Microbial warfare: B. subtilis antagonizes E. coli biofilm formation.

Biofilm formation helps bacteria to survive environmental challenges. Biofilm development often involves multiple genetic pathways that can be regulated by external signals. Diego Serra and his team (Cordisco et al.) explore how Bacillus subtilis can antagonize Escherichia coli macrocolony biofilm formation via the metabolite bacillaene.

Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity.

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites colonize the large intestine, primarily feeding on bacteria. However, in the gastrointestinal tract, bacterial cells form aggregates or structured communities called biofilms too large for phagocytosis. Remarkably, trophozoites are still able to invade and degrade established biofilms, utilizing a mechanism that mimics digestive exophagy. Digestive exophagy refers to the secretion of digestive enzymes that promote the digestion of objects too large for direct phagocytosis by phagocytes. E. histolytica cysteine proteinases (CPs) play a crucial role in the degradation process of Bacillus subtilis biofilm. These proteinases target TasA, a major component of the B. subtilis biofilm matrix, also contributing to the adhesion of the parasite to the biofilm. In addition, they are also involved in the degradation of biofilms formed by Gram-negative and Gram-positive enteric pathogens. Furthermore, biofilms also play an important role in protecting trophozoites against oxidative stress. This specific mechanism suggests that the amoeba has adapted to prey on biofilms, potentially serving as an untapped reservoir for novel therapeutic approaches to treat biofilms. Consistently, products derived from the amoeba have been shown to restore antibiotic sensitivity to biofilm cells. In addition, our findings reveal that probiotic biofilms can act as a protective shield for mammalian cells, hindering the progression of the parasite towards them.

Oral versus intravenous acetaminophen for perioperative pain management in adult patients undergoing non-cardiac surgery: A quantile segmented regression analysis.

STUDY OBJECTIVE: Determine whether preferential use of perioperative enteral acetaminophen is associated with changes in perioperative pain, narcotic administration, or time to meeting criteria for post anesthesia care unit (PACU) discharge, compared to preferential parenteral administration. DESIGN: Retrospective Cohort with quantile segmented regression analysis. Groups determined by date of surgery, one year pre-initiative and one year post-initiative. SETTING: Operating room and PACU of a tertiary academic medical center. PATIENTS: Adult (age > 18 years), ASA status 1-5, non-pregnant patients undergoing non-cardiac surgery of less than six hours duration admitted to the PACU postoperatively. INTERVENTIONS: A multidisciplinary initiative to preferentially utilize enteral over parenteral acetaminophen. MEASUREMENTS: The primary outcome was narcotic consumption in the PACU. Secondary outcomes were intraoperative narcotic administration, pain score on PACU admission and discharge, and time to meeting criteria for PACU discharge. RESULTS: 24,701 patients were included in the analysis; 12,379 had surgery prior to the initiative and 12,322 after. Enteral acetaminophen administration increased preoperatively from 13.49% to 26.84%, and postoperatively from 43.16% to 51.45%, while intraoperative parenteral APAP use dropped from 43.23% to 6.81%. Quantile Segmented regression analysis after adjusting for period (pre versus postintervention), day, age, gender, inpatient status, and ASA class demonstrated a decrease in adjusted median perioperative acetaminophen dose (-175 mg P < 0.001), with no significant difference in level change of intraoperative or PACU narcotic administration. There was no significant difference in median time to meet criteria for PACU discharge, though there was a significant change in the slope, (-0.36, p = 0.007.) Median pain scores measured on a standard 0-10 numeric rating scale at PACU admission did not change, while median pain scores at PACU discharge decreased slightly (-0.24 p < 0.001). There was no change in the probability of PONV. CONCLUSION: In adult patients undergoing non-cardiac surgery of <6 h duration, preferential use of enteral rather than parenteral acetaminophen is associated with non-inferior outcomes in narcotic requirements, pain scores, time to PACU discharge, and probability of PONV when compared with routine parenteral administration. Further studies are needed to validate these findings.

Cell surface-localized CsgF condensate is a gatekeeper in bacterial curli subunit secretion.

Curli are functional amyloids present on the outer membrane of E. coli. CsgF is required for the proper assembly of curli. Here, we found that the CsgF phase separates in vitro and that the ability of CsgF variants to phase-separate is tightly correlated with CsgF function during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli assembly. Exogenous addition of purified CsgF complemented csgF - cells. This exogenous addition assay was used to assess the ability of CsgF variants to complement csgF ‒ cells. CsgF on the cell surface modulated the secretion of CsgA, the curli major subunit, to the cell surface. We also found that the CsgB nucleator protein can form SDS-insoluble aggregates within the dynamic CsgF condensate. We propose that these multicomponent CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface.

Electrostatic interactions mediate the nucleation and growth of a bacterial functional amyloid.

Bacterial biofilm formation can have severe impacts on human and environmental health. Enteric bacteria produce functional amyloid fibers called curli that aid in biofilm formation and host colonization. CsgA is the major proteinaceous component of curli amyloid fibers and is conserved in many gram-negative enteric bacteria. The CsgA amyloid core consists of five imperfect repeats (R1-R5). R2, R3, and R4 have aspartic acid (D) and glycine (G) residues that serve as "gatekeeper" residues by modulating the intrinsic aggregation propensity of CsgA. Here, using mutagenesis, salt-mediated charge screening, and by varying pH conditions, we show that the ability of CsgA variants to nucleate and form amyloid fibers is dictated by the charge state of the gatekeeper residues. We report that in Citrobacter youngae CsgA, certain arginine (R) and lysine (K) residues also act as gatekeeper residues. A mechanism of gatekeeping is proposed wherein R and K residues electrostatically interact with negatively charged D residues, tempering CsgA fiber formation.

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids.

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson's disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.

Bacterial Amyloid Curli Associated Gut Epithelial Neuroendocrine Activation Predominantly Observed in Alzheimer's Disease Mice with Central Amyloid-ß Pathology.

BACKGROUND: Substantial evidence from recent research suggests an influential and underappreciated force in Alzheimer's disease (AD) pathogenesis: the pathological signals originate from outside the brain. Pathogenic bacteria produce amyloid-like proteins "curli" that form biofilms and show functional similarities to human amyloid-ß (Aß). These proteins may contribute to neurological disease progression via signaling cascade from the gut to the brain. OBJECTIVE: We propose that curli causes neuroendocrine activation from the gut to brain that promotes central Aß pathology. METHODS: PGP9.5 and TLR2 levels in response to curli in the lumen of Tg2576 AD mice were analyzed by immunohistochemical and qRT-PCR analysis. Western blot and human 3D in vitro enteroids culture systems were also used. 16S rRNA gene sequencing was used to investigate bacterial dysbiosis. RESULTS: We found significant increase in bacterial-amyloid curli with elevated TLR2 at the mRNA level in the pre- and symptomatic Tg-AD gut compared to littermate WT controls. This data associates with increased gram-positive bacterial colonization in the ileum of the symptomatic AD mice. We found fundamental evidence for vagus nerve activation in response to bacterial curli. Neuroendocrine marker PGP9.5 was significantly elevated in the gut epithelium of symptomatic AD mice, and this was colocalized with increased TLR2 expression. Enteroids, 3D-human ileal mini-gut monolayer in vitro model system also revealed increase levels of TLR2 upon stimulation with purified bacterial curli fibrils. CONCLUSION: These findings reveal the importance of pathological changes within the gut-vagus-brain signaling in response to luminal bacterial amyloid that might play a vital role in central Aß pathogenesis seen in the AD brain.

MRI T2/STIR epaxial muscle hyperintensity in some dogs with intervertebral disc extrusion corresponds to histologic patterns of muscle degeneration and inflammation.

Magnetic resonance imaging hyperintensity on T2-weighted turbo SE and STIR sequences of the paraspinal musculature in canine patients being imaged for thoracolumbar intervertebral disc extrusion is frequently observed but poorly understood in veterinary medicine. The objective of this prospective analytical study was to describe the histopathology of muscle hyperintensity in dogs with thoracolumbar intervertebral disc extrusions and to determine if a relationship exists between the presence of this hyperintensity and various patient factors. Twenty privately owned dogs who underwent surgical decompression of intervertebral disc extrusions diagnosed on MRI were enrolled (10 normal "control or nonaffected cases" without MRI paraspinal musculature hyperintensity and 10 "affected cases" with hyperintensity). Surgical biopsies of the epaxial musculature at the region of hyperintensity (affecteds) and at the site of the disc herniation (controls) were submitted for histopathology. The degree of myofiber degeneration and necrosis was scored using an ordinal scoring system: absent (0), minimal (10), mild (20), moderate (30), marked/severe (40), and massive (50). Associations between hyperintensity presence and patient age, weight, body condition, neurologic status, acuteness of onset, number of disc herniation sites, degree of spinal cord compression, and volume of herniated material were investigated. Nonaffected patients were significantly older (median age = 9.4 years) than affected patients (median age = 3.5 years), but no other significant associations were found. Acute myofiber degeneration/necrosis and intramuscular inflammation were observed in half of affected patients. Therefore, T2/STIR muscle hyperintensity in some patients with intervertebral disc extrusion may represent muscle degeneration and inflammation.

Functional Amyloids Are the Rule Rather Than the Exception in Cellular Biology.

Amyloids are a class of protein aggregates that have been historically characterized by their relationship with human disease. Indeed, amyloids can be the result of misfolded proteins that self-associate to form insoluble, extracellular plaques in diseased tissue. For the first 150 years of their study, the pathogen-first definition of amyloids was sufficient. However, new observations of amyloids foster an appreciation for non-pathological roles for amyloids in cellular systems. There is now evidence from all domains of life that amyloids can be non-pathogenic and functional, and that their formation can be the result of purposeful and controlled cellular processes. So-called functional amyloids fulfill an assortment of biological functions including acting as structural scaffolds, regulatory mechanisms, and storage mechanisms. The conceptual convergence of amyloids serving a functional role has been repeatedly confirmed by discoveries of additional functional amyloids. With dozens already known, and with the vigorous rate of discovery, the biology of amyloids is robustly represented by non-pathogenic amyloids.

Half a century of amyloids: past, present and future.

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

Tuning Functional Amyloid Formation Through Disulfide Engineering.

Many organisms produce "functional" amyloid fibers, which are stable protein polymers that serve many roles in cellular biology. Certain Enterobacteriaceae assemble functional amyloid fibers called curli that are the main protein component of the biofilm extracellular matrix. CsgA is the major protein subunit of curli and will rapidly adopt the polymeric amyloid conformation in vitro. The rapid and irreversible nature of CsgA amyloid formation makes it challenging to study in vitro. Here, we engineered CsgA so that amyloid formation could be tuned to the redox state of the protein. A double cysteine variant of CsgA called CsgACC was created and characterized for its ability to form amyloid. When kept under oxidizing conditions, CsgACC did not adopt a ß-sheet rich structure or form detectable amyloid-like aggregates. Oxidized CsgACC remained in a soluble, non-amyloid state for at least 90 days. The addition of reducing agents to CsgACC resulted in amyloid formation within hours. The amyloid fibers formed by CsgACC were indistinguishable from the fibers made by CsgA WT. When measured by thioflavin T fluorescence the amyloid formation by CsgACC in the reduced form displayed the same lag, fast, and plateau phases as CsgA WT. Amyloid formation by CsgACC could be halted by the addition of oxidizing agents. Therefore, CsgACC serves as a proof-of-concept for capitalizing on the convertible nature of disulfide bonds to control the aggregation of amyloidogenic proteins.

A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice.

Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.

Shotgun metagenomic analysis of microbial communities from the Loxahatchee nature preserve in the Florida Everglades.

BACKGROUND: Currently, much is unknown about the taxonomic diversity and the mechanisms of methane metabolism in the Florida Everglades ecosystem. The Loxahatchee National Wildlife Refuge is a section of the Florida Everglades that is almost entirely unstudied in regard to taxonomic profiling. This short report analyzes the metagenome of soil samples from this Refuge to investigate the predominant taxa, as well as the abundance of genes involved in environmentally significant metabolic pathways related to methane production (nitrogen fixation and dissimilatory sulfite reduction). METHODS: Shotgun metagenomic sequencing using the Illumina platform was performed on 17 soil samples from four different sites within the Loxahatchee National Wildlife Refuge, and underwent quality control, assembly, and annotation. The soil from each sample was tested for water content and concentrations of organic carbon and nitrogen. RESULTS: The three most common phyla of bacteria for every site were Actinobacteria, Acidobacteria, and Proteobacteria; however, there was variation in relative phylum composition. The most common phylum of Archaea was Euryarchaeota for all sites. Alpha and beta diversity analyses indicated significant congruity in taxonomic diversity in most samples from Sites 1, 3, and 4 and negligible congruity between Site 2 and the other sites. Shotgun metagenomic sequencing revealed the presence of biogeochemical biomarkers of particular interest (e.g., mrcA, nifH, and dsrB) within the samples. The normalized abundances of mcrA, nifH, and dsrB exhibited a positive correlation with nitrogen concentration and water content, and a negative correlation with organic carbon concentration. CONCLUSION: This Everglades soil metagenomic study allowed examination of wetlands biological processes and showed expected correlations between measured organic constituents and prokaryotic gene frequency. Additionally, the taxonomic profile generated gives a basis for the diversity of prokaryotic microbial life throughout the Everglades.

Physician Suicide: A Call to Action.

Physician suicide is topic of growing professional and public health concern. Despite working to improve the health of others, physicians often sacrifice their own well-being to do so. Furthermore, there are systemic barriers in place that discourage self-care and help-seeking behaviors among physicians. This article will discuss the relevant epidemiology, risk factors, and barriers to treatment, then explore solutions to address this alarming trend.

Bacterial functional amyloids: Order from disorder.

The discovery of intrinsic disorderness in proteins and peptide regions has given a new and useful insight into the working of biological systems. Due to enormous plasticity and heterogeneity, intrinsically disordered proteins or regions in proteins can perform myriad of functions. The flexibility in disordered proteins allows them to undergo conformation transition to form homopolymers of proteins called amyloids. Amyloids are highly structured protein aggregates associated with many neurodegenerative diseases. However, amyloids have gained much appreciation in recent years due to their functional roles. A functional amyloid fiber called curli is assembled on the bacterial cell surface as a part of the extracellular matrix during biofilm formation. The extracellular matrix that encases cells in a biofilm protects the cells and provides resistance against many environmental stresses. Several of the Csg (curli specific genes) proteins that are required for curli amyloid assembly are predicted to be intrinsically disordered. Therefore, curli amyloid formation is highly orchestrated so that these intrinsically disordered proteins do not inappropriately aggregate at the wrong time or place. The curli proteins are compartmentalized and there are chaperone-like proteins that prevent inappropriate aggregation and allow the controlled assembly of curli amyloids. Here we review the biogenesis of curli amyloids and the role that intrinsically disordered proteins play in the process.

Defining a Theory-Driven Ultrasound Curriculum for Prehospital Providers.

Advances in point-of-care ultrasound technology have allowed for the extension of emergency medicine ultrasound beyond the walls of the emergency department. Emergency medical system providers may benefit from the use of ultrasound. It has previously been shown that with a brief introductory course, novices can obtain and correctly interpret focused ultrasound examinations. The purpose of this study was to design a theory-driven point-of-care ultrasound curriculum to assess and develop ultrasound skill in prehospital providers. The resultant curriculum outlined in this paper encompasses a large array of skills that may be useful for different prehospital services to use to develop curriculum for their own needs.

Curli Biogenesis: Bacterial Amyloid Assembly by the Type VIII Secretion Pathway.

In 1989, Normark and coworkers reported on fibrous surface structures called curli on strains of Escherichia coli that were suspected of causing bovine mastitis. Subsequent work by many groups has revealed an elegant and highly regulated curli biogenesis pathway also referred to as the type VIII secretion system. Curli biogenesis is governed by two divergently transcribed operons, csgBAC and csgDEFG. The csgBAC operon encodes the structural subunits of curli, CsgA and CsgB, along with a chaperone-like protein, CsgC. The csgDEFG operon encodes the accessory proteins required for efficient transcription, secretion, and assembly of the curli fiber. CsgA and CsgB are secreted as largely unstructured proteins and transition to ß-rich structures that aggregate into regular fibers at the cell surface. Since both of these proteins have been shown to be amyloidogenic in nature, the correct spatiotemporal synthesis of the curli fiber is of paramount importance for proper functioning and viability. Gram-negative bacteria have evolved an elegant machinery for the safe handling, secretion, and extracellular assembly of these amyloidogenic proteins.