Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Extracellular matrix viscoelasticity regulates TGFß1-induced epithelial-mesenchymal transition and apoptosis via integrin linked kinase.

Transforming growth factor (TGF)-ß1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFß1 activation, signaling, and downstream cell responses including epithelial-mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFß1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFß1-induced cell spreading, F-actin stress fiber formation, and EMT-associated gene expression changes, and promotes TGFß1-induced apoptosis in mammary epithelial cells. Furthermore, TGFß1-induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFß1-induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFß1 in response to matrix viscoelasticity.

Dynamic Structural Change of Plant Epidermal Cell Walls under Strain.

The molecular foundations of epidermal cell wall mechanics are critical for understanding structure-function relationships of primary cell walls in plants and facilitating the design of bioinspired materials. To uncover the molecular mechanisms regulating the high extensibility and strength of the cell wall, the onion epidermal wall is stretched uniaxially to various strains and cell wall structures from mesoscale to atomic scale are characterized. Upon longitudinal stretching to high strain, epidermal walls contract in the transverse direction, resulting in a reduced area. Atomic force microscopy shows that cellulose microfibrils exhibit orientation-dependent rearrangements at high strains: longitudinal microfibrils are straightened out and become highly ordered, while transverse microfibrils curve and kink. Small-angle X-ray scattering detects a 7.4 nm spacing aligned along the stretch direction at high strain, which is attributed to distances between individual cellulose microfibrils. Furthermore, wide-angle X-ray scattering reveals a widening of (004) lattice spacing and contraction of (200) lattice spacing in longitudinally aligned cellulose microfibrils at high strain, which implies longitudinal stretching of the cellulose crystal. These findings provide molecular insights into the ability of the wall to bear additional load after yielding: the aggregation of longitudinal microfibrils impedes sliding and enables further stretching of the cellulose to bear increased loads.

Crosstalk between ERK and MRTF-A signaling regulates TGFß1-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell-cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast-like properties with enhanced expression of the mesenchymal protein marker α-smooth muscle actin and contractile activity. Transforming growth factor (TGF)-ß1 is a well-known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal-regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFß1-induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin-related transcription factor-A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes.

Community and shotgun metagenomic analysis of Alligator mississippiensis oral cavity and GI tracts reveal complex ecosystems and potential reservoirs of antibiotic resistance.

We report here the community structure and functional analysis of the microbiome of the Alligator mississippiensis GI tract from the oral cavity through the entirety of the digestive tract. Although many vertebrate microbiomes have been studied in recent years, the archosaur microbiome has only been given cursory attention. In the oral cavity we used amplicon-based community analysis to examine the structure of the oral microbiome during alligator development. We found a community that diversified over time and showed many of the hallmarks we would expect of a stable oral community. This is a bit surprising given the rapid turnover of alligator teeth but suggests that the stable gumline microbes are able to rapidly colonize the emerging teeth. As we move down the digestive tract, we were able to use both long and short read sequencing approaches to evaluate the community using a shotgun metagenomics approach. Long read sequencing was applied to samples from the stomach/duodenum, and the colorectal region, revealing a fairly uniform and low complexity community made up primarily of proteobacteria at the top of the gut and much more diversity in the colon. We used deep short read sequencing to further interrogate this colorectal community. The two sequencing approaches were concordant with respect to community structure but substantially more detail was available in the short read data, in spite of high levels of host DNA contamination. Using both approaches we were able to show that the colorectal community is a potential reservoir for antibiotic resistance, human pathogens such as Clostridiodes difficile and a possible source of novel antimicrobials or other useful secondary metabolites.

Orthopedic Surgeons' Accuracy When Orienting an Acetabular Cup. A Comparison with Untrained Individuals.

Background and Objectives: Previous studies demonstrated a huge variability among surgeons when it comes to reproducing the position of an acetabular cup in total hip arthroplasty. Our main objective is to determine if orthopedic surgeons can replicate a given orientation on a pelvic model better than untrained individuals. Our secondary objective is to determine if experience has any influence on their ability for this task. Materials and Methods: A group of specialist orthopedic hip surgeons and a group of volunteers with no medical training were asked to reproduce three given (randomly generated) acetabular cup orientations (inclination and anteversion) on a pelvic model. Error was measured by means of a hip navigation system and comparisons between groups were made using the appropriate statistical methods. Results: The study included 107 individuals, 36 orthopedic surgeons and 71 untrained volunteers. The mean error among surgeons was slightly greater as regards both inclination (7.84 ± 5.53 vs. 6.70 ± 4.03) and anteversion (5.85 ± 4.52 vs. 5.48 ± 3.44), although statistical significance was not reached (p = 0.226 and p = 0.639, respectively). Similarly, although surgeons with more than 100 procedures a year obtained better results than those with less surgical experience (8.01 vs. 7.67 degrees of error in inclination and 5.83 vs. 5.87 in anteversion), this difference was not statistically significant, either (p = 0.852 and p = 0.981). Conclusions: No differences were found in the average error made by orthopedic surgeons and untrained individuals. Furthermore, the surgeons' cup orientation accuracy was not seen to improve significantly with experience.

Rapid preparation of nanodiscs for biophysical studies.

Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample. A panoply of biophysical techniques including analytical ultracentrifugation, dynamic light scattering, gel filtration chromatography, circular dichroism spectroscopy, and cryogenic electron microscopy were employed to unequivocally confirm that aggregates formed by this method are indeed nanodiscs. We believe that this method will be attractive for time-sensitive and high-throughput experiments.

Complete puborectalis, puboperinealis muscle and urethral rhabdomyosphincter preservation in laparoscopic radical prostatectomy: Anatomical landmarks to achieve early urinary continence.

OBJECTIVES: To describe our surgical technique of "muscle-sparing" laparoscopic radical prostatectomy and to review relevant anatomical landmarks during the procedure. METHODS: This was a prospective non-controlled case series of 120 consecutive patients who underwent laparoscopic radical prostatectomy, always carried out by the same surgeon (OL). The median follow-up period was 33 months. Dissection of the puboperinealis and puborectalis muscle consists of the precise dissection of the puborectalis and puboperinealis muscles from the periprostatic fascia. Rhabdomyo-dissection consists of an approach that spares the external urethral sphincter from the ventral surface of the prostate and membranous urethra. Clinical data were collected in a dedicated database. Intraoperative variables, postoperative complications and outcomes of urinary continence were assessed. A descriptive statistical analysis was carried out. RESULTS: Continence rates were 70.8%, 83.3% and 92.5%, at 0-2, 3-4 and 5-8 weeks after removal of the urethral catheter, respectively; 96.6% and 98.3% at 6 and 12 months after surgery. The positive surgical margin rate associated with rhabdomyo-dissection was 8.3%. CONCLUSIONS: Laparoscopic radical prostatectomy with dissection of the puboperinealis and puborectalis muscle, and rhabdomyo-dissection is an oncologically safe procedure, associated with very early recovery urinary continence in most patients. It is a technique that can be applied in most cases, as long as there is no invasion of the ventral side of the prostate.

A rare case of bacteremia caused by Propionibacterium (Propionimicrobium) lymphophilum.

Propionibacterium (Propionimicrobium) lymphophilum is a Gram-positive anaerobic rod involved in few human diseases. We report a rare case of bacteremia due to this microorganism in an elderly patient. A 95-year-old woman without a remarkable medical history presented with dyspnea, chest pain and fever for seven days. Blood cultures resulted in isolation of P. lymphophilum. Resistance only to metronidazole was found. Treatment with amoxicillin-clavulanic acid was established, and the patient was discharged and improvement of her general condition was documented.

Becoming Stressed: Does the Age Matter? Reviewing the Neurobiological and Socio-Affective Effects of Stress throughout the Lifespan.

Social and affective relations occur at every stage of our lives. Impairments in the quality of this "social world" can be exceptionally detrimental and lead to psychopathology or pathological behavior, including schizophrenia, autism spectrum disorder, affective disorders, social phobia or violence, among other things. Exposure to highly stressful or traumatic events, depending on the stage of life in which stress exposure occurs, could severely affect limbic structures, including the amygdala, and lead to alterations in social and affective behaviors. This review summarizes recent findings from stress research and provides an overview of its age-dependent effects on the structure and function of the amygdala, which includes molecular and cellular changes, and how they can trigger deviant social and affective behaviors. It is important to highlight that discoveries in this field may represent a breakthrough both for medical science and for society, as they may help in the development of new therapeutic approaches and prevention strategies in neuropsychiatric disorders and pathological behaviors.

MAP/ERK Signaling in Developing Cognitive and Emotional Function and Its Effect on Pathological and Neurodegenerative Processes.

The signaling pathway of the microtubule-associated protein kinase or extracellular regulated kinase (MAPK/ERK) is a common mechanism of extracellular information transduction from extracellular stimuli to the intracellular space. The transduction of information leads to changes in the ongoing metabolic pathways and the modification of gene expression patterns. In the central nervous system, ERK is expressed ubiquitously, both temporally and spatially. As for the temporal ubiquity, this signaling system participates in three key moments: (i) Embryonic development; (ii) the early postnatal period; and iii) adulthood. During embryonic development, the system is partly responsible for the patterning of segmentation in the encephalic vesicle through the FGF8-ERK pathway. In addition, during this period, ERK directs neurogenesis migration and the final fate of neural progenitors. During the early postnatal period, ERK participates in the maturation process of dendritic trees and synaptogenesis. During adulthood, ERK participates in social and emotional behavior and memory processes, including long-term potentiation. Alterations in mechanisms related to ERK are associated with different pathological outcomes. Genetic alterations in any component of the ERK pathway result in pathologies associated with neural crest derivatives and mental dysfunctions associated with autism spectrum disorders. The MAP-ERK pathway is a key element of the neuroinflammatory pathway triggered by glial cells during the development of neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as prionic diseases. The process triggered by MAPK/ERK activation depends on the stage of development (mature or senescence), the type of cellular element in which the pathway is activated, and the anatomic neural structure. However, extensive gaps exist with regards to the targets of the phosphorylated ERK in many of these processes.

Creating cross-linked lamellar block copolymer supporting layers for biomimetic membranes.

The long-standing goal in membrane development is creating materials with superior transport properties, including both high flux and high selectivity. These properties are common in biological membranes, and thus mimicking nature is a promising strategy towards improved membrane design. In previous studies, we have shown that artificial water channels can have excellent water transport abilities that are comparable to biological water channel proteins, aquaporins. In this study, we propose a strategy for incorporation of artificial channels that mimic biological channels into stable polymeric membranes. Specifically, we synthesized an amphiphilic triblock copolymer, poly(isoprene)-block-poly(ethylene oxide)-block-poly(isoprene), which is a high molecular weight synthetic analog of naturally occurring lipids in terms of its self-assembled structure. This polymer was used to build stacked membranes composed of self-assembled lamellae. The resulting membranes resemble layers of natural lipid bilayers in living systems, but with superior mechanical properties suitable for real-world applications. The procedures used to synthesize the triblock copolymer resulted in membranes with increased stability due to the crosslinkability of the hydrophobic domains. Furthermore, the introduction of bridging hydrophilic domains leads to the preservation of the stacked membrane structure when the membrane is in contact with water, something that is challenging for diblock lamellae that tend to swell, and delaminate in aqueous solutions. This new method of membrane fabrication offers a practical model for making channel-based biomimetic membranes, which may lead to technological applications in reverse osmosis, nanofiltration, and ultrafiltration membranes.

[Prevalence and characterization of overactive bladder detected in a population in Madrid with self-administered OAB-V3 questionnaire in Primary Care]. / Prevalencia y caracterización de la vejiga hiperactiva detectada en una población de Madrid con el cuestionario OAB-V3 autoadministrado en atención primaria.

OBJECTIVE: Determining the prevalence of symptoms suggestive of overactive bladder (OAB) in a Spanish population and evaluate the impact of these symptoms on well-being and labour productivity in this population. DESIGN: Transversal study. LOCATION: Primary health care, Madrid, Spain. PARTICIPANTS: Males and females >30 years. INTERVENTIONS: Classification by primary care physicians with the Overactive Bladder Awareness Tool abbreviated version (OAB-V3). Subjects with score ≥3 and a similarly balanced control population with score <3 were clinically investigated. PRINCIPAL MEASUREMENTS: History, physical examination, urinalysis, sonography, general well-being scale and the questionnaires PPBC, OAB-q y WPAI-SHP. RESULTS: A total 923 subjects were screened, of which 209 (22.6%), 35% males and 65% females, had probable OAB. Age distribution increased from 11.1% in 4th decade to 44.4% in 9th decade. Kappa coefficient between suspected OAB and definite diagnosis was .83. The area under ROC curve for diagnosis based on OAB-V3 questionnaire and the presence of perceived bother and coping strategies was 92%. Subjects classified by score ≥3 had worse well-being, higher PPBC score and worse parameters on total OAB-q and transformed scores for each OAB-q subscale (P<.0001). In these subjects labour productivity was not affected (P=.14) but the capacity to perform regular activities was (P<.0001). CONCLUSIONS: OAB-V3 is a simple questionnaire to screen OAB with good predictive accuracy in a primary care setting and reveals important implications on health related quality of life issues.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

The brain as immunoprecipitator of serum autoantibodies against N-Methyl-D-aspartate receptor subunit NR1.

Autoantibodies (AB) against N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) are highly seroprevalent in health and disease. Symptomatic relevance may arise upon compromised blood-brain barrier (BBB). However, it remained unknown whether circulating NMDAR1 AB appear in the cerebrospinal fluid (CSF). Of n = 271 subjects with CSF-serum pairs, 26 were NMDAR1 AB seropositive, but only 1 was CSF positive. Contrariwise, tetanus AB (non-brain-binding) were present in serum and CSF of all subjects, with CSF levels higher upon BBB dysfunction. Translational mouse experiments proved the hypothesis that the brain acts as an 'immunoprecipitator'; simultaneous injection of NMDAR1 AB and the non-brain-binding green fluorescent protein AB resulted in high detectability of the former in brain and the latter in CSF.

Cyanomargarita gen. nov. (Nostocales, Cyanobacteria): convergent evolution resulting in a cryptic genus.

Two populations of Rivularia-like cyanobacteria were isolated from ecologically distinct and biogeographically distant sites. One population was from an unpolluted stream in the Kola Peninsula of Russia, whereas the other was from a wet wall in the Grand Staircase-Escalante National Monument, a desert park-land in Utah. Though both were virtually indistinguishable from Rivularia in field and cultured material, they were both phylogenetically distant from Rivularia and the Rivulariaceae based on both 16S rRNA and rbcLX phylogenies. We here name the new cryptic genus Cyanomargarita gen. nov., with type species C. melechinii sp. nov., and additional species C. calcarea sp. nov. We also name a new family for these taxa, the Cyanomargaritaceae.

Macrochaete gen. nov. (Nostocales, Cyanobacteria), a taxon morphologically and molecularly distinct from Calothrix.

Historically, the genus Calothrix included all noncolonial, tapered, heterocytous filaments within the cyanobacteria. However, recent molecular phylogenies show that "Calothrix" defined in this sense represents five distinct clades. The type species of Calothrix is marine, with solitary basal heterocytes, no akinetes, and distal ends tapering abruptly into short hairs. We examined the morphology and phylogeny of 45 tapering cyanobacteria in the Rivulariaceae, including freshwater and marine representatives of both Calothrix (35 strains) and its sister taxon Rivularia (10 strains). The marine Calothrix fall into two lineages, but we lack the generitype and so cannot identify the clade corresponding to the type species. The freshwater and soil Calothrix fall into the C. parietina clade and are characterized by having a basal heterocyte, no akinetes, and gradual tapering-but not into a long hyaline hair. Macrochaete gen. nov. is a freshwater taxon sister to the Calothrix lineages but clearly separated from Rivularia. The species in this genus differ morphologically from Calothrix by their ability to produce two heteromorphic basal heterocytes and specific secondary structures of the 16S-23S ITS. An additional feature present in most species is the presence of a distal, long hyaline hair, but this character has incomplete penetrance due to its expression only under specific environmental conditions (low phosphate), and in one species appears to be lost. We recognize three species: M. psychrophila (type species) from cold environments (high mountains, Antarctica), M. santannae from wet walls of subtropical South America, and M. lichenoides, a phycobiont of lichens from Europe.

Effects of Chronic Dopamine D2R Agonist Treatment and Polysialic Acid Depletion on Dendritic Spine Density and Excitatory Neurotransmission in the mPFC of Adult Rats.

Dopamine D2 receptors (D2R) in the medial prefrontal cortex (mPFC) are key players in the etiology and therapeutics of schizophrenia. The overactivation of these receptors contributes to mPFC dysfunction. Chronic treatment with D2R agonists modifies the expression of molecules implicated in neuronal structural plasticity, synaptic function, and inhibitory neurotransmission, which are also altered in schizophrenia. These changes are dependent on the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, but nothing is known about the effects of D2R and PSA-NCAM on excitatory neurotransmission and the structure of mPFC pyramidal neurons, two additional features affected in schizophrenia. To evaluate these parameters, we have chronically treated adult rats with PPHT (a D2R agonist) after enzymatic removal of PSA with Endo-N. Both treatments decreased spine density in apical dendrites of pyramidal neurons without affecting their inhibitory innervation. Endo-N also reduced the expression of vesicular glutamate transporter-1. These results indicate that D2R and PSA-NCAM are important players in the regulation of the structural plasticity of mPFC excitatory neurons. This is relevant to our understanding of the neurobiological basis of schizophrenia, in which structural alterations of pyramidal neurons and altered expression of D2R and PSA-NCAM have been found.

Matrix Rigidity Mediates TGFß1-Induced Epithelial-Myofibroblast Transition by Controlling Cytoskeletal Organization and MRTF-A Localization.

Myofibroblasts mediate normal wound healing and upon chronic activation can contribute to the development of pathological conditions including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells through an epithelial-mesenchymal transition (EMT) during which epithelial cells exhibit drastic morphological changes and upregulate cytoskeletal associated proteins that enable exertion of large contractile forces and remodeling of the surrounding microenvironment. Increased matrix rigidity is a hallmark of fibrosis and tumor progression and mechanical tension has been identified as a regulator of EMT; however, the mechanisms governing the mechanical regulation of EMT are not completely understood. Here, we find that matrix rigidity regulates transforming growth factor (TGF)-ß1-induced EMT, with rigid substrata enabling increased myofibroblast marker expression, cell morphology changes, and cytoskeletal reorganization while soft matrices block these changes. Furthermore, we find that matrix rigidity controls the subcellular localization of myocardin related transcription factor (MRTF)-A, a regulator of cytoskeletal protein expression that contributes to the acquisition of myogenic features during EMT. Results from these studies provide insight into how biophysical cues contribute to myofibroblast development from epithelial cells and may suggest ways to enhance wound healing or to engineer therapeutic solutions for fibrosis and cancer.