Environmental purines decrease Pseudomonas aeruginosa biofilm formation by disrupting c-di-GMP metabolism.

Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling.

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Active pH regulation facilitates Bacillus subtilis biofilm development in a minimally buffered environment.

Biofilms provide individual bacteria with many advantages, yet dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress can be masked in buffered laboratory media-such as MSgg commonly used to study Bacillus subtilis biofilms-it is not always clear how such biofilms cope with minimally buffered natural environments. Here, we report how B. subtilis biofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that these biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development when grown in minimally buffered conditions. Our findings reveal an active pH regulation mechanism in B. subtilis biofilms that could lead to new targets to control unwanted biofilm growth.IMPORTANCEpH is known to influence microbial growth and community dynamics in multiple bacterial species and environmental contexts. Furthermore, in many bacterial species, rapid cellular proliferation demands the use of overflow metabolism, which can often result in excessive acidification. However, in the case of bacterial communities known as biofilms, these acidification challenges can be masked when buffered laboratory media are employed to stabilize the pH environment for optimal growth. Our study reveals that B. subtilis biofilms use an active pH regulation mechanism to mitigate both growth-associated acidification and external pH challenges. This discovery provides new opportunities for understanding microbial communities and could lead to new methods for controlling biofilm growth outside of buffered laboratory conditions.

Engineered calprotectin-sensing probiotics for IBD surveillance in humans.

Inflammatory bowel disease (IBD) is a spectrum of autoimmune diseases affecting the gastrointestinal tract characterized by a relapsing and remitting course of gut mucosal inflammation. Disease flares can be difficult to predict, and the current practice of IBD disease activity surveillance through endoscopy is invasive and requires medical expertise. Recent advancements in synthetic biology raise the possibility that symbiotic microbes can be engineered to selectively detect disease biomarkers used in current clinical practice. Here, we introduce an engineered probiotic capable of detecting the clinical gold standard IBD biomarker, calprotectin, with sensitivity and specificity in IBD patients. Specifically, we identified a bacterial promoter in the probiotic strain Escherichia coli Nissle 1917 (EcN) which exhibits a specific expression increase in the presence of calprotectin. Using murine models of colitis, we show that the reporter signal is activated in vivo during transit of the GI tract following oral delivery. Furthermore, our engineered probiotic can successfully discriminate human patients with active IBD from those in remission and without IBD using patient stool samples, where the intensity of reporter signal quantitatively tracks with clinical laboratory-measured levels of calprotectin. Our pilot study sets the stage for probiotics that can be engineered to detect fecal calprotectin for precise noninvasive disease activity monitoring in IBD patients.

Risk factors for microcystic macular oedema in glaucoma.

BACKGROUND/AIMS: To identify clinical characteristics and factors associated with microcystic macular edema (MME) in patients with primary open-angle glaucoma (POAG). METHODS: We included 315 POAG eyes between 2010 and 2019 with good-quality macular volume scans that had reliable visual fields (VF) available within 6 months in this observational retrospective cohort study. Eyes with retinal pathologies except for epiretinal membrane (ERM) were excluded. The inner nuclear layer was qualitatively assessed for the presence of MME. Global mean deviation (MD) and Visual Field Index (VFI) decay rates, superior and inferior MD rates and pointwise total deviation rates of change were estimated with linear regression. Logistic regression was performed to identify baseline factors associated with the presence of MME and to determine whether MME is associated with progressive VF loss. RESULTS: 25 out of 315 eyes (7.9%) demonstrated MME. The average (±SD) age and MD in eyes with and without MME was 57.2 (±8.7) versus 62.0 (±9.9) years (p=0.02) and -9.8 (±5.7) versus -4.9 (±5.3) dB (p<0.001), respectively. Worse global MD at baseline (p=0.001) and younger age (p=0.02) were associated with presence of MME. ERM was not associated with the presence of MME (p=0.84) in this cohort. MME was not associated with MD and VFI decay rates (p>0.49). CONCLUSIONS: More severe glaucoma and younger age were associated with MME. MME was not associated with faster global VF decay in this cohort. MME may confound monitoring of glaucoma with full macular thickness.

Toward manipulating serotonin signaling via the microbiota-gut-brain axis.

It is now well established in humans that there is a bidirectional pathway of communication between the central and enteric nervous systems in which members of the microbiome participate. This microbiota-gut-brain axis (MGBA) is crucial for normal development and physiology, and its dysregulation has been implicated in a range of neurological and intestinal disorders. Investigations into the mechanistic underpinnings of the MGBA have identified serotonin as a molecule of particular interest. In this review, we highlight recent advances toward understanding the role of endogenous serotonin in microbial communities, how microbial communities bidirectionally interact with host serotonin, and potential future engineering opportunities to leverage these novel mechanisms for biomedical applications.

Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool.

Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.

Automated Segmentation and Connectivity Analysis for Normal Pressure Hydrocephalus.

Objective and Impact Statement. We propose an automated method of predicting Normal Pressure Hydrocephalus (NPH) from CT scans. A deep convolutional network segments regions of interest from the scans. These regions are then combined with MRI information to predict NPH. To our knowledge, this is the first method which automatically predicts NPH from CT scans and incorporates diffusion tractography information for prediction. Introduction. Due to their low cost and high versatility, CT scans are often used in NPH diagnosis. No well-defined and effective protocol currently exists for analysis of CT scans for NPH. Evans' index, an approximation of the ventricle to brain volume using one 2D image slice, has been proposed but is not robust. The proposed approach is an effective way to quantify regions of interest and offers a computational method for predicting NPH. Methods. We propose a novel method to predict NPH by combining regions of interest segmented from CT scans with connectome data to compute features which capture the impact of enlarged ventricles by excluding fiber tracts passing through these regions. The segmentation and network features are used to train a model for NPH prediction. Results. Our method outperforms the current state-of-the-art by 9 precision points and 29 recall points. Our segmentation model outperforms the current state-of-the-art in segmenting the ventricle, gray-white matter, and subarachnoid space in CT scans. Conclusion. Our experimental results demonstrate that fast and accurate volumetric segmentation of CT brain scans can help improve the NPH diagnosis process, and network properties can increase NPH prediction accuracy.

Vesicle-Based Sensors for Extracellular Potassium Detection.

INTRODUCTION: The design of sensors that can detect biological ions in situ remains challenging. While many fluorescent indicators exist that can provide a fast, easy readout, they are often nonspecific, particularly to ions with similar charge states. To address this issue, we developed a vesicle-based sensor that harnesses membrane channels to gate access of potassium (K+) ions to an encapsulated fluorescent indicator. METHODS: We assembled phospholipid vesicles that incorporated valinomycin, a K+ specific membrane transporter, and that encapsulated benzofuran isophthalate (PBFI), a K+ sensitive dye that nonspecifically fluoresces in the presence of other ions, like sodium (Na+). The specificity, kinetics, and reversibility of encapsulated PBFI fluorescence was determined in a plate reader and fluorimeter. The sensors were then added to E. coli bacterial cultures to evaluate K+ levels in media as a function of cell density. RESULTS: Vesicle sensors significantly improved specificity of K+ detection in the presence of a competing monovalent ion, sodium (Na+), and a divalent cation, calcium (Ca2+), relative to controls where the dye was free in solution. The sensor was able to report both increases and decreases in K+ concentration. Finally, we observed our vesicle sensors could detect changes in K+ concentration in bacterial cultures. CONCLUSION: Our data present a new platform for extracellular ion detection that harnesses ion-specific membrane transporters to improve the specificity of ion detection. By changing the membrane transporter and encapsulated sensor, our approach should be broadly useful for designing biological sensors that detect an array of biological analytes in traditionally hard-to-monitor environments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00688-7.

Mechanical properties of human patellar tendon collagen fibrils. An exploratory study of aging and sex.

Tendons are connective tissues that transmit mechanical forces from muscle to bone and consist mainly of nano-scale fibrils of type I collagen. Aging has been associated with reduced mechanical function of tendons at the whole-tendon level and also with increased glycation of tendon collagen fibrils. Yet, the mechanical effects of aging at the fibril level remain unknown. In vitro glycation has previously been reported to substantially increase fibril strength and stiffness in young rats, suggesting a potentially large effect of aging through the glycation mechanism. We therefore expected that aging would have a similar major impact on fibril mechanical properties. In addition, differences in fibril mechanical properties between men and women have never been studied. This study investigated human patellar tendon biopsies from young (26 ± 4 years) and elderly (66 ± 1 years), men and women by measuring the mechanical properties of individual collagen fibrils using a custom nano-mechanical device. There were no major mechanical differences with either age or sex, but there were modestly greater failure stress (22%) and tensile modulus at both low and high strain (16% and 26% respectively) in the elderly group. No significant differences in mechanical properties were observed between men and women. The slightly greater strength and stiffness in the elderly group are in contrasts to the age-related deficits observed for whole-tendons in vivo, although the study was not designed to investigate these minor differences.

Cancer-related Emergency Department Visits: Comparing Characteristics and Outcomes.

INTRODUCTION: There is increasing appreciation of the challenges of providing safe and appropriate care to cancer patients in the emergency department (ED). Our goal here was to assess which patient characteristics are associated with more frequent ED revisits. METHODS: This was a retrospective cohort study of all ED visits in California during the 2016 calendar year using data from the California Office of Statewide Health Planning and Development. We defined revisits as a return visit to an ED within seven days of the index visit. For both index and return visits, we assessed various patient characteristics, including age, cancer type, medical comorbidities, and ED disposition. RESULTS: Among 12.9 million ED visits, we identified 73,465 adult cancer patients comprising 103,523 visits that met our inclusion criteria. Cancer patients had a 7-day revisit rate of 17.9% vs 13.2% for non-cancer patients. Cancer patients had a higher rate of admission upon 7-day revisit (36.7% vs 15.6%). Patients with cancers of the small intestine, stomach, and pancreas had the highest rate of 7-day revisits (22-24%). Cancer patients younger than 65 had a higher 7-day revisit rate than the elderly (20.0% vs 16.2%). CONCLUSION: In a review of all cancer-related ED visits in the state of California, we found a variety of characteristics associated with a higher rate of 7-day ED revisits. Our goal in this study was to inform future research to identify interventions on the index visit that may improve patient outcomes.

Potential Roles for Gamma-Aminobutyric Acid Signaling in Bacterial Communities.

It is now established that the gut microbiome influences human neurology and behavior, and vice versa. Distinct mechanisms underlying this bidirectional communication pathway, termed the gut-brain axis, are becoming increasingly uncovered. This review summarizes recent interkingdom signaling research focused on gamma-aminobutyric acid (GABA), a human neurotransmitter and ubiquitous signaling molecule found in bacteria, fungi, plants, invertebrates, and mammals. We detail how GABAergic signaling has been shown to be a crucial component of the gut-brain axis. We further describe how GABA is also being found to mediate interkingdom signaling between algae and invertebrates, plants and invertebrates, and plants and bacteria. Based on these emerging results, we argue that obtaining a complete understanding of GABA-mediated communication in the gut-brain axis will involve deciphering the role of GABA signaling and metabolism within bacterial communities themselves.

Magnetic Resonance T2 * Is Increased in Patients With Early-Stage Achilles and Patellar Tendinopathy.

BACKGROUND: T2 * mapping has proven useful in tendon research and may have the ability to detect subtle changes at an early stage of tendinopathy. PURPOSE: To investigate the difference in T2 * between patients with early tendinopathy and healthy controls, and to investigate the relationship between T2 * and clinical outcomes, tendon size, and mechanical properties. STUDY TYPE: Prospective cross-sectional. SUBJECTS: Sixty-five patients with early tendinopathy and 25 healthy controls. FIELD STRENGTH/SEQUENCE: Three Tesla, ultrashort time to echo magnetic resonance imaging. ASSESSMENT: Tendon T2 * was quantified using a monoexponential fitting algorithm. Clinical symptoms were evaluated using the Victorian Institute of Sports Assessment-Achilles/Patella (VISA-A/VISA-P). In vivo mechanical properties were measured using an ultrasound-based method that determined force and deformation simultaneously in tendons of patellar tendinopathy patients. STATISTICAL TESTS: A generalized linear model adjusted for age was applied to investigate the difference between patients and controls. In the two patient groups, linear regressions were applied to investigate the association between T2 * and tendon size, clinical outcomes, and biomechanical properties. RESULTS: There was a significant difference in T2 * between patients and healthy controls (204.8 [95% CI: 44.5-365.0] µsec, P < 0.05). There was a positive correlation between tendon size and T2 * for both Achilles (r = 0.72; P < 0.05) and patellar tendons (r = 0.53; P < 0.05). There was no significant correlation between VISA-A and T2 * (r = -0.2; P = 0.17) or VISA-P and T2 * (r = -0.5; P = 0.0504). Lastly, there was a negative correlation between modulus and T2 * (r = -0.51; P < 0.05). DATA CONCLUSIONS: T2 * mapping can detect subtle structural changes that translate to altered mechanical properties in early-phase tendinopathy. However, T2 * did not correlate with clinical scores in patients with early-phase Achilles and patellar tendinopathy. Thus, T2 * mapping may serve as a tool for early detection of structural changes in tendinopathy but does not necessarily describe the clinical severity of disease. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 2.

Synthetic biology in biofilms: Tools, challenges, and opportunities.

The field of synthetic biology seeks to program living cells to perform novel functions with applications ranging from environmental biosensing to smart cell-based therapeutics. Bacteria are an especially attractive chassis organism due to their rapid growth, ease of genetic manipulation, and ability to persist across many environmental niches. Despite significant progress in bacterial synthetic biology, programming bacteria to perform novel functions outside the well-controlled laboratory context remains challenging. In contrast to planktonic laboratory growth, bacteria in nature predominately reside in the context of densely packed communities known as biofilms. While biofilms have historically been considered environmental and biomedical hazards, their physiology and emergent behaviors could be leveraged for synthetic biology to engineer more capable and robust bacteria. Specifically, bacteria within biofilms participate in complex emergent behaviors such as collective organization, cell-to-cell signaling, and division of labor. Understanding and utilizing these properties can enable the effective deployment of engineered bacteria into natural target environments. Toward this goal, this review summarizes the current state of synthetic biology in biofilms by highlighting new molecular tools and remaining biological challenges. Looking to future opportunities, advancing synthetic biology in biofilms will enable the next generation of smart cell-based technologies for use in medicine, biomanufacturing, and environmental remediation.

Colorectal cancer cells from patients treated with FOLFOX or CAPOX are resistant to oxaliplatin.

BACKGROUND: Numerous studies have suggested benefit for heated intraperitoneal chemotherapy (HIPEC) in the treatment of peritoneal metastases from colon cancer. However, the PRODIGE 7 trial that randomized 265 colon cancer patients to surgery plus HIPEC vs. surgery alone after neoadjuvant chemotherapy (NACT) did not confirm benefit. These data were published as an abstract and not as a peer-reviewed manuscript. One concern is that prior drug exposure may select for drug resistance and blunt HIPEC efficacy. METHODS: A database query identified colon cancer specimens evaluated for chemotherapy sensitivity by ex-vivo analysis of programmed cell death (EVA/PCD), a primary culture platform that examines drug-induced cell death (apoptotic & non-apoptotic) by morphologic, metabolic and histologic endpoints. RESULTS: Of 87 fresh colon cancer specimens, 54 (62%) were untreated and 33 (38%) had received prior folinic acid, 5-fluorouracil, oxaliplatin (FOLFOX) or capecitabine and oxaliplatin (CAPOX). In an apoptosis assay, the lethal concentration of 50% (LC50) in untreated patients was significantly lower than in patients treated by FOLFOX (p = 0.002). Then to approximate PRODIGE 7, treated patients were separated by having received oxaliplatin treatment less than or greater than 2 months before EVA/PCD analysis. The degree of resistance increasing significantly for patients who received treatment less than 2 months prior to EVA/PCD (p < 0.002). Activity for mitomycin and irinotecan was not significantly different for untreated vs. treated patients, but 5-FU was more resistant (P = 0.048). CONCLUSIONS: The failure of PRODIGE 7 to improve survival with surgery plus HIPEC following NACT may reflect diminished oxaliplatin cytotoxicity in patients whose residual disease has been selected for oxaliplatin and 5-FU resistance.

Bioelectrical understanding and engineering of cell biology.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.

Early development of tendinopathy in humans: Sequence of pathological changes in structure and tissue turnover signaling.

Overloading of tendon tissue with resulting chronic pain (tendinopathy) is a common disorder in occupational-, leisure- and sports-activity, but its pathogenesis remains poorly understood. To investigate the very early phase of tendinopathy, Achilles and patellar tendons were investigated in 200 physically active patients and 50 healthy control persons. Patients were divided into three groups: symptoms for 0-1 months (T1), 1-2 months (T2) or 2-3 months (T3). Tendinopathic Achilles tendon cross-sectional area determined by ultrasonography (US) was ~25% larger than in healthy control persons. Both Achilles and patellar anterior-posterior diameter were elevated in tendinopathy, and only later in Achilles was the width increased. Increased tendon size was accompanied by an increase in hypervascularization (US Doppler flow) without any change in mRNA for angiogenic factors. From patellar biopsies taken bilaterally, mRNA for most growth factors and tendon components remained unchanged (except for TGF-beta1 and substance-P) in early tendinopathy. Tendon stiffness remained unaltered over the first three months of tendinopathy and was similar to the asymptomatic contra-lateral tendon. In conclusion, this suggests that tendinopathy pathogenesis represents a disturbed tissue homeostasis with fluid accumulation. The disturbance is likely induced by repeated mechanical overloading rather than a partial rupture of the tendon.

The influence of fibrillin-1 and physical activity upon tendon tissue morphology and mechanical properties in mice.

Fibrillin-1 mutations cause pathological changes in connective tissue that constitute the complex phenotype of Marfan syndrome. In this study, we used fibrillin-1 hypomorphic and haploinsufficient mice (Fbn1mgr/mgR and Fbn1+/- mice, respectively) to investigate the impact of fibrillin-1 deficiency alone or in combination with regular physical activity on tendon tissue morphology and mechanical properties. Morphological and biomechanical analyses revealed that Fbn1mgr/mgR but not Fbn1+/- mice displayed smaller tendons with physical properties that were unremarkable when normalized to tendon size. Fbn1mgR/mgR mice (n = 43) Fbn1+/- mice (n = 27) and wild-type mice (WT, n = 25) were randomly assigned to either control cage conditions (n = 54) or to a running on a running wheel for 4 weeks (n = 41). Both fibrillin-1-deficient mice ran voluntarily on the running wheel in a manner similar to WT mice (3-4 km/24 h). Regular exercise did not mitigate aneurysm progression in Fbn1mgR/mgR mice (P < 0.05) as evidenced by unmodified median survival. In spite of the smaller size, tendons of fibrillin-1-deficient mice subjected to regular exercise showed no evidence of overt histopathological changes or tissue overload. We therefore concluded that lack of optimal fibrillin-1 synthesis leads to a down regulation of integrated tendon formation, rather than to a loss of tendon quality, which also implies that fibrillin-1 deficiency in combination with exercise is not a suitable animal model for studying the development of tendon overuse (tendinopathy).