The enhancer RNA, AANCR, regulates APOE expression in astrocytes and microglia.

Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.

Risk of infections in multiple myeloma. A population-based study on 8672 multiple myeloma patients diagnosed 2008-2021 from the Swedish Myeloma Registry.

In multiple myeloma (MM), advancements in treatments and toxicity management have enhanced survival rates. This, coupled with shifting age demographics in MM, necessitates an updated understanding of infection risks in MM patients compared to the general population. Using Swedish population-based registries, we investigated the incidence of infections in 8,672 Swedish symptomatic MM patients diagnosed 2008-2021 and 34,561 matched controls. Overall, MM patients had a 5-fold risk (hazard ratio (HR) = 5.30; 95%, Confidence Interval = CI 5.14-5.47) of developing any clinically significant infection compared to matched controls. Bacterial infections represented a 5-fold (HR 4.88; CI 4.70-5.07) increased risk, viral and fungal infections 7-fold compared to controls. The 1st year after MM diagnosis the risk of infections compared to controls was 7 -fold (HR 6.95; CI 6.61-7.30) and remained elevated up to 5 years after the myeloma diagnosis. The risk of infection compared to controls remained 5-fold in MM patients with follow-up till 2022. Preceding MM diagnosis, the risk compared to matched controls was significantly increased up to four years before MM diagnosis (HR1.16; CI 1.05-1.28). Among MM patients, 8% had died within 2 months of diagnosis and infection contributed to 32% of all deaths. After 1 year, 20% MM patients had died, and infection-related mortality was 27%. Our data constitute the largest population-based study to date on the risk of infections compared to the normal population in the era of modern MM therapies and confirms that infections still represent a major threat to patients and underscores importance of preventive strategies.

A Possible Mechanism of Laryngohyoid Fractures in Hanging: A Preliminary Observation.

ABSTRACT: Fractures of the hyoid bone, particularly the greater horns, and thyroid cartilage (superior horns) are known to be associated with hanging deaths. Depending on the literature, the frequency of these fractures varies from 0% to 83%. The mechanism underlying these fractures is believed to be direct compression or indirect traction from the ligature. The relationship of these structures with the cervical spine cannot be visualized with traditional internal examination, due to obstruction by surrounding soft tissue. Postmortem computed tomography scan offers an unobscured view of the relationship of the laryngohyoid structures with the cervical spine.We aim to illustrate the phenomenon of displacement of the laryngohyoid structures associated with fractures of the horns. In our case reports, the laryngohyoid structures were displaced, not only superiorly and posteriorly, but also in 2 of the cases, by tilting, when the suspension point was at the posterior or posterolateral aspect of the neck. This displacement had caused the greater horns of the hyoid bone and superior horns of the thyroid cartilage to be approximated against the cervical spine, particularly the transverse processes. We believe that, in these circumstances, the fractures were caused by pressure of the horns of the laryngohyoid structures against the cervical spine.

A polygenic risk score for alcohol-associated cirrhosis among heavy drinkers with European ancestry.

BACKGROUND: Polygenic Risk Scores (PRS) based on results from genome-wide association studies offer the prospect of risk stratification for many common and complex diseases. We developed a PRS for alcohol-associated cirrhosis by comparing single-nucleotide polymorphisms among patients with alcohol-associated cirrhosis (ALC) versus drinkers who did not have evidence of liver fibrosis/cirrhosis. METHODS: Using a data-driven approach, a PRS for ALC was generated using a meta-genome-wide association study of ALC (N=4305) and an independent cohort of heavy drinkers with ALC and without significant liver disease (N=3037). It was validated in 2 additional independent cohorts from the UK Biobank with diagnosed ALC (N=467) and high-risk drinking controls (N=8981) and participants in the Indiana Biobank Liver cohort with alcohol-associated liver disease (N=121) and controls without liver disease (N=3239). RESULTS: A 20-single-nucleotide polymorphisms PRS for ALC (PRSALC) was generated that stratified risk for ALC comparing the top and bottom deciles of PRS in the 2 validation cohorts (ORs: 2.83 [95% CI: 1.82 -4.39] in UK Biobank; 4.40 [1.56 -12.44] in Indiana Biobank Liver cohort). Furthermore, PRSALC improved the prediction of ALC risk when added to the models of clinically known predictors of ALC risk. It also stratified the risk for metabolic dysfunction -associated steatotic liver disease -cirrhosis (3.94 [2.23 -6.95]) in the Indiana Biobank Liver cohort -based exploratory analysis. CONCLUSIONS: PRSALC incorporates 20 single-nucleotide polymorphisms, predicts increased risk for ALC, and improves risk stratification for ALC compared with the models that only include clinical risk factors. This new score has the potential for early detection of heavy drinking patients who are at high risk for ALC.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

Arterio-Ureteral Fistula: A Rare Elusive Cause of Significant Hematuria.

Arterioureteral fistula (AUF) is a direct communication between the ureter and an artery and is a rare cause of catastrophic, life-threatening haematuria. Fistulation may occur between the ureter and the abdominal aorta, common iliac, external and internal iliac, and inferior mesenteric arteries, and is typically observed in patients with a prior history of pelvic radiotherapy, oncological pelvic surgeries, aortoiliac vascular procedures, and pelvic exenteration. There is also an increased frequency of cases amongst patients who have undergone urological diversion surgeries and in those with chronic indwelling ureteric stents requiring repeated exchange. As AUF is so rarely encountered in clinical practice, the urologist may fail to appreciate its presence until late in the patient's presentation; such diagnostic delay is associated with high mortality and thus rapid clinical suspicion and investigative action are necessary. There are sporadic cases of this rare entity mentioned in literature. In this report, we present two cases as well as a review of the literature. A 73-year-old female presented with repeated episodic haematuria for a week in whom the cause of symptoms remained persistently elusive despite repeated imaging and operative approaches. An eventual diagnosis of a secondary right internal iliac-ureteral fistula was ascertained on a subsequent digital subtraction angiography of the renal tract. The fistula was embolised using an endovascular approach. The patient remained stable post emobilisation and was successfully discharged shortly after the procedure. In the second case, a 51-year-old female, presented with hematuria from her ileal conduit for a few days. Initially, the cause of symptoms was thought to be due to ureteric stents. During a change in her stents, brisk bleeding led to further investigation including an iliac angiogram confirming bleeding from the left common iliac artery. She had a covered common iliac artery stent, which successfully controlled her bleeding This report emphasizes the diagnostic difficulty of AUF, outlines the management principles of this rare disease, and aims to increase awareness of this rare yet potentially lethal phenomenon among practitioners of urology and interventional radiology.

Impact of COVID-19 on Traffic Signal Systems: Survey of Agency Interventions and Observed Changes in Pedestrian Activity.

The COVID-19 pandemic, the most significant public health crisis since the 1918-1919 influenza epidemic, is the first such event to occur since the development of modern transportation systems in the twentieth century. Many states across the U.S. imposed lockdowns in early spring 2020, which reduced demand for trips of various types and affected transportation systems. In urban areas, the shift resulted in a reduction in traffic volumes and an increase in bicycling and walking in certain land use contexts. This paper seeks to understand the changes occurring at signalized intersections as a result of the lockdown and the ongoing pandemic, as well as the actions taken in response to these impacts. The results of a survey of agency reactions to COVID-19 with respect to traffic signal operations and changes in pedestrian activity during the spring 2020 lockdown using two case study examples in Utah are presented. First, the effects of placing intersections on pedestrian recall (with signage) to stop pedestrians from pushing the pedestrian button are examined. Next, the changes in pedestrian activity at Utah signalized intersections between the first 6 months of both 2019 and 2020 are analyzed and the impact of land use characteristics is explored. Survey results reveal the importance of using technologies such as adaptive systems and automated traffic signal performance measures to drive decisions. While pedestrian pushbutton actuations decreased in response to the implementation of pedestrian recalls, many pedestrians continued to use the pushbutton. Pedestrian activity changes were also largely driven by surrounding land uses.

Elsberg Syndrome with Mixed Presentation as Meningitis Retention Syndrome: A Pediatric Case Report and Comprehensive Review of the Literature.

Elsberg syndrome is a typically infectious syndrome that may cause acute or subacute bilateral lumbosacral radiculitis and sometimes lower spinal cord myelitis. Patients often present with various neurological symptoms involving the lower extremities, including numbness, weakness, and urinary disturbances such as retention. A 9-year-old girl with no significant past medical history presented with altered mental status, fever, urinary retention, and anuria and was found to have encephalomyelitis. An extensive diagnostic workup led to ruling out possible etiologies until identifying Elsberg syndrome. In this report, we describe a case of Elsberg syndrome caused by West Nile virus (WNV). To the best of our knowledge, this is the first reported case of its kind in the pediatric population. Utilizing PubMed and Web of Science databases, we reviewed the literature to describe the neurogenic control of the urinary system in correlation to a multitude of neurologic pathologies.

New Perspectives on Escherichia coli Signal Peptidase I Substrate Specificity: Investigating Why the TasA Cleavage Site Is Incompatible with LepB Cleavage.

Escherichia coli signal peptidase I (LepB) has been shown to inefficiently cleave secreted proteins with aromatic amino acids at the second position after the signal peptidase cleavage site (P2'). The Bacillus subtilis exported protein TasA contains a phenylalanine at P2', which in B. subtilis is cleaved by a dedicated archaeal-organism-like signal peptidase, SipW. We have previously shown that when the TasA signal peptide is fused to maltose binding protein (MBP) up to the P2' position, the TasA-MBP fusion protein is cleaved very inefficiently by LepB. However, the precise reason why the TasA signal peptide hinders cleavage by LepB is not known. In this study, a set of 11 peptides were designed to mimic the inefficiently cleaved secreted proteins, wild-type TasA and TasA-MBP fusions, to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB were assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modeling of the interaction between TasA signal peptide and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Replacing the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when the TasA-MBP fusion was expressed in E. coli. The importance of this residue to inhibit signal peptide cleavage and the potential to design LepB inhibitors based on the TasA signal peptide are discussed. IMPORTANCE Signal peptidase I is an important drug target, and understanding its substrate is critically important to develop new bacterium-specific drugs. To that end, we have a unique signal peptide that we have shown is refractory to processing by LepB, the essential signal peptidase I in E. coli, but previously has been shown to be processed by a more human-like signal peptidase found in some bacteria. In this study, we demonstrate how the signal peptide can bind but is unable to be processed by LepB, using a variety of methods. This can inform the field on how to better design drugs that can target LepB and understand the differences between bacterial and human-like signal peptidases.

Comparison of Clinical and Functional Outcomes in One versus Two Component Revision for Total Knee Arthroplasty.

BACKGROUND: Revisions of total knee arthroplasties (TKAs) may require revision of one or both tibial and femoral components. Our purpose was to examine the clinical and functional outcomes in 1- versus 2-component TKA revisions. METHODS: We identified 92 1-component (tibial or femoral) revisions at a single center. Our inclusion criteria were isolated revision of the tibial or femoral components with a minimum 2-year follow-up. The included cases were matched 1:2 with a control group of 2-component revisions (tibial and femoral) by age, body mass index, American Society of Anesthesiologists score, and indication for revision. We collected demographics, complications, operative times, any subsequent rerevisions, and functional outcome scores. RESULTS: The median follow-up time for the 1- and 2-component revision groups were 10 years (range, 3 to 17) and 8 years (range, 2 to 18), respectively. The most common complication after rerevision in both groups was stiffness at 9 of 92 (9.8%) and 9 of 170 (5.3%) in the 1- and 2-component groups, respectively (P = .20). The overall complication prevalence in the 1- and 2- component revision groups was similar 20 of 92 (22%) and 35 of 170 (21%), respectively (P = .87). Subsequent rerevisions for any indication were encountered in 12 of 92 (13.0%) of the 1-component and 18 of 170 (11%) in the 2-component groups (P = .69). There was no statistical difference in survivorship or functional outcomes scores between the groups. CONCLUSION: Our results showed that isolated revision of a single TKA component is an acceptable option, with comparable functional outcomes, complications, and survivorships when compared with both-component revision. As such, a 1-component revision should be considered where appropriate.

N-glycolylneuraminic acid as a carbohydrate cancer biomarker.

One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5'-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.

A cross-sectional analysis of Yelp and Google reviews of hospitals in the United States.

Objective: Patient satisfaction is now an important metric in emergency medicine, but the means by which satisfaction is assessed is evolving. We sought to examine hospital ratings on Google and Yelp as compared to those on Medicare's Care Compare (CC) and to determine if certain hospital characteristics are associated with crowdsourced ratings. Methods: We performed a cross-sectional analysis of hospital ratings on Google and Yelp as compared to those on CC using data collected between July 8 and August 2, 2021. For each hospital, we recorded the CC ratings, Yelp ratings, Google ratings, and each hospital's characteristics. Using multivariable linear regression, we assessed for associations between hospital characteristics and crowdsourced ratings. We calculated Spearman's correlation coefficients for CC ratings versus crowdsourced ratings. Results: Among 3000 analyzed hospitals, the median hospital ratings on Yelp and Google were 2.5 stars (interquartile ratio [IQR], 2-3) and 3 stars (IQR, 2.7-3.5), respectively. The median number of Yelp and Google reviews per hospital was 13 and 150, respectively. The correlation coefficients for Yelp and Google ratings with CC's overall star ratings were 0.19 and 0.20, respectively. For Yelp and Google ratings with CC's patient survey ratings, correlation coefficients were 0.26 and 0.22, respectively. On multivariable analysis, critical access hospitals had 0.22 (95% confidence interval [CI], 0.14-0.30) more Google stars and hospitals in the West had 0.12 (95% CI, 0.05-0.18) more Google stars than references standard hospitals. Conclusion: Patients use Google more frequently than Yelp to review hospitals. Median UnS hospital ratings on Yelp and Google are 2.5 and 3 stars, respectively. Crowdsourced reviews weakly correlate with CC ratings. Critical access hospitals and hospitals in the West have higher crowdsourced ratings.

Repurposing Carbamazepine To Treat Gonococcal Infection in Women: Oral Delivery for Control of Epilepsy Generates Therapeutically Effective Levels in Vaginal Secretions.

Neisseria gonorrhoeae has developed resistance to all previous antibiotics used for treatment. This highlights a crucial need for novel antimicrobials to treat gonococcal infections. We previously showed that carbamazepine (Cz), one of the most commonly prescribed antiepileptic drugs, can block the interaction between gonococcal pili and the I-domain region of human complement receptor 3 (CR3)-an interaction that is vital for infection of the female cervix. We also show that Cz can completely clear an established N. gonorrhoeae infection of primary human cervical cells. In this study, we quantified Cz in serum, saliva, and vaginal fluid collected from 16 women who were, or were not, regularly taking Cz. We detected Cz in lower reproductive tract mucosal secretions in the test group (women taking Cz) at potentially therapeutic levels using a competitive ELISA. Furthermore, we found that Cz concentrations present in vaginal fluid from women taking this drug were sufficient to result in a greater than 99% reduction (within 24 h) in the number of viable gonococci recovered from ex vivo, human, primary cervical cell infections. These data provide strong support for the further development of Cz as a novel, host-targeted therapy to treat gonococcal cervicitis.

Serum Neu5Gc biomarkers are elevated in primary cutaneous melanoma.

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.

Diverse Sensory Repertoire of Paralogous Chemoreceptors Tlp2, Tlp3, and Tlp4 in Campylobacter jejuni.

Campylobacter jejuni responds to extracellular stimuli via transducer-like chemoreceptors (Tlps). Here, we describe receptor-ligand interactions of a unique paralogue family of dCache_1 (double Calcium channels and chemotaxis) chemoreceptors: Tlp2, Tlp3, and Tlp4. Phylogenetic analysis revealed that Tlp2, Tlp3, and Tlp4 receptors may have arisen through domain duplications, followed by a divergent evolutionary drift, with Tlp3 emerging more recently, and unexpectedly, responded to glycans, as well as multiple organic and amino acids with overlapping specificities. All three Tlps interacted with five monosaccharides and complex glycans, including Lewis's antigens, P antigens, and fucosyl GM1 ganglioside, indicating a potential role in host-pathogen interactions. Analysis of chemotactic motility of single, double, and triple mutants indicated that these chemoreceptors are likely to work together to balance responses to attractants and repellents to modulate chemotaxis in C. jejuni. Molecular docking experiments, in combination with saturation transfer difference nuclear magnetic resonance spectroscopy and competition surface plasmon resonance analysis, illustrated that the ligand-binding domain of Tlp3 possess one major binding pocket with two overlapping, but distinct binding sites able to interact with multiple ligands. A diverse sensory repertoire could provide C. jejuni with the ability to modulate responses to attractant and repellent signals and allow for adaptation in host-pathogen interactions. IMPORTANCE Campylobacter jejuni responds to extracellular stimuli via transducer-like chemoreceptors (Tlps). This remarkable sensory perception mechanism allows bacteria to sense environmental changes and avoid unfavorable conditions or to maneuver toward nutrient sources and host cells. Here, we describe receptor-ligand interactions of a unique paralogue family of chemoreceptors, Tlp2, Tlp3, and Tlp4, that may have arisen through domain duplications, followed by a divergent evolutionary drift, with Tlp3 emerging more recently. Unlike previous reports of ligands interacting with sensory proteins, Tlp2, Tlp3, and Tlp4 responded to many types of chemical compounds, including simple and complex sugars such as those present on human blood group antigens and gangliosides, indicating a potential role in host-pathogen interactions. Diverse sensory repertoire could provide C. jejuni with the ability to modulate responses to attractant and repellent signals and allow for adaptation in host-pathogen interactions.

A Review of the Multi-Systemic Complications of a Ketogenic Diet in Children and Infants with Epilepsy.

Ketogenic diets (KDs) are highly effective in the treatment of epilepsy. However, numerous complications have been reported. During the initiation phase of the diet, common side effects include vomiting, hypoglycemia, metabolic acidosis and refusal of the diet. While on the diet, the side effects involve the following systems: gastrointestinal, hepatic, cardiovascular, renal, dermatological, hematologic and bone. Many of the common side effects can be tackled easily with careful monitoring including blood counts, liver enzymes, renal function tests, urinalysis, vitamin levels, mineral levels, lipid profiles, and serum carnitine levels. Some rare and serious side effects reported in the literature include pancreatitis, protein-losing enteropathy, prolonged QT interval, cardiomyopathy and changes in the basal ganglia. These serious complications may need more advanced work-up and immediate cessation of the diet. With appropriate monitoring and close follow-up to minimize adverse effects, KDs can be effective for patients with intractable epilepsy.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

CRISPR in Your Kitchen: an At-Home CRISPR Kit to Edit Genes in Saccharomyces cerevisiae Used during a Remote Lab Course.

The use of CRISPR-based experiments in an undergraduate course is appealing because of the ease of editing, and the relevance of CRISPR to current research. Before the COVID-19 pandemic, we developed an in-person lab for a high-enrollment course that allowed students to design and conduct CRISPR editing experiments in budding yeast, Saccharomyces cerevisiae. Post pandemic, the lab course moved online, and we lost the hands-on component. We subsequently developed an at-home kit that contained all the necessary materials for students to grow and transform S. cerevisiae with the DNA molecules necessary for CRISPR-Cas9 induced editing. Our at-home kits cost approximately $70 each to produce and were shipped to over 600 students during the 2020 to 2021 academic year. By adding the at-home experimental work to our remote, online lab course, students were able to generate loss-of-function mutants in ADE2 (causing a red color phenotype). Students were able to send edited yeast samples back to campus for sequencing, allowing for characterization of the different mutations that can occur due to CRISPR-Cas9 induced editing. Here, we described the protocol to produce and use the kits and summarized the student experience of using the at-home kit in a large enrollment, remote lab course. These kits provided opportunities to engage students in hands-on experimentation during a remote course and could also be used to reach learners in other domains, such as high schools and outreach programs.