A meta-analysis of the gut microbiome in inflammatory bowel disease patients identifies disease-associated small molecules.

Changes in the gut microbiome have been associated with several human diseases, but the molecular and functional details underlying these associations remain largely unknown. Here, we performed a multi-cohort analysis of small molecule biosynthetic gene clusters (BGCs) in 5,306 metagenomic samples of the gut microbiome from 2,033 Inflammatory Bowel Disease (IBD) patients and 833 matched healthy subjects and identified a group of Clostridia-derived BGCs that are significantly associated with IBD. Using synthetic biology, we discovered and solved the structures of six fatty acid amides as the products of the IBD-enriched BGCs. Using two mouse models of colitis, we show that the discovered small molecules disrupt gut permeability and exacerbate inflammation in chemically and genetically susceptible mice. These findings suggest that microbiome-derived small molecules may play a role in the etiology of IBD and represent a generalizable approach for discovering molecular mediators of microbiome-host interactions in the context of microbiome-associated diseases.

Understanding the effects of a sudden directional shift in somatosensory feedback and increasing task complexity on postural adaptation in individuals with and without chronic ankle instability.

BACKGROUND: Individuals with chronic ankle instability (CAI) present somatosensory dysfunction following an initial ankle sprain. However, little is known about how individuals with CAI adapt to a sudden sensory perturbation of instability with increasing task and environmental constraints to maintain postural stability. METHODS: Forty-four individuals with and without unilateral CAI performed the Adaptation Test to a sudden somatosensory inversion and plantarflexion perturbations (environment) in double-, injured-, and uninjured- limbs. Mean sway energy scores were analyzed using 2 (group) × 2 (somatosensory perturbations) × 3 (task) repeated measures analysis of variance. RESULTS: There were significant interactions between the group, environment, and task (P=.025). The CAI group adapted faster than healthy controls to a sudden somatosensory inversion perturbation in the uninjured- (P=.002) and injured- (P<.001) limbs, as well as a sudden somatosensory plantarflexion perturbation in the double- (P=.033) and uninjured- (P=.035) limbs. The CAI and healthy groups presented slower postural adaptation to a sudden inversion perturbation than a sudden somatosensory plantarflexion perturbation in double-limb (P<.001). Whereas both groups demonstrated faster postural adaptation to a sudden somatosensory inversion perturbation compared to somatosensory plantarflexion perturbation while maintaining posture in the injured- (P<.001) and uninjured- (P<.001) limbs. The CAI and healthy groups adapted faster to a sudden somatosensory inversion perturbation in the injured- (P<.001) and uninjured- (P<.001) limbs than in double-limb, respectively. DISCUSSION: Postural adaptation in individuals with and without CAI depended on environmental (somatosensory perturbations) and task constraints. The CAI group displayed comparable and faster postural adaptation to a sudden somatosensory inversion and plantarflexion in double-, injured-, and uninjured- limbs, which may reflect a centrally mediated alteration in neuromuscular control in CAI.

Sensory Reweighting System Differences on Vestibular Feedback with Increased Task Constraints in Individuals with Chronic Ankle Instability Compared to Healthy Controls.

CONTEXT: Chronic ankle instability (CAI) is associated with a less flexibly adaptable sensorimotor system. Thus, individuals with CAI may present an inadequate sensory reweighting system inhibiting the ability to emphasize weight on reliable sensory feedback to control posture. However, how individuals with CAI reweight sensory feedback to maintain postural control in bilateral and unilateral stances has yet to be established. OBJECTIVES: The primary purpose was to examine group differences in how the sensory reweighting system changes to control posture in a simple double-limb stance and a more complex single-limb stance (uninjured-limb, injured-limb) under increased environmental constraints manipulating somatosensory and visual information for individuals with and without CAI. The secondary purpose was to examine the effect of environmental and task constraints on postural control. STUDY DESIGN: Case-control study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: 21 individuals with CAI (26.4±5.7years, 171.2±9.8cm, 76.6±15.17kg) and 21 healthy controls (25.8±5.7years, 169.5±9.5cm, 72.4±15.0kg) participated in the study. MAIN OUTCOME MEASURE(S): Equilibrium10 were examined while completing 6 environmental conditions of the Sensory Organization Test (SOT) during 3 tasks (double-limb and single-limb [uninjured, injured] stances). Sensory reweighting ratios for sensory systems (somatosensory, vision, vestibular) were computed from paired Equilibrium10. RESULTS: Significant 3-factor interactions were found between group, sensory systems, and tasks (P=0.006) and for groups, task, and environment (P=0.007). The CAI group failed to downweight vestibular feedback compared to healthy controls while maintaining posture in the injured-limb (P=0.030). The CAI group displayed better postural stability than healthy controls while standing with absent vision, fixed surroundings, and a moving platform in the injured-limb (P=0.032). CONCLUSIONS: The CAI group relied on vestibular feedback while maintaining better postural stability than healthy controls in the injured-limb. Group differences in postural control depended on both environmental (absent vision, moving platform) and task (injured-limb) constraints.

Modified proximal thigh kinematics captured with a novel smartphone app in individuals with a history of recurrent ankle sprains and altered dorsiflexion with walking.

BACKGROUND: We examined sagittal-plane thigh angular kinematics in individuals with and without recurrent ankle sprains using a clinical smartphone app called AccWalker. Sagittal-plane ankle kinematics were also compared to ascertain that altered ankle dorsiflexion, which is typically displayed with chronic ankle instability, is also present in individuals with recurrent ankle sprains. METHODS: Participants with (n = 22) and without (n = 22) recurrent ankle sprains were evaluated on average sagittal-plane ankle kinematics during walking and average sagittal-plane thigh angular kinematics during stepping-in-place with AccWalker. FINDINGS: Significant group-by-limb interactions were found for sagittal-plane ankle kinematics (F(1,42) = 63.786, P < .010) during walking and sagittal-plane average thigh angular range-of-motion (F(1,42) = 6.166, P = .017) with AccWalker. Individuals with recurrent ankle sprains displayed more ankle dorsiflexion in affected (P < .001) and unaffected (P = .001) limbs during walking than healthy controls and exhibited more ankle dorsiflexion in their affected-limb compared to their unaffected-limb (P < .001). The average sagittal-plane thigh angular range-of-motion was lower in the unaffected-limb for recurrent ankle sprains compared to their affected-limb (P = .038) and the assigned unaffected-limb of healthy controls (P = .035). INTERPRETATION: Increased dorsiflexion was present in both limbs of the recurrent ankle sprain group with walking. AccWalker does not assess ankle movement, but uniquely identified thigh motion impairments associated with recurrent ankle sprains in their unaffected-limb, potentially identifying central deficits associated with recurrent ankle sprains. This app has clinical implications for assessing potential pathological movement that can be corrected through rehabilitation.

An Extension of Reversible Image Enhancement Processing for Saturation and Brightness Contrast.

This paper proposes a reversible image processing method for color images that can independently improve saturation and enhance brightness contrast. Image processing techniques have been popularly used to obtain desired images. The existing techniques generally do not consider reversibility. Recently, many reversible image processing methods have been widely researched. Most of the previous studies have investigated reversible contrast enhancement for grayscale images based on data hiding techniques. When these techniques are simply applied to color images, hue distortion occurs. Several efficient methods have been studied for color images, but they could not guarantee complete reversibility. We previously proposed a new method that reversibly controls not only the brightness contrast, but also saturation. However, this method cannot fully control them independently. To tackle this issue, we extend our previous work without losing its advantages. The proposed method uses the HSV cone model, while our previous method uses the HSV cylinder model. The experimental results demonstrate that our method flexibly controls saturation and brightness contrast reversibly and independently.

Pseudomonas aeruginosa detachment from surfaces via a self-made small molecule.

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal.

In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803.

Phototaxis is a phenomenon where cyanobacteria move toward a light source. Previous studies have shown that the blue-light-using-flavin (BLUF)-type photoreceptor PixD and the response regulator-like protein PixE control the phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. The pixD-null mutant moves away from light, whereas WT, pixE mutant, and pixD pixE double mutant move toward the light. This indicates that PixE functions downstream of PixD and influences the direction of movement. However, it is still unclear how the light signal received by PixD is transmitted to PixE, and then subsequently transmitted to the type IV pili motor mechanism. Here, we investigated intracellular localization and oligomerization of PixD and PixE to elucidate mechanisms of phototaxis regulation. Blue-native PAGE analysis, coupled with western blotting, indicated that most PixD exist as a dimer in soluble fractions, whereas PixE localized in ~250 kDa and ~450 kDa protein complexes in membrane fractions. When blue-native PAGE was performed after illuminating the membrane fractions with blue light, PixE levels in the ~250 kDa and ~450 kDa complexes were reduced and increased, respectively. These results suggest that PixE, localized in the ~450 kDa complex, controls activity of the motor ATPase PilB1 to regulate pilus motility.

The (PATAN)-CheY-Like Response Regulator PixE Interacts with the Motor ATPase PilB1 to Control Negative Phototaxis in the Cyanobacterium Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 can move directionally on a moist surface toward or away from a light source to reach optimal light conditions for its photosynthetic lifestyle. This behavior, called phototaxis, is mediated by type IV pili (T4P), which can pull a single cell into a certain direction. Several photoreceptors and their downstream signal transduction elements are involved in the control of phototaxis. However, the critical steps of local pilus assembly in positive and negative phototaxis remain elusive. One of the photoreceptors controlling negative phototaxis in Synechocystis is the blue-light sensor PixD. PixD forms a complex with the CheY-like response regulator PixE that dissociates upon illumination with blue light. In this study, we investigate the phototactic behavior of pixE deletion and overexpression mutants in response to unidirectional red light with or without additional blue-light irradiation. Furthermore, we show that PixD and PixE partly localize in spots close to the cytoplasmic membrane. Interaction studies of PixE with the motor ATPase PilB1, demonstrated by in vivo colocalization, yeast two-hybrid and coimmunoprecipitation analysis, suggest that the PixD-PixE signal transduction system targets the T4P directly, thereby controlling blue-light-dependent negative phototaxis. An intriguing feature of PixE is its distinctive structure with a PATAN (PatA N-terminus) domain. This domain is found in several other regulators, which are known to control directional phototaxis. As our PilB1 coimmunoprecipitation analysis revealed an enrichment of PATAN domain response regulators in the eluate, we suggest that multiple environmental signals can be integrated via these regulators to control pilus function.

A metagenomic strategy for harnessing the chemical repertoire of the human microbiome.

Extensive progress has been made in determining the effects of the microbiome on human physiology and disease, but the underlying molecules and mechanisms governing these effects remain largely unexplored. Here, we combine a new computational algorithm with synthetic biology to access biologically active small molecules encoded directly in human microbiome-derived metagenomic sequencing data. We discover that members of a clinically used class of molecules are widely encoded in the human microbiome and that they exert potent antibacterial activities against neighboring microbes, implying a possible role in niche competition and host defense. Our approach paves the way toward a systematic unveiling of the chemical repertoire encoded by the human microbiome and provides a generalizable platform for discovering molecular mediators of microbiome-host and microbiome-microbiome interactions.

Emulating evolutionary processes to morph aureothin-type modular polyketide synthases and associated oxygenases.

Polyketides produced by modular type I polyketide synthases (PKSs) play eminent roles in the development of medicines. Yet, the production of structural analogs by genetic engineering poses a major challenge. We report an evolution-guided morphing of modular PKSs inspired by recombination processes that lead to structural diversity in nature. By deletion and insertion of PKS modules we interconvert the assembly lines for related antibiotic and antifungal agents, aureothin (aur) and neoaureothin (nor) (aka spectinabilin), in both directions. Mutational and functional analyses of the polyketide-tailoring cytochrome P450 monooxygenases, and PKS phylogenies give contradictory clues on potential evolutionary scenarios (generalist-to-specialist enzyme evolution vs. most parsimonious ancestor). The KS-AT linker proves to be well suited as fusion site for both excision and insertion of modules, which supports a model for alternative module boundaries in some PKS systems. This study teaches important lessons on the evolution of PKSs, which may guide future engineering approaches.

Paravertebral block catheter breakage by electrocautery during thoracic surgery.

Advantages of thoracic paravertebral analgesia (TPA) include placement of the catheter closer to the surgical field; however, the catheter can become damaged during the operation. We experienced a case of intraoperative TPA catheter breakage that prompted us to perform an experiment to investigate possible causes. A 50-year-old male underwent a thoracoscopic lower lobectomy under general anesthesia with TPA via an intercostal approach. Following surgery, it was discovered that the catheter had become occluded, as well as cut and fused, so we reopened the incision and removed the residual catheter. From that experience, we performed an experiment to examine electrocautery-induced damage in normal (Portex™, Smith's Medical), radiopaque (Perifix SoftTip™, BBraun), and reinforced (Perifix FX™, BBraun) epidural catheters (n = 8 each). Chicken meat was penetrated by each catheter and then cut by electrocautery. In the normal group, breakage occurred in 8 and occlusion in 6 of the catheters, and in the radiopaque group breakage occurred in 8 and occlusion in 7. In contrast, breakage occurred in only 3 and occlusion in none in the reinforced group, with the 5 without breakage remaining connected only by the spring coil. Furthermore, in 7 of the reinforced catheters, electric arc-induced thermal damage was observed at the tip of the catheter. A TPA catheter for thoracic surgery should be inserted via the median approach, or it should be inserted after surgery to avoid catheter damage during surgery.

Genetics of the Blue Light-Dependent Signal Cascade That Controls Phototaxis in the Cyanobacterium Synechocystis sp. PCC6803.

The Synechocystis sp. PCC6803 can move on a solid surface in response to light, a phenomenon called phototaxis. Although many of the photoreceptors involved in phototaxis have been identified, the mechanisms that regulate directional motility of Synechocystis are not well understood. Previous studies showed that a mutant lacking the blue light-using flavin (BLUF) photoreceptor PixD exhibits negative phototaxis under conditions where the wild type responds positively. PixD interacts with the pseudo-response regulator-like protein PixE in a light-dependent manner, suggesting that this intermolecular interaction is important for phototaxis regulation, although genetic evidence has been lacking. To gain further insight into phototaxis regulation by PixD-PixE signaling, we constructed the deletion mutants ΔPixE and ΔPixD-ΔPixE, and characterized their phenotypes, which matched those of the wild type (positive phototaxis). Because ΔPixD exhibited negative phototaxis, PixE must function downstream of PixD. Under intense blue light (>100 µmol m-2 s-1; 470 nm) the wild type exhibited negative phototaxis, but ΔPixD-PixE exhibited positive phototaxis toward low-intensity blue light (∼0.8 µmol m-2 s-1; 470 nm). These results suggest that an unknown light-sensing system(s), that is necessary for directional cell movement, can be activated by low-intensity blue light; on the other hand, PixD needs high-intensity blue light to be activated. We also isolated spontaneous mutants that compensated for the pixE deletion. Genome-wide sequencing of the mutants revealed that the uncharacterized gene sll2003 regulates positive and negative phototaxis in response to light intensity.

Cross-linking analysis reveals the putative dimer structure of the cyanobacterial BLUF photoreceptor PixD.

PixD is a blue light using flavin (BLUF)-type blue-light photoreceptor controlling phototaxis in the cyanobacterium Synechocystis sp. PCC6803. The crystal structure of PixD shows a decamer, although in solution an equilibrium is maintained between the dimer and decamer. Because the ratio of these two conformers is altered by illumination, the equilibrium state determines photosensitivity. However, no structural information is available for the PixD dimer. Here, we report a predicted structure for the dimer based on docking simulation, mutagenesis, and mass spectrometry-based cross-linking analyses. The results indicate the importance of the PixD C-terminus for dimer preference and photosensitivity.

Freedom and constraint in engineered noncolinear polyketide assembly lines.

Many pharmacologically important natural products are assembled by modular type I polyketide synthases (PKS), which typically act in a unidirectional fashion. The synthases producing the unusual nitro-substituted polyketides neoaureothin (nor, also called spectinabilin) and aureothin (aur) are exceptional, as they employ individual modules iteratively. Here, we investigate the plasticity of the nor PKS and the factors governing the number of elongations catalyzed by the noncanonical module. Surprisingly, we observe that the nor PKS can mediate an additional chain elongation to yield the higher homolog homoneoaureothin. Furthermore, we design several truncated variants of the nor PKS to use them in the context of artificial assembly lines for aureothin and homoaureothin. The resulting polypropionate derivatives provide valuable insights into chain length control and reveal structure-activity relationships relating to the size of the polypropionate backbones. Overall, we show that iterative modules are remarkably adaptable while downstream modules are gatekeepers that select for correct polyketide chain length.

Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line.

The unusual nitro-substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context-dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non-colinear behavior of PKS domains.

Intrathecal adrenomedullin modulates acute inflammatory pain in the rat formalin test.

Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator. This study was undertaken to investigate the role of AM in acute inflammatory pain induced by formalin injection in rats. Interestingly Cerebrospinal fluid (CSF) levels of AM increased 45 min after formalin injection and a selective AM receptor antagonist, AM22-52, administered intrathecally (i.t.) decreased phase 2 flinching in a dose-dependent manner but not phase 1 flinching during the formalin test. This anti-hyperalgesic effect of i.t. AM22-52 lasted for 4 h or more. AM in the CSF contributes to the modulation of acute inflammatory pain in the formalin test, and blocking downstream signaling effects of the AM receptor has the potential to relieve pain associated with acute inflammation.

Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx(+)/Sox9(+) progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx(+) domain revealed that Scx(+) progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. Tenocytes are derived from Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx(+)/Sox9(+) progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx(+)/Sox9(+) progenitors. Conditional inactivation of Sox9 in Scx(+)/Sox9(+) cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx(+)/Sox9(+) progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.