How to inhibit transforming growth factor beta safely in diabetic kidney disease.

PURPOSE OF REVIEW: Diabetic kidney disease (DKD) is a leading cause of mortality and morbidity in diabetes. This review aims to discuss the major features of DKD, to identify the difficult barrier encountered in developing a therapeutic strategy and to provide a potentially superior novel approach to retard DKD. RECENT FINDINGS: Renal inflammation and fibrosis are prominent features of DKD. Transforming growth factor beta (TGFß) with its activity enhanced in DKD plays a key pathological profibrotic role in promoting renal fibrosis. However, TGFß is a difficult drug target because it has multiple important physiological functions, such as immunomodulation. These physiological functions of TGFß can be interrupted as a result of complete blockade of the TGFß pathway if TGFß is directly targeted, leading to catastrophic side-effects, such as fulminant inflammation. Cell division autoantigen 1 (CDA1) is recently identified as an enhancer of profibrotic TGFß signaling and inhibitor of anti-inflammatory SIRT1. Renal CDA1 expression is elevated in human DKD as well as in rodent models of DKD. Targeting CDA1, by either genetic approach or pharmacological approach in mice, leads to concurrent attenuation of renal fibrosis and inflammation without any deleterious effects observed. SUMMARY: Targeting CDA1, instead of directly targeting TGFß, represents a superior approach to retard DKD.

Glucose and Blood Pressure-Dependent Pathways-The Progression of Diabetic Kidney Disease.

The major clinical associations with the progression of diabetic kidney disease (DKD) are glycemic control and systemic hypertension. Recent studies have continued to emphasize vasoactive hormone pathways including aldosterone and endothelin which suggest a key role for vasoconstrictor pathways in promoting renal damage in diabetes. The role of glucose per se remains difficult to define in DKD but appears to involve key intermediates including reactive oxygen species (ROS) and dicarbonyls such as methylglyoxal which activate intracellular pathways to promote fibrosis and inflammation in the kidney. Recent studies have identified a novel molecular interaction between hemodynamic and metabolic pathways which could lead to new treatments for DKD. This should lead to a further improvement in the outlook of DKD building on positive results from RAAS blockade and more recently newer classes of glucose-lowering agents such as SGLT2 inhibitors and GLP1 receptor agonists.

Insertion/deletion-activated frame-shift fluorescence protein is a sensitive reporter for genomic DNA editing.

BACKGROUND: Reporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as "Insertion/deletion-activated frame-shift fluorescence protein". To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency. RESULTS: To test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location. CONCLUSION: The insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.

Annexin A1 is a new functional linker between actin filaments and phagosomes during phagocytosis.

Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.

The UTH-UMB Global Health Education Collaboration: Building a Bidirectional Exchange Based on Equity and Reciprocity.

The global health exchange program between the University Teaching Hospitals (UTH) of Lusaka, Zambia and the University of Maryland, Baltimore (UMB) has been operating since 2015. As trainees and facilitators of this exchange program, we describe our experiences working in Lusaka and Baltimore, and strengths and challenges of the partnership. Since 2015, we have facilitated rotations for 71 UMB trainees, who spent four weeks on the Infectious Disease (ID) team at UTH. Since 2019 with funding from UMB, nine UTH ID trainee physicians spent up to six weeks each rotating on various ID consult services at University of Maryland Medical Center (UMMC). Challenges in global health rotations can include inadequate preparation or inappropriate expectations among high-income country trainees, low-value experiences for low- and middle-income country trainees, lack of appropriate mentorship at sites, and power imbalances in research collaborations. We try to mitigate these issues by ensuring pre-departure and on-site orientation for UMB trainees, cross-cultural mentored experiences for all trainees, and intentional sharing of authorship and credit on scientific collaborations. We present a description of our medical education collaboration as a successful model for building equitable and reciprocal collaborations between low- and middle-income countries and high-income countries, and offer suggestions for future program initiatives to enhance global health education equity among participants and organizations.

The evolutionarily conserved arginyltransferase 1 mediates a pVHL-independent oxygen-sensing pathway in mammalian cells.

The response to oxygen availability is a fundamental process concerning metabolism and survival/death in all mitochondria-containing eukaryotes. However, the known oxygen-sensing mechanism in mammalian cells depends on pVHL, which is only found among metazoans but not in other species. Here, we present an alternative oxygen-sensing pathway regulated by ATE1, an enzyme ubiquitously conserved in eukaryotes that influences protein degradation by posttranslational arginylation. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by preferentially arginylating HIF1α that is hydroxylated by PHD in the presence of oxygen. Furthermore, the degradation of arginylated HIF1α is independent of pVHL E3 ubiquitin ligase but dependent on the UBR family proteins. Bioinformatic analysis of human tumor data reveals that the ATE1/UBR and pVHL pathways jointly regulate oxygen sensing in a transcription-independent manner with different tissue specificities. Phylogenetic analysis suggests that eukaryotic ATE1 likely evolved during mitochondrial domestication, much earlier than pVHL.

Beginning a new medical school curriculum amidst a global pandemic.

The University of Maryland School of Medicine embarked on our first major curriculum revision since 1994 with a plan to implement this Renaissance Curriculum in August 2020. However, in the Spring of 2020, the coronavirus disease (COVID-19) pandemic disrupted clinical care and medical education on a large scale requiring expeditious modifications to our Renaissance Curriculum as well as our traditional Legacy Curriculum in order to meet our goal of educating the next-generation of physicians. The rippling effects of the COVID-19 pandemic led to major changes in the delivery of the pre-clerkship curriculum, the way we assessed and evaluated students, entry into the clinical environment, length of clinical rotations, and orientation for our new medical students. We relied on "new" technology, digital medical resources, and the creativity of our educators to ensure that our learners continue to acquire the skills necessary to become skilled clinicians in these unprecedented times.

Subacute Polymicrobial Bacterial Pericarditis Mimicking Tuberculous Pericarditis: A Case Report.

BACKGROUND Bacterial pericarditis can present a diagnostic challenge due to the difficulty of obtaining tissue for bacterial identification. This report is of a 34-year-old man who presented with fever and cough. Diagnosis was initially delayed without a tissue sample, but the patient was later found to have polymicrobial bacterial pericarditis. CASE REPORT A 34-year-old man from the Democratic Republic of Congo presented to the emergency room with cough, fever, and night sweats. He was admitted and found to have pericardial thickening and fluid collection with calcifications. A tissue sample was not obtained for diagnosis, and he was discharged on RIPE (rifampin, isoniazid, pyrazinamide, and ethambutol) and steroids for presumed tuberculosis pericarditis. He worsened clinically and was readmitted to the hospital with evolving pericardial effusion with air present, in addition to new pleural effusion and parenchymal consolidation. He subsequently underwent thoracotomy and pericardial biopsy. Tissue cultures and sequence-based bacterial analysis eventually revealed the presence of Prevotella oris and Fusobacterium nucleatum. He improved dramatically with appropriate antibiotic therapy. CONCLUSIONS This report demonstrates the importance of undergoing further diagnostic work-up for bacterial pericarditis, especially in resource-rich settings. Although tuberculosis pericarditis should remain high on the differential, it is imperative not to anchor on that diagnosis. Instead, when feasible and safe, tissue biopsy should be obtained and sent for organism identification. AFB smears and cultures, Xpert MTB/RIF, and sequence-based bacterial analysis have all been used for identification. Delay in diagnosis can lead to progression of disease and unnecessary incorrect therapies.

Antibiogram Development in the Setting of a High Frequency of Multi-Drug Resistant Organisms at University Teaching Hospital, Lusaka, Zambia.

Antimicrobial resistance is a global challenge requiring reliable surveillance data collection and use. Prior studies on resistance in Zambia depended on laboratory methods with limited standardization. Since 2015, the University Teaching Hospital (UTH) microbiology laboratory has used the Vitek 2 Compact (bioMerieux, Inc., Marcy-l'Étoile, France) for standardized identification and susceptibility testing. We conducted a cross-sectional study of 2019 bacterial isolates collected from July 2015 to April 2017 to identify bacterial causes of infections, their susceptibility to commonly used antibiotics at UTH, and develop hospital antibiograms with a multidisciplinary team using World Health Organization guidance. We found high levels of antibiotic resistance among Gram negative bacteria. Escherichia coli and Klebsiella pneumoniae were highly resistant to all antibiotics except amikacin and carbapenems. E. coli had susceptibilities of 42.4% to amoxicillin/clavulanic acid, 41.4% to ceftriaxone, 40.2% to ciprofloxacin, and 10.4% to trimethoprim/sulfamethoxazole (TMP/SMX). K. pneumoniae had susceptibilities of 20.7% to amoxicillin/clavulanic acid, 15.6% to ceftriaxone, 48.5% to ciprofloxacin, and 12.3% to TMP/SMX. The high resistance to 3rd generation cephalosporins indicates high rates of beta-lactamase production. This is information that clinicians need to inform clinical decision making and choice of empiric antibiotics and that UTH requires to inform antimicrobial stewardship such as improvements in antibiotic use.

Discoidin domain receptor 1 activation links extracellular matrix to podocyte lipotoxicity in Alport syndrome.

BACKGROUND: Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is activated by collagens that is involved in the pathogenesis of fibrotic disorders. Interestingly, de novo production of the collagen type I (Col I) has been observed in Col4a3 knockout mice, a mouse model of Alport Syndrome (AS mice). Deletion of the DDR1 in AS mice was shown to improve survival and renal function. However, the mechanisms driving DDR1-dependent fibrosis remain largely unknown. METHODS: Podocyte pDDR1 levels, Collagen and cluster of differentiation 36 (CD36) expression was analyzed by Real-time PCR and Western blot. Lipid droplet accumulation and content was determined using Bodipy staining and enzymatic analysis. CD36 and DDR1 interaction was determined by co-immunoprecipitation. Creatinine, BUN, albuminuria, lipid content, and histological and morphological assessment of kidneys harvested from AS mice treated with Ezetimibe and/or Ramipril or vehicle was performed. FINDINGS: We demonstrate that Col I-mediated DDR1 activation induces CD36-mediated podocyte lipotoxic injury. We show that Ezetimibe interferes with the CD36/DDR1 interaction in vitro and prevents lipotoxicity in AS mice thus preserving renal function similarly to ramipril. INTERPRETATION: Our study suggests that Col I/DDR1-mediated lipotoxicity contributes to renal failure in AS and that targeting this pathway may represent a new therapeutic strategy for patients with AS and with chronic kidney diseases (CKD) associated with Col4 mutations. FUNDING: This study is supported by the NIH grants R01DK117599, R01DK104753, R01CA227493, U54DK083912, UM1DK100846, U01DK116101, UL1TR000460 (Miami Clinical Translational Science Institute, National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities), F32DK115109, Hoffmann-La Roche and Alport Syndrome Foundation.

Mycobacterium szulgai pulmonary infection in a woman with anorexia nervosa.

A 40-year-old woman with severe anorexia nervosa was found to have a bilateral pulmonary infection with rare atypical mycobacterium Mycobacterium szulgai. Of note, she had no preexisting structural lung disease or history of tuberculosis, smoking, or HIV. Current data suggest that both impaired cell-mediated immunity and altered respiratory mechanics are risk factors for mycobacterial infection in patients with anorexia nervosa.

Regulation of Mitochondrial Respiratory Chain Complex Levels, Organization, and Function by Arginyltransferase 1.

Arginyltransferase 1 (ATE1) is an evolutionary-conserved eukaryotic protein that localizes to the cytosol and nucleus. It is the only known enzyme in metazoans and fungi that catalyzes posttranslational arginylation. Lack of arginylation has been linked to an array of human disorders, including cancer, by altering the response to stress and the regulation of metabolism and apoptosis. Although mitochondria play relevant roles in these processes in health and disease, a causal relationship between ATE1 activity and mitochondrial biology has yet to be established. Here, we report a phylogenetic analysis that traces the roots of ATE1 to alpha-proteobacteria, the mitochondrion microbial ancestor. We then demonstrate that a small fraction of ATE1 localizes within mitochondria. Furthermore, the absence of ATE1 influences the levels, organization, and function of respiratory chain complexes in mouse cells. Specifically, ATE1-KO mouse embryonic fibroblasts have increased levels of respiratory supercomplexes I+III2+IVn. However, they have decreased mitochondrial respiration owing to severely lowered complex II levels, which leads to accumulation of succinate and downstream metabolic effects. Taken together, our findings establish a novel pathway for mitochondrial function regulation that might explain ATE1-dependent effects in various disease conditions, including cancer and aging, in which metabolic shifts are part of the pathogenic or deleterious underlying mechanism.

Reduced Arginyltransferase 1 is a driver and a potential prognostic indicator of prostate cancer metastasis.

Most prostate cancer cases remain indolent for long periods of time, but metastatic progression quickly worsens the prognosis and leads to mortality. However, little is known about what promotes the metastasis of prostate cancer and there is a lack of effective prognostic indicators, making it immensely difficult to manage options for treatment or surveillance. Arginyltransferase 1 (Ate1) is the enzyme mediating post-translational protein arginylation, which has recently been identified as a master regulator affecting many cancer-relevant pathways including stress response, cell cycle checkpoints, and cell migration/adhesion. However, the precise role of Ate1 in cancer remains unknown. In this study, we found the occurrence of metastasis of prostate cancer is inversely correlated with the levels of Ate1 protein and mRNA in the primary tumor. We also found that metastatic prostate cancer cell lines have a reduced level of Ate1 protein compared to non-metastatic cell lines, and that a depletion of Ate1 drives prostate cancer cells towards more aggressive pro-metastatic phenotypes without affecting proliferation rates. Furthermore, we demonstrated that a reduction of Ate1 can result from chronic stress, and that shRNA-reduced Ate1 increases cellular resistance to stress, and drives spontaneous and stress-induced genomic mutations. Finally, by using a prostate orthotropic xenograft mouse model, we found that a reduction of Ate1 was sufficient to enhance the metastatic phenotypes of prostate cancer cell line PC-3 in vivo. Our study revealed a novel role of Ate1 in suppressing prostate cancer metastasis, which has a profound significance for establishing metastatic indicators for prostate cancer, and for finding potential treatments to prevent its metastasis.

Treatment outcomes after implementation of an adapted WHO protocol for severe sepsis and septic shock in Haiti.

PURPOSE: The World Health Organization (WHO) has developed a simplified algorithm specific to resource-limited settings for the treatment of severe sepsis emphasizing early fluids and antibiotics. However, this protocol's clinical effectiveness is unknown. We describe patient outcomes before and after implementation of an adapted WHO severe sepsis protocol at a community hospital in Haiti. MATERIALS AND METHODS: Using a before-and-after study design, we retrospectively enrolled 99 adult Emergency Department patients with severe sepsis from January through March 2012. After protocol implementation in January 2014, we compared outcomes to 67 patients with severe sepsis retrospectively enrolled from February to April 2014. We defined sepsis according to the WHO's Integrated Management of Adult Illness guidelines and severe sepsis as sepsis plus organ dysfunction. RESULTS: After protocol implementation, quantity of fluid administered increased and the physician's differential diagnoses more often included sepsis. Patients were more likely to have follow-up vital signs taken sooner, a radiograph performed, and a lactic acid tested. There were no improvements in mortality, time to fluids or antimicrobials. CONCLUSIONS: Use of a simplified sepsis protocol based primarily on physiologic parameters allows for substantial improvements in process measures in the care of severely septic patients in a resource-constrained setting.

Sepsis in Haiti: Prevalence, treatment, and outcomes in a Port-au-Prince referral hospital.

PURPOSE: Developing countries carry the greatest burden of sepsis, yet few descriptive data exist from the Western Hemisphere. We conducted a retrospective cohort study to elucidate the presentation, treatment, and outcomes of sepsis at an urban referral hospital in Port-au-Prince, Haiti. MATERIALS AND METHODS: We studied all adult emergency department patient encounters from January through March 2012. We characterized presentation, management, and outcomes using univariable and multivariable analyses. RESULTS: Of 1078 adult patients, 224 (20.8%) had sepsis and 99 (9.2%) had severe sepsis. In-hospital mortality for severe sepsis was 24.2%. Encephalopathy was a predictor of intravenous fluid administration (adjusted odds ratio [OR], 5.63; 95% confidence interval [CI], 1.46-21.76; P=.01), and lower blood pressures predicted shorter time to fluid administration. Increasing temperature and lower blood pressures predicted antibiotic administration. Encephalopathy at presentation (adjusted OR, 6.92; 95% CI, 1.94-24.64; P=.003), oxygen administration (adjusted OR, 15.96; 95% CI, 3.05-83.59; P=.001), and stool microscopy (adjusted OR, 45.84; 95% CI, 1.43-1469.34; P=.03) predicted death in severe sepsis patients. CONCLUSIONS: This is the first descriptive study of sepsis in Haiti. Our findings contribute to the knowledge base of global sepsis and reveal similarities in independent predictors of mortality between high- and low-income countries.

Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response.

Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.

Arginylation regulates purine nucleotide biosynthesis by enhancing the activity of phosphoribosyl pyrophosphate synthase.

Protein arginylation is an emerging post-translational modification that targets a number of metabolic enzymes; however, the mechanisms and downstream effects of this modification are unknown. Here we show that lack of arginylation renders cells vulnerable to purine nucleotide synthesis inhibitors and affects the related glycine and serine biosynthesis pathways. We show that the purine nucleotide biosynthesis enzyme PRPS2 is selectively arginylated, unlike its close homologue PRPS1, and that arginylation of PRPS2 directly facilitates its biological activity. Moreover, selective arginylation of PRPS2 but not PRPS1 is regulated through a coding sequence-dependent mechanism that combines elements of mRNA secondary structure with lysine residues encoded near the N-terminus of PRPS1. This mechanism promotes arginylation-specific degradation of PRPS1 and selective retention of arginylated PRPS2 in vivo. We therefore demonstrate that arginylation affects both the activity and stability of a major metabolic enzyme.

Fever in immunocompromised hosts.

Fever is one of the most common reasons for the emergency department presentation of immunocompromised patients. Their differential diagnosis can be broad and includes rare or unexpected pathogens. Certain infectious causes of fever portend true emergencies; if they are not managed appropriately, rapid progression and death may ensue. This article reviews the diagnosis and management of fevers in patients immunocompromised by human immunodeficiency virus/AIDS, solid-organ and hematopoietic transplants, chemotherapy-induced neutropenia, and tumor necrosis factor-α inhibitors. Prompt recognition of the type of immunosuppression and delineation of possible causes of fever are critical for management of these complex patients.