefore, synergistically enhancing the anti-MRSA activities of TPPC and beta-lactams by combining them is a novel and potentially powerful therapeutic strategy for MRSA infections.

Tripropeptins, novel antimicrobial agents produced by Lysobacter sp.

Planar structures of tripropeptins (TPPs) were elucidated by spectroscopic studies including various NMR measurements. Stereochemistry of constituent amino acids of tripropeptin C (TPPC) (3) was identified by marfey's method except hydroxyproline which was determined by studies of NMR and CD spectra. The absolute structure of 3 was determined by analyses of the fragments obtained by Birch reduction and LiBH4 reduction of 3. The configuration of the fatty acid, isolated from acid hydrolysate of 3, was determined to be (3R)-hydroxy-13-methyltetradecanoic acid from MS, NMR spectra and negative sign of the optical rotation.

Biosynthesis of the cyclitol moiety of pyralomicin 1a in Nonomuraea spiralis MI178-34F18.

The biosynthetic pathway leading to the cyclitol moiety of pyralomicin 1a (1) in Nonomuraea spiralis MI178-34F18 has been studied using a series of 2H-labeled potential precursors. The results demonstrate that 2-epi-5-epi-valiolone (7), a common precursor for acarbose (4) and validamycin A (5) biosynthesis, is an immediate precursor of pyralomicin 1a. 5-epi-Valiolone (8) was also incorporated into 1, albeit less efficiently than 7. Other potential intermediates, such as valiolone (9), valienone (10), valienol (11), 1-epi-valienol (12), 5-epi-valiolol (13), and 1-epi-5-epi-valiolol (14) were not incorporated into pyralomicin 1a. To explain this surprising observation, it is proposed that either 2-epi-5-epi-valiolone (7) is specifically activated (e.g., to its phosphate) and that the further transformations take place on activated intermediates (which can not be generated directly from their unactivated counterparts), or that the transformation of 7 into 1 involves a substrate-channeling mechanism in which enzyme-bound intermediates are directly transferred from one enzyme active site to the next in a multi-enzyme complex.

[Studies for the development of novel anti-MRSA/VRE drugs].

The widespread emergence of multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) is a high threat for human health. In the course of screening for active compounds against the above drug-resistant bacteria from microbial metabolites, we discovered three kinds of novel compounds designated tripropeptins, pargamicin, and amycolamicin. Tripropeptin C (TPPC), major component of tripropeptins, is the most promising compound because it is efficacious against MRSA and VRE both in vitro and in a mouse septicemia model, and shows no cross-resistance to available drugs including vancomycin. Studies of incorporation of radioactive macromolecular precursors and accumulation of UDP-MurNAc-pentapeptide in the cytoplasm in S. aureus Smith revealed that TPPC is a cell wall synthesis inhibitor. Antimicrobial activity of TPPC was weakened by addition of prenylpyrophosphates but not with prenylphosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. Direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin layer chromatography, and TPPC inhibits C(55)-PP phosphatase, which plays a crucial role in peptidoglycan synthesis at an IC(50) of 0.03-0.1 µM in vitro. From the analysis of accumulation of lipid carrier-related compounds, TPPC caused accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-added lipid intermediate, suggesting a distinct mode of action from that of clinically important drugs such as vancomycin, daptomycin, and bacitracin. TPPC might represent a promising novel class of antibiotic against MRSA and VRE infections.

Tumor necrosis factor α stimulates expression and secretion of urokinase plasminogen activator in human dental pulp cells.

Plasminogen activator (PA) is the enzyme responsible for converting plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. PA exists in two forms: urokinase-type PA (uPA) and tissue-type PA (tPA). Here we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNF-α) on PA production and secretion in human dental pulp cells. When the cells were stimulated with TNF-α (10 ng/mL), PA activity in the medium clearly increased in a time-dependent manner, and this activity was reduced after immunoprecipitation with anti-uPA antibody, but not with anti-tPA antibody. In TNF-α-stimulated cells, the expression of uPA mRNA was enhanced, but was lower than that of tPA mRNA. The expression of uPA mRNA and PA secretion stimulated by TNF-α were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFκB inhibitor pyrrolidine dithiocarbamate, but were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. In the presence of another inflammatory cytokine, interleukin 1ß (IL-1ß, 100 pg/mL), TNF-α-stimulated expression of uPA mRNA and secretion of uPA were enhanced. These observations suggest that TNF-α stimulates uPA production and secretion, and that this effect is regulated via activation of NFκB and tyrosine phosphorylation, apparently in conjunction with IL-1ß, during inflammation in human dental pulp.