Heme alters biofilm formation in .

(Mabs) is commonly found in the cystic fibrosis (CF) lung. During infection, Mabs can form biofilms in the lung which reduce both the ability of the immune response to clear infection and the effectiveness of antibiotic therapy. In the CF lung, heme and hemoglobin levels are increased and may provide both iron and heme to Mabs cells.

Fluorometric Methods to Measure Bioavailable and Total Heme.

Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme.

Regulation of heme biosynthesis via the coproporphyrin dependent pathway in bacteria.

Heme biosynthesis in the Gram-positive bacteria occurs mostly via a pathway that is distinct from that of eukaryotes and Gram-negative bacteria in the three terminal heme synthesis steps. In many of these bacteria heme is a necessary cofactor that fulfills roles in respiration, gas sensing, and detoxification of reactive oxygen species. These varying roles for heme, the requirement of iron and glutamate, as glutamyl tRNA, for synthesis, and the sharing of intermediates with the synthesis of other porphyrin derivatives necessitates the need for many points of regulation in response to nutrient availability and metabolic state.

The role of host heme in bacterial infection.

Heme is an indispensable cofactor for almost all aerobic life, including the human host and many bacterial pathogens. During infection, heme and hemoproteins are the largest source of bioavailable iron, and pathogens have evolved various heme acquisition pathways to satisfy their need for iron and heme. Many of these pathways are regulated transcriptionally by intracellular iron levels, however, host heme availability and intracellular heme levels have also been found to regulate heme uptake in some species.

Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

Candida albicans is a major fungal pathogen that can utilise hemin and haemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilise external heme as a cellular heme source.

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages.

A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages.

Handling heme: The mechanisms underlying the movement of heme within and between cells.

Heme is an essential cofactor and signaling molecule required for virtually all aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely transported and trafficked from the site of synthesis in the mitochondria or uptake at the cell surface, to hemoproteins in most subcellular compartments.

Heme and hemoglobin suppress amyloid β-mediated inflammatory activation of mouse astrocytes.

Glial immune activity is a key feature of Alzheimer's disease (AD). Given that the blood factors heme and hemoglobin (Hb) are both elevated in AD tissues and have immunomodulatory roles, here we sought to interrogate their roles in modulating β-amyloid (Aβ)-mediated inflammatory activation of astrocytes. We discovered that heme and Hb suppress immune activity of primary mouse astrocytes by reducing expression of several proinflammatory cytokines ( RANTES (regulated on activation normal T cell expressed and secreted)) and the scavenger receptor CD36 and reducing internalization of Aβ(1-42) by astrocytes.

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin.

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues.

Discovery of Molecular Therapeutics for Glaucoma: Challenges, Successes, and Promising Directions.

Glaucoma, a heterogeneous ocular disorder affecting ∼60 million people worldwide, is characterized by painless neurodegeneration of retinal ganglion cells (RGCs), resulting in irreversible vision loss. Available therapies, which decrease the common causal risk factor of elevated intraocular pressure, delay, but cannot prevent, RGC death and blindness. Notably, it is changes in the anterior segment of the eye, particularly in the drainage of aqueous humor fluid, which are believed to bring about changes in pressure.

Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies.

Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H.

Structural basis for misfolding in myocilin-associated glaucoma.

Olfactomedin (OLF) domain-containing proteins play roles in fundamental cellular processes and have been implicated in disorders ranging from glaucoma, cancers and inflammatory bowel disorder, to attention deficit disorder and childhood obesity. We solved crystal structures of the OLF domain of myocilin (myoc-OLF), the best studied such domain to date. Mutations in myoc-OLF are causative in the autosomal dominant inherited form of the prevalent ocular disorder glaucoma.

The glaucoma-associated olfactomedin domain of myocilin forms polymorphic fibrils that are constrained by partial unfolding and peptide sequence.

The glaucoma-associated olfactomedin domain of myocilin (myoc-OLF) is a recent addition to the growing list of disease-associated amyloidogenic proteins. Inherited, disease-causing myocilin variants aggregate intracellularly instead of being secreted to the trabecular meshwork, which is a scenario toxic to trabecular meshwork cells and leads to early onset of ocular hypertension, the major risk factor for glaucoma. Here we systematically structurally and biophysically dissected myoc-OLF to better understand its amyloidogenesis.

The glaucoma-associated olfactomedin domain of myocilin is a novel calcium binding protein.

Myocilin is a protein found in the trabecular meshwork extracellular matrix tissue of the eye that plays a role in regulating intraocular pressure. Both wild-type and certain myocilin variants containing mutations in the olfactomedin (OLF) domain are linked to the optic neuropathy glaucoma. Because calcium ions are important biological cofactors that play numerous roles in extracellular matrix proteins, we examined the calcium binding properties of the myocilin OLF domain (myoc-OLF).