Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.

Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

Butyrate-producing colonic clostridia: picky glycan utilization specialists.

Butyrate-producing human gut microbiota members are recognized for their strong association with a healthy immune-homeostasis and protection from inflammatory disorders and colorectal cancer. These effects are attributed to butyrate, the terminal electron sink of glycan fermentation by prevalent and abundant colonic Firmicutes from the Lachnospiraceae and Oscillospiraceae families. Remarkably, our insight into the glycan utilization mechanisms and preferences of butyrogenic Firmicutes remains very limited as compared with other gut symbionts, especially from the Bacteroides, Bifidobacterium, and Lactobacillus genera. Here, we summarize recent findings on the strategies that colonic butyrate producers have evolved to harvest energy from major dietary fibres, especially plant structural and storage glycans, such as resistant starch, xylans, and mannans. Besides dietary fibre, we also present the unexpected discovery of a conserved protein apparatus that confers the growth of butyrate producers on human milk oligosaccharides (HMOs), which are unique to mother's milk. The dual dietary fibre/HMO utilization machinery attests the adaptation of this group to both the infant and adult guts. These finding are discussed in relation to the early colonization of butyrogenic bacteria and the maturation of the microbiota during the transition from mother's milk to solid food. To date, the described butyrogenic Firmicutes are glycan utilization specialists that target only a few glycans in a highly competitive manner relying on co-regulated glycan utilization loci. We describe the common pillars of this machinery, highlighting butyrate producers as a source for discovery of biochemically and structurally novel carbohydrate active enzymes.

Biopsy-Proven Amyloid-Beta Related Angiitis with Unusual Presentation and Long-Term Follow-Up.

Amyloid-beta related angiitis (ABRA) is a rare central nervous system inflammatory and vasculitic process. It is seen in patients with cerebral amyloid angiopathy (CAA) and thought to be mediated by an autoimmune reaction against cerebrovascular ß-amyloid. We describe the case of a patient with ABRA with clinical information and brain imaging over a 10-year period. The patient was hospitalized in 2018 for altered mental status, paranoia and hallucinations. Her symptoms started in 2009 with an episode of vertigo and loss of consciousness. From 2011-2019, she had multiple episodes of transient focal neurological deficits with overall cumulative progressive decline in cognition and functional status. Retrospective and comparative reviews of brain magnetic resonance imaging (MRI) from 2009-2019 showed waxing and waning vasogenic cerebral edema with overall progression of white matter hyperintensities and peripheral micro-hemorrhages consistent with inflammatory CAA. Re-examination of a brain biopsy from 2009 showed ABRA, and immunostaining was positive for ß-amyloid. She was treated with intravenous steroids with minimal symptomatic improvement. She was lost to our follow-up after hospital discharge. We describe the temporal progression of ABRA through serial brain imaging over a 10-year period. To our knowledge, this is the longest published follow-up duration of ABRA. The patient in our case had severe cognitive impairment and disability despite treatment with steroids.

Orally active bivalent VHH construct prevents proliferation of F4+ enterotoxigenic Escherichia coli in weaned piglets.

A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent VHH-based protein BL1.2 that cross-links a F4+ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4+ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4+ ETEC proliferation when administrated as a feed additive to weaned F4+ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent VHH-based feed additive in effectively delimiting pathogenic F4+ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study.

Pan-immunoglobulin assays can simultaneously detect IgG, IgM and IgA directed against the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 S1-RBD Ig). In this work, we aim to evaluate a quantitative SARS-CoV-2 S1-RBD Ig electrochemiluminescence immunoassay (ECLIA) regarding analytical, diagnostic, operational and clinical characteristics. Our work takes the form of a population-based study in the principality of Liechtenstein, including 125 cases with clinically well-described and laboratory confirmed SARS-CoV-2 infection and 1159 individuals without evidence of coronavirus disease 2019 (COVID-19). SARS-CoV-2 cases were tested for antibodies in sera taken with a median of 48 days (interquartile range, IQR, 43-52) and 139 days (IQR, 129-144) after symptom onset. Sera were also tested with other assays targeting antibodies against non-RBD-S1 and -S1/S2 epitopes. Sensitivity was 97.6% (95% confidence interval, CI, 93.2-99.1), whereas specificity was 99.8% (95% CI, 99.4-99.9). Antibody levels linearly decreased from hospitalized patients to symptomatic outpatients and SARS-CoV-2 infection without symptoms (p < 0.001). Among cases with SARS-CoV-2 infection, smokers had lower antibody levels than non-smokers (p = 0.04), and patients with fever had higher antibody levels than patients without fever (p = 0.001). Pan-SARS-CoV-2 S1-RBD Ig in SARS-CoV-2 infection cases significantly increased from first to second follow-up (p < 0.001). A substantial proportion of individuals without evidence of past SARS-CoV-2 infection displayed non-S1-RBD antibody reactivities (248/1159, i.e., 21.4%, 95% CI, 19.1-23.4). In conclusion, a quantitative SARS-CoV-2 S1-RBD Ig assay offers favorable and sustained assay characteristics allowing the determination of quantitative associations between clinical characteristics (e.g., disease severity, smoking or fever) and antibody levels. The assay could also help to identify individuals with antibodies of non-S1-RBD specificity with potential clinical cross-reactivity to SARS-CoV-2.

Butyrate producing colonic Clostridiales metabolise human milk oligosaccharides and cross feed on mucin via conserved pathways.

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.

An 1,4-α-Glucosyltransferase Defines a New Maltodextrin Catabolism Scheme in Lactobacillus acidophilus.

The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs): a putative maltogenic α-amylase of family 13, subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65), and a family 13, subfamily 31, member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS with a degree of polymerization of ≥2, with a preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus revealed that the substrate preference of LaGH13_31B toward maltotriose complements the ~40-fold lower kcat of LaGH13_20 toward this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolyzed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop that is unique for a previously unexplored clade of GH13_31, represented by LaGH13_31B. The identified loop insertion harbors a conserved aromatic residue that modulates the activity and substrate affinity of the enzyme, thereby offering a functional signature of this clade, which segregates from 1,6-α-glucosidases and sucrose isomerases previously described within GH13_31. Genomic analyses revealed that the LaGH13_31B gene is conserved in the MOS utilization loci of lactobacilli, including acidophilus cluster members that dominate the human small intestine.IMPORTANCE The degradation of starch in the small intestine generates short linear and branched α-glucans. The latter are poorly digestible by humans, rendering them available to the gut microbiota, e.g., lactobacilli adapted to the small intestine and considered beneficial to health. This study unveils a previously unknown scheme of maltooligosaccharide (MOS) catabolism via the concerted activity of an 1,4-α-glucosyltransferase together with a classical hydrolase and a phosphorylase. The intriguing involvement of a glucosyltransferase likely allows the fine-tuning of the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities that have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.

Design, Implementation, and Evaluation of a Head and Neck MRI RF Array Integrated with a 511 keV Transmission Source for Attenuation Correction in PET/MR.

The goal of this work is to further improve positron emission tomography (PET) attenuation correction and magnetic resonance (MR) sensitivity for head and neck applications of PET/MR. A dedicated 24-channel receive-only array, fully-integrated with a hydraulic system to move a transmission source helically around the patient and radiofrequency (RF) coil array, is designed, implemented, and evaluated. The device enables the calculation of attenuation coefficients from PET measurements at 511 keV including the RF coil and the particular patient. The RF coil design is PET-optimized by minimizing photon attenuation from coil components and housing. The functionality of the presented device is successfully demonstrated by calculating the attenuation map of a water bottle based on PET transmission measurements; results are in excellent agreement with reference values. It is shown that the device itself has marginal influence on the static magnetic field B0 and the radiofrequency transmit field B1 of the 3T PET/MR system. Furthermore, the developed RF array is shown to outperform a standard commercial 16-channel head and neck coil in terms of signal-to-noise ratio (SNR) and parallel imaging performance. In conclusion, the presented hardware enables accurate calculation of attenuation maps for PET/MR systems while improving the SNR of corresponding MR images in a single device without degrading the B0 and B1 homogeneity of the scanner.

Flexible 23-channel coil array for high-resolution magnetic resonance imaging at 3 Tesla.

PURPOSE: The purpose of this work is the design, implementation and evaluation of a mechanically flexible receive-only coil array for magnetic resonance imaging (MRI) at 3 T that can be applied to various target organs and provides high parallel imaging performance. METHODS: A 23-channel array was designed based on a rigid-flex printed circuit board (PCB). The flexible multi-layer part contains the copper traces forming the coil elements. The rigid part of the PCB houses the solder joints and lumped elements. The coil housing consists of rigid caps mounted above the rigid parts. Adhesive PTFE sheets cover all flexible parts. The developed array was tested on the bench as well as in phantom and in vivo MRI experiments employing parallel imaging acceleration factors up to six. RESULTS: Efficient mutual decoupling between receive elements and detuning between receive array and body coil was achieved. An increased signal-to-noise ratio in comparison to commercial reference coils is demonstrated, especially in regions close to the developed array and for high parallel imaging acceleration factors. Exemplary in vivo images of head, ankle, knee, shoulder and hand are presented. CONCLUSION: Based on high sensitivity close to the array and low g-factors, this flexible coil is well suited for studies of occipital and temporal cortex, as well as musculoskeletal targets like knee, ankle, elbow and wrist.

A flexible 12-channel transceiver array of transmission line resonators for 7 T MRI.

A flexible transceiver array based on transmission line resonators (TLRs) combining the advantages of coil arrays with the possibility of form-fitting targeting cardiac MRI at 7 T is presented. The design contains 12 elements which are fabricated on a flexible substrate with rigid PCBs attached on the center of each element to place the interface components, i.e. transmit/receive (T/R) switch, power splitter, pre-amplifier and capacitive tuning/matching circuitry. The mutual coupling between elements is cancelled using a decoupling ring-based technique. The performance of the developed array is evaluated by 3D electromagnetic simulations, bench tests, and MR measurements using phantoms. Efficient inter-element decoupling is demonstrated in flat configuration on a box-shaped phantom (Sij < -19 dB), and bent on a human torso phantom (Sij < -16 dB). Acceleration factors up to 3 can be employed in bent configuration with reasonable g-factors (<1.7) in an ROI at the position of the heart. The array enables geometrical conformity to bodies within a large range of size and shape and is compatible with parallel imaging and parallel transmission techniques.

Proton-decoupled carbon magnetic resonance spectroscopy in human calf muscles at 7 T using a multi-channel radiofrequency coil.

13C magnetic resonance spectroscopy is a viable, non-invasive method to study cell metabolism in skeletal muscles. However, MR sensitivity of 13C is inherently low, which can be overcome by applying a higher static magnetic field strength together with radiofrequency coil arrays instead of single loop coils or large volume coils, and 1H decoupling, which leads to a simplified spectral pattern. 1H-decoupled 13C-MRS requires RF coils which support both, 1H and 13C, Larmor frequencies with sufficient electromagnetic isolation between the pathways of the two frequencies. We present the development, evaluation, and first in vivo measurement with a 7 T 3-channel 13C and 4-channel 1H transceiver array optimized for 1H-decoupled 13C-MRS in the posterior human calf muscles. To ensure minimal cross-coupling between 13C and 1H arrays, several strategies were combined: mutual magnetic flux was minimized by coil geometry, two LCC traps were inserted into each 13C element, and band-pass and low-pass filters were integrated along the signal pathways. The developed coil array was successfully tested in phantom and in vivo MR experiments, showing a simplified spectral pattern and increase in signal-to-noise ratio of approximately a factor 2 between non-decoupled and 1H-decoupled spectra in a glucose phantom and the human calf muscle.

Prevalence and Natural History of Superficial Siderosis: A Population-Based Study.

BACKGROUND AND PURPOSE: Superficial siderosis (SS) is characterized by hemosiderin deposition in the superficial layers of the central nervous system and can be seen during postmortem examination or with iron-sensitive magnetic resonance imaging techniques. The distribution of SS may predict the probable underlying cause. This study aimed to report the prevalence and natural history of SS in a population-based study. METHODS: Brain magnetic resonance imaging scans from the MCSA (Mayo Clinic Study of Aging), a population-based study of residents 50 to 89 years of age in Olmsted County, Minnesota, were reviewed. Participants with imaging consistent with SS were identified from 2011 through 2016. An inverse probability weighting approach was used to convert our observed frequencies to population prevalence of SS. Additional data abstracted included amyloid positron emission tomography, Apolipoprotein E genotype, coexisting cerebral microbleeds, and extent of SS. RESULTS: A total of 1412 participants had eligible magnetic resonance imaging scans. Two participants had infratentorial SS, restricted to the posterior fossa. Thirteen participants had cortical SS involving the cerebral convexities (7 focal and 6 disseminated). Only 3 of the participants with cortical SS (23%) also had cerebral microbleeds. The population prevalence of SS was 0.21% (95% confidence interval, 0-0.45) in those 50 to 69 years old and 1.43% (confidence interval, 0.53-2.34) in those over 69 years old. Apolipoprotein E ε2 allele was more common in those with SS (57.1% versus 15.0%; P<0.001). Compared with participants without SS, those with SS were also more likely to have a positive amyloid positron emission tomographic scan (76.9% versus 29.8%; P<0.001). CONCLUSIONS: SS may be encountered in the general elderly population. The association with increased amyloid burden and Apolipoprotein E ε2 genotype supports cerebral amyloid angiopathy as the most common mechanism. Longitudinal follow-up is needed to evaluate the risk of subsequent hemorrhage in cases of incidentally discovered SS.

RhoA activation and nuclearization marks loss of chondrocyte phenotype in crosstalk with Wnt pathway.

De-differentiation comprises a major drawback for the use of autologous chondrocytes in cartilage repair. Here, we investigate the role of RhoA and canonical Wnt signaling in chondrocyte phenotype. Chondrocyte de-differentiation is accompanied by an upregulation and nuclear localization of RhoA. Effectors of canonical Wnt signaling including ß-catenin and YAP/TAZ are upregulated in de-differentiating chondrocytes in a Rho-dependent manner. Inhibition of Rho activation with C3 transferase inhibits nuclear localization of RhoA, induces expression of chondrogenic markers on 2D and enhances the chondrogenic effect of 3D culturing. Upregulation of chondrogenic markers by Rho inhibition is accompanied by loss of canonical Wnt signaling markers in 3D or on 2D whereas treatment of chondrocytes with Wnt-3a abrogates this effect. However, induction of canonical Wnt signaling inhibits chondrogenic markers on 2D but enhances chondrogenic re-differentiation on 2D with C3 transferase or in 3D. These data provide insights on the context-dependent role of RhoA and Wnt signaling in de-differentiation and on mechanisms to induce chondrogenic markers for therapeutic approaches.

Hypoxia regulates RhoA and Wnt/ß-catenin signaling in a context-dependent way to control re-differentiation of chondrocytes.

Cartilage tissue is avascular and hypoxic which regulates chondrocyte phenotype via stabilization of HIFs. Here, we investigated the role of hypoxia and HIFs in regulation of Rho and canonical Wnt signaling in chondrocytes. Our data demonstrates that hypoxia controls the expression of RhoA in chondrocytes in a context-dependent manner on the culturing conditions. Within a 3D microenvironment, hypoxia suppresses RhoA on which hypoxia-driven expression of chondrogenic markers depends. Conversely, hypoxia leads to upregulation of RhoA in chondrocytes on 2D with a failure in re-expression of chondrogenic markers. Similarly to RhoA, hypoxic regulation of Wnt/ß-catenin signaling depends on the microenvironment. Hypoxia downregulates ß-catenin within 3D hydrogels whereas it causes a potent increase on 2D. Hypoxia-induced suppression of canonical Wnt signaling in 3D contributes to the promotion of chondrogenic phenotype as induction of Wnt signaling abrogates the hypoxic re-differentiation of chondrocytes. Inhibiting Wnt/ß-catenin signaling via stabilization of Axin2 leads to a synergistic enhancement of hypoxia-induced expression of chondrogenic markers. The effects of hypoxia on Rho and Wnt/ß-catenin signaling are HIF-dependent as stabilizing HIFs under normoxia revealed similar effects on chondrocytes. The study reveals important insights on hypoxic signaling of chondrocytes and how hypoxia regulates cellular mechanisms depending on the cellular microenvironment.

A Woman in Her 60s With Chronic Meningitis.

A renal transplant recipient in her 60s presented with a history of chronic headaches and progressive encephalopathy. Serial cerebrospinal fluid examinations were consistent with chronic lymphocytic meningitis, but no definitive cause was identified. Imaging studies showed the development of an enhancing suprasellar mass lesion and hydrocephalus. Symptoms progressed despite empiric antimicrobial therapy, and the patient died after an acute large-volume subarachnoid hemorrhage. A complete autopsy was performed. The differential diagnosis, pathologic findings, and diagnosis are discussed.

Conjunctival biopsy to diagnose neurosarcoidosis in patients with inflammatory nervous system disease of unknown etiology.

Neurosarcoidosis mimics many neurologic diseases and poses a major diagnostic challenge. Blind conjunctival biopsy is often used to help diagnose neurosarcoidosis when biopsy of affected nervous system tissue is not feasible. While this test is relatively inexpensive and well-tolerated, the diagnostic yield in patients with inflammatory nervous system disease of unknown etiology remained uncertain. We evaluated 440 patients who underwent conjunctival biopsy due to concern for neurosarcoidosis. Only a small minority of patients (3%) had positive conjunctival biopsies consistent with sarcoidosis, and some patients (1%) with positive biopsies were found to have a cause for their neurologic disease other than neurosarcoidosis. Many patients (14%) had negative conjunctival biopsies but were later confirmed to have neurosarcoidosis after additional evaluations. This study demonstrates that conjunctival biopsy has a low diagnostic yield for neurosarcoidosis in patients with inflammatory nervous system disease and suggests that alternative diagnostic means should be pursued.