Michaelis-like complex of mouse ketohexokinase isoform C.

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Šresolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the ß-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

UNITED KINGDOM DATABASE STUDY OF INTRAVITREAL DEXAMETHASONE IMPLANT (OZURDEX) FOR MACULAR EDEMA RELATED TO RETINAL VEIN OCCLUSION.

PURPOSE: To study the treatment patterns, visual outcomes and safety profile of intravitreal dexamethasone implant (IDI) used for the treatment of macular edema secondary to retinal vein occlusion. METHODS: Up to 2 years of routinely collected anonymized data within electronic medical record systems were remotely extracted from 16 centers. The outcome measures include visual outcome, number of injections, and safety measures, including the rate of intraocular pressure (IOP) rise, frequency of IOP-lowering medication usage, and cataract surgery rates. RESULTS: The study included 688 eyes (44.4%) with central retinal vein occlusion and 862 eyes (55.6%) with branch retinal vein occlusion; 1,250 eyes (80.6%) were treatment naive and 28% (275/989) had high IOP or were on IOP-lowering medications before IDI use. It was found that 31% (476) of eyes received two injections, and 11.7% (182) and 3.7% (58) of eyes received three and four injections, respectively. The mean baseline Snellen visual acuity improved from 20/125 to 20/40 after the first injection. The probability of cataract surgery was 15% at 24 months. The proportion of eyes with ≥10 mmHg change from baseline was higher in phakic (14.2%) compared with pseudophakic eyes (5.4%, P = 0.004). Three eyes required IOP filtering surgery (0.2%). CONCLUSION: The visual results of IDI in eyes with macular edema secondary to retinal vein occlusion in the real world are comparable to those of clinical trial setting. Increased IOP in eyes with preexisting ocular hypertension or glaucoma can be controlled with additional medical treatment. Intraocular pressure rise with IDI may be more frequent in phakic than in pseudophakic eyes.

Aligning an emergency department hepatitis C and human immunodeficiency virus testing quality improvement initiative with universal screening recommendations.

Objective: The interactions among hepatitis C virus (HCV), human immunodeficiency virus (HIV), and the ongoing injection drug epidemic have created a syndemic that significantly affects the Appalachian region of the United States. The purpose of this work is to describe a successful Kentucky program that aimed to increase HCV and HIV testing for people visiting an urban emergency department (ED) who were screened, diagnosed, and linked to care after diagnosis with special consideration for substance use disorder. Methods: The Plan-Do-Study-Act model for quality improvement was used to create a streamlined process for testing, reporting results, and linking people to care. The program was refined and expanded across 3 phases. Results: Across all phases, a total of 25,685 patients were eligible for testing and did not opt out. Of those, 17,090 had HCV antibody (Ab) testing; 3460 (20.2%) had HCV Ab; 1750 (50.8%) had HCV RNA, and an average of 31% of patients were linked to care within 30 days. The program found 54 new cases of HIV infection. Conclusions: Universal HCV and HIV testing and linkage to care is possible within an ED. In areas affected by the syndemic, EDs may serve as a public health safety net to identify affected individuals and ensure they receive follow-up care. Testing in this center uncovered an exceptionally high prevalence of HCV infection and new HIV case identification.

Pseudohyperkalemia and the Need for Imperative Caution With the Newly Introduced Potent Potassium Binders: Two Cases.

Pseudohyperkalemia was first reported in 1955 by Hartmann and Mellinkoff, as a marked elevation of serum potassium in the absence of clinical evidence of electrolyte imbalance - simultaneous serum potassium exceeds plasma potassium by >0.4 mmol/L. We describe two patients with pseudohyperkalemia who inadvertently received inappropriate potassium binder therapy for weeks to months before the diagnosis of pseudohyperkalemia was entertained and subsequently confirmed. Potassium binders ultimately were promptly discontinued once the diagnosis of pseudohyperkalemia was confirmed. Physicians' attention must be drawn to the availability of the new potent oral potassium binders, patiromer and sodium zirconium cyclosilicate. We strongly advocate for imperative caution with these new binders. Iatrogenic life-threatening hypokalemia remains a real concern and must be avoided. Our patients highlighted the importance of caution in the use of the newer potent potassium binders to mitigate against the causation of iatrogenic hypokalemia. Also as important is the observation that in the same patient, with changing clinical scenarios, a patient might exhibit true hyperkalemia that alternated with pseudohyperkalemia, the first of such a report.

The reliability of saliva for the detection of SARS-CoV-2 in symptomatic and asymptomatic patients: Insights on the diagnostic performance and utility for COVID-19 screening.

Current literature has focused on testing saliva in symptomatic patients, and little information is available regarding saliva performance in asymptomatic severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. We compared paired saliva and nasopharyngeal swabs (NPS) collected from 33 symptomatic and 12 asymptomatic known SARS-CoV-2-positive patients. Saliva had an overall sensitivity of 59%, a specificity of 95%, and a negative predictive value of 98%. Saliva demonstrated higher sensitivity in symptomatic (80%) vs. asymptomatic individuals (36%) (P = 0.006), and in high-risk (symptomatic, febrile and/or with comorbidities) (82%) vs. low-risk (asymptomatic, afebrile, and no comorbidities) (22%) patients (P = 0.0002). Cycle threshold (Ct) values in NPS specimens were higher in saliva-negative vs. saliva-positive cases (P = 0.02 and <0.001). Overall, these findings show that despite saliva's low sensitivity in asymptomatic SARS-CoV-2 infections, it can detect infections with lower Ct values and a potentially higher chance of viral transmission. Additional studies are warranted to fully evaluate saliva as a screening test for coronavirus disease-2019.

Float like a Butterfly, Sting like One Too.

Rash after contact with butterflies has not been previously reported in the medical literature to our knowledge. We describe potentially the first suspected case of a cutaneous reaction to the Compton tortoiseshell butterfly (Nymphalis vaualbum) in a young boy who developed urticaria after the modified hairs of the butterfly embedded within his finger. His urticaria improved through treatment with oral and topical steroids as well as systemic antihistamines. This case report expands the variety of insect species that may cause human disease and should raise awareness for this possible reaction.

Systemic corticosteroid use in UK Uveitis practice: results from the ocular inflammation steroid toxicity risk (OSTRICH) study.

OBJECTIVES: To ascertain adherence to an international consensus target of ≤7.5 mg/day of prednisolone for maintenance systemic corticosteroid (CS) prescribing in uveitis and report the frequency of courses of high-dose systemic CS in the UK. METHODS: We conducted a national, multicentre audit of systemic CS prescribing for uveitis at 11 UK sites between November 2018 and March 2019. High-dose CS was defined as (1) maintenance >7.5 mg prednisolone for >3 consecutive months, or (2) >1 course ≥40 mg oral CS or ≥500 mg intravenous (IV) methylprednisolone in the past 12 months. Case notes of patients exceeding threshold CS doses were reviewed by an independent uveitis specialist and judged as avoidable or not, based upon a scoring matrix. RESULTS: Of 667 eligible patients, 285 (42.7%) were treated with oral or IV CS over the preceding 12 months; 96 (33.7%) of these exceeded the threshold for high-dose CS. Twenty-five percent of prescribing in patients on excess CS was judged avoidable; attributed to either prescribing long-term CS without evidence of consideration of alternative strategies, prescribing error or miscommunication. More patients received immunomodulatory therapy (IMT) in the group treated with CS above threshold than below threshold (p < 0.001) but there was no significant difference in doses of IMT. CONCLUSION: 33% of patients had been prescribed excessive corticosteroid when compared to the reference standard. An analysis of decision-making suggests there may be opportunity to reduce excess CS prescribing in 25% of these patients.

Oncogenic N-Ras Mitigates Oxidative Stress-Induced Apoptosis of Hematopoietic Stem Cells.

Leukemic relapse is believed to be driven by transformed hematopoietic stem cells (HSC) that harbor oncogenic mutations or have lost tumor suppressor function. Recent comprehensive sequencing studies have shown that mutations predicted to activate Ras signaling are highly prevalent in hematologic malignancies and, notably, in refractory and relapsed cases. To better understand what drives this clinical phenomenon, we expressed oncogenic NrasG12D within the hematopoietic system in mice and interrogated its effects on HSC survival. N-RasG12D conferred a survival benefit to HSCs and progenitors following metabolic and genotoxic stress. This effect was limited to HSCs and early progenitors and was independent of autophagy and cell proliferation. N-RasG12D-mediated HSC survival was not affected by inhibition of canonical Ras effectors such as MEK and PI3K. However, inhibition of the noncanonical Ras effector pathway protein kinase C (PKC) ameliorated the protective effects of N-RasG12D. Mechanistically, N-RasG12D lowered levels of reactive oxygen species (ROS), which correlated with reduced mitochondrial membrane potential and ATP levels. Inhibition of PKC restored the levels of ROS to that of control HSCs and abrogated the protective effects granted by N-RasG12D. Thus, N-RasG12D activation within HSCs promotes cell survival through the mitigation of ROS, and targeting this mechanism may represent a viable strategy to induce apoptosis during malignant transformation of HSCs. SIGNIFICANCE: Targeting oncogenic N-Ras-mediated reduction of ROS in hematopoietic stem cells through inhibition of the noncanonical Ras effector PKC may serve as a novel strategy for treatment of leukemia and other Ras-mutated cancers.

COVID-19 and emergency department volume: The patients return but have different characteristics.

BACKGROUND: The COVID-19 pandemic has altered behaviors in the general population, as well as processes in the healthcare industry. Patients may be afraid to pursue care in the emergency department (ED) due to perceived risk of infection. The objective of this study was to determine the impact of COVID-19 on ED metrics. METHODS: At one metropolitan trauma center ED, we conducted a review of all visits from February to May in 2020 and compared findings with the same months from 2019. RESULTS: A total of 34,213 ED visits occurred during the study periods (18,471 in 2019 and 15,742 in 2020), with a decline in patient visits occurring after state emergency declarations. In 2020, patients were less likely to be female and more likely to arrive by ambulance. Diagnoses in the musculoskeletal, neurologic, and genitourinary categories occurred in lower proportions in 2020; toxicology, psychiatry, and infectious diseases occurred in higher proportions. In contrast to other insurance categories, Medicare patients comprised a larger share of ED visits in 2020 compared to 2019. DISCUSSION: Despite relatively low local prevalence of COVID-19, we report decreases in ED volume for some medical diagnosis categories. A volume rebound occurred in May 2020, but did not reach 2019 levels. Public health officials should encourage local populations to seek emergency care when concerned, and could consider programs to provide transportation. Patients should continue to protect themselves with social distancing and masks.

Pak1ip1 Loss-of-Function Leads to Cell Cycle Arrest, Loss of Neural Crest Cells, and Craniofacial Abnormalities.

Neural crest cells (NCCs) comprise a transient progenitor cell population of neuroepithelial origin that contributes to a variety of cell types throughout vertebrate embryos including most mesenchymal cells of the cranial and facial structures. Consequently, abnormal NCC development underlies a variety of craniofacial defects including orofacial clefts, which constitute some of the most common birth defects. We previously reported the generation of manta ray (mray) mice that carry a loss-of-function allele of the gene encoding the preribosomal factor Pak1ip1. Here we describe cranioskeletal abnormalities in homozygous mray mutants that arise from a loss of NCCs after their specification. Our results show that the localized loss of cranial NCCs in the developing frontonasal prominences is caused by cell cycle arrest and cell death. In addition, and consistent with deficits in ribosome biosynthesis, homozygous mray mutants display decreased protein biosynthesis, further linking Pak1ip1 to a role in ribosome biogenesis.

Patients with Abrasion or Ecchymosis Seat Belt Sign Have High Risk for Abdominal Injury, but Initial Computed Tomography is 100% Sensitive.

BACKGROUND: Trauma providers seek to accurately assess the risk of patients with abdominal seat belt sign (ASBS). As hospital costs continue to rise, identification of strategies to safely discharge emergency department (ED) patients has become crucial. OBJECTIVES: The purpose of this study is to 1) describe a large cohort of patients by type of ASBS and 2) determine the value of computed tomography (CT) of the abdomen and pelvis as a screening tool to rule out intra-abdominal injury (IAI) and support discharge of stable patients. METHODS: We conducted a retrospective case series of all patients presenting to our urban, Level I trauma center from 2013-2015. We studied motor vehicle collision patients who presented with ASBS. We further classified individuals into ASBS groups: Abrasion, Ecchymosis, Abrasion + Ecchymosis, or Unknown ASBS to examine differences between groups. RESULTS: In one of the largest described cohorts, the ASBS remained associated with IAI, most commonly, solid organ injury. Of 425 patients, 36.1% had some IAI on CT, but only 13.6% required laparotomy. Categorizing the type of skin injury in ASBS, we found that both abrasion and ecchymosis were associated with IAI. Initial CT performed with 100% sensitivity. CONCLUSIONS: This study shows that ED trauma patients with significant seat belt abrasion or contusion can have IAI. With the very high sensitivity of modern abdominal CT scanners, clinicians could consider safe ED discharge of stable ASBS patients while providing strong return precautions. Our large cohort strengthens the evidence on decision-making in ASBS patients to ensure outcomes and use of health care resources.

Recommendations by a UK expert panel on an aflibercept treat-and-extend pathway for the treatment of neovascular age-related macular degeneration.

OBJECTIVES: This report aims to provide clear recommendations and practical guidance from a panel of UK retinal experts on an aflibercept treat-and-extend (T&E) pathway that can be implemented in clinical practice. These recommendations may help service providers across the NHS intending to implement a T&E approach, with the aim of effectively addressing the capacity and resource issues putting strain on UK neovascular age-related macular degeneration (nAMD) services while promoting patients' best interests throughout. METHODS: Two structured roundtable meetings of retinal specialists were held in London, UK on 7 December 2018 and 1 March 2019. These meetings were organised and funded by Bayer. RESULTS: The panel provided recommendations for an aflibercept T&E pathway and developed specific criteria based on visual acuity, retinal morphology and optical coherence tomography imaging to guide reduction, maintenance and extension of injection intervals. They also discussed the extension of treatment intervals by 2- or 4-week adjustments to a maximum treatment interval of 16 weeks, the management of retinal fluid and the stopping of treatment. CONCLUSIONS: The long-term benefits of implementing a T&E pathway may include superior visual outcomes compared with a pro re nata (PRN; as needed) protocol, and a lower treatment burden compared with a fixed protocol, which is likely to improve service capacity. Furthermore, the predictable nature of a T&E approach compared with a PRN service may aid capacity planning for the future nAMD treatment demand.

Action on neovascular age-related macular degeneration (nAMD): recommendations for management and service provision in the UK hospital eye service.

This report by a group of UK retina specialists and health professionals considers best practice recommendations for the management of sight-threatening neovascular age-related macular degeneration (nAMD), based on collective experience and expertise in routine clinical practice. The authors provide an update for ophthalmologists, allied healthcare professionals and commissioners on practice principles for optimal patient care and service provision standards. Refinement of care pathways for nAMD has improved access to intravitreal anti-vascular endothelial growth factor therapy but there are still variations in care and reported outcomes between clinic centres. Innovative organisational models of service provision allow providers to better match capacity with increasing demand. The authors review the recent NICE guideline for diagnosis and management of AMD, considerations for switching therapies and stopping treatment and need for regular monitoring of non-affected fellow eyes in patients with unilateral nAMD. Actions for delivery of high-quality care and to improve long-term patient outcomes are discussed. Local pathways need to detail nAMD target time to treat, maintenance of review intervals to ensure proactive treatment regimens are delivered on time and appropriate discharge for patients deemed low risk or no longer benefiting from treatment. Actual visual acuity outcomes achieved and maintenance of the level of vision when disease stability is achieved are considered good measures for judging the quality of care in the treatment of patients with nAMD. Robust community referral pathways must be in place for suspected reactivation of choroidal neovascularisation and rapid referral for second eye involvement. Practical considerations for intravitreal injection therapy are outlined.

Assessment of Physician Well-being, Part One: Burnout and Other Negative States.

Physician well-being is a complex and multifactorial issue. A large number of tools have been developed in an attempt to measure the nature, severity, and impact of both burnout and well-being in a range of clinical populations. This two-article series provides a review of relevant tools and offers guidance to clinical mentors and researchers in choosing the appropriate instrument to suit their needs, whether assessing mentees or testing interventions in the research setting. Part One begins with a discussion of burnout and focuses on assessment tools to measure burnout and other negative states. Part Two of the series examines the assessment of well-being, coping skills, and other positive states.

A review and update on the ophthalmic implications of Susac syndrome.

Susac syndrome is a rare condition presumed to be immune-mediated occlusion of small arterial vasculature principally of the brain, inner ear, and retina. Clinically, the syndrome manifests as a pathognomonic triad of encephalopathy, hearing loss, and branch retinal artery occlusion. Early recognition and diagnosis is important as delayed treatment may be profound and result in deafness, blindness, dementia, and other neurological deficits. The plethora of imaging technology, including magnetic resonance imaging, retinal fluorescein angiography, optical coherence tomography, and optical coherence tomography angiography, allows deeper and more discrete anatomical-physiological correlation of underlying pathology, early diagnosis, and imaging biomarkers for early detection of relapse during follow-up. We highlight the current clinical classification of Susac syndrome, available investigations, treatment, and care pathways.

Sestd1 Encodes a Developmentally Dynamic Synapse Protein That Complexes With BCR Rac1-GAP to Regulate Forebrain Dendrite, Spine and Synapse Formation.

SEC14 and Spectrin domain-1 (Sestd1) is a synapse protein that exhibits a striking shift from the presynaptic to postsynaptic space as neurons mature postnatally in the mouse hippocampus. Hippocampal pyramidal neurons from mice with global genetic deletion of Sestd1 have reduced dendrite arbors, spines, and excitatory synapses. Electrophysiologically this correlates with cell-autonomous reductions in both AMPA- and NMDA-excitatory postsynaptic currents in individual hippocampal neurons from which Sestd1 has been deleted in vivo. These neurodevelopmental and functional deficits are associated with increased activation of the Rho family GTPases Rac1 and RhoA. Co-immunoprecipitation and mass spectrometry reveal that the Breakpoint Cluster Region protein, a Rho GTPase activating protein (GAP), forms complexes with Sestd1 in brain tissue. This complements earlier findings that Sestd1 can also partner with other Rho family GAPs and guanine nucleotide exchange factors. Our findings demonstrate that Sestd1 is a developmentally dynamic synaptic regulator of Rho GTPases that contributes to dendrite and excitatory synapse formation within differentiating pyramidal neurons of the forebrain.

A non-canonical autophagy-dependent role of the ATG16L1T300A variant in urothelial vesicular trafficking and uropathogenic Escherichia coli persistence.

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.