Within the ultraviolet C (UVC) spectrum, the wavelengths in the 200-235 nm range, here named far-UVC, have been shown to effectively inactivate a variety of pathogens. Because of their limited penetration in biological materials, far-UVC wavelengths are anticipated to be minimally damaging to human skin and eyes. The germicidal efficacy of these wavelengths combined with a predicted low health hazard for humans suggests that far-UVC sources could operate continuously in indoor locations to reduce the risk of transmission of airborne diseases among occupants. While it is well-established that exposure to far-UVC light is minimally damaging to skin, concerns remain on the safety of exposed eyes. Scientific bodies overseeing UV radiation protection recommend eye safety limits based on published peer-reviewed data. To support this goal, our previous work used a 3D model of the human cornea to assess the wavelength dependence of corneal damage induced by UVC radiation; unlike relatively longer wavelengths, far-UVC wavelengths induced DNA dimers only in the uppermost layers of the corneal epithelium. Here, similar eye safety studies were extended to excised human corneas. The depth of DNA photodamage into the corneal epithelium was evaluated after exposure of the anterior surface to 50 mJ/cm2 or 100 mJ/cm2 from 222 nm or 254 nm light.

BACKGROUND:Platelet transfusions are increasing with advances in medical care. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. To address this, we developed a novel platelet transfusion model that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detected in clots in vivo. STUDY DESIGN AND METHODS:Platelet units stored in mouse plasma were prepared using a modified platelet rich plasma collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTS:Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and ADP, aggregable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. DISCUSSION:We developed mouse platelets for transfusion analogous to human platelet units using a modified platelet rich plasma collection protocol with maximum storage of 1 day for an "old" unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for more consistent expression compared to plasmid-borne constructs. mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

"Standardized Digital Method for Histological Evaluation of Experimental Acute Lung Injury." American Journal of Respiratory Cell and Molecular Biology, 69(5), pp. 596–598

Aggregation of mutant Huntingtin protein (mHtt) leads to neuronal cell death and human disease. We investigated the effect of inclusion formation on yeast cells. Previous work indicates that mHtt protein moves both in and out of inclusions, potentially undergoing refolding in the inclusion. However, the sustained influx of unfolded protein into an inclusion leads to a dramatic change from a phase-separated body to an irregular, less soluble form at a threshold inclusion size. Altered morphology was associated with a prion-like seeding that accelerated inclusion growth despite loss of soluble cytoplasmic protein. The structural change abolished exchange of material between the inclusion and the cytosol and resulted in early cell death. Affected cells continued to divide occasionally, giving rise to daughters with a similar phenotype. Most newly born cells were able to reverse the prion-like aggregation, restoring both soluble cytoplasmic protein and a normal inclusion structure.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for more consistent expression compared to plasmid-borne constructs. mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Background/Case Studies: Manufacturers of Food and Drug Administration-approved chimeric antigen receptor T-cell (CAR-T) therapies specify apheresis collection parameters (ACP) for harvest of mononuclear cells (MNCs). With the growing number of products, the varying requirements for ACP are increasing apheresis unit operations complexity and decreasing efficiency. Given the lack of ACP standardization, we investigated the rationale for the ACPs requested by the manufacturers. We assessed if ACPs were based on the pivotal study protocols or publications and whether physician authors involved in the publications were transfusion medicine (TM) affiliated. Study Design/Methods: Pivotal study protocols and publications for the 6 approved CAR-T products were reviewed to search for ACP. The standard operating procedure (SOP) provided by each company was reviewed to obtain ACP. ACP were compared for alignment between the SOP and protocols or publications. Study author affiliations were identified through PubMed and verified on department websites or LinkedIn if available. Results/Findings: Study results are summarized in Table 1. All (100%) of the products request specific ACP in the collection SOPs. ACP were stated in study protocols for three of six products (50%). When stated in protocols, ACP matched those requested in collection SOPs. Only one of six (16.7%) products specified target ACP in the study publication and this product, which is the most recently approved, required only a target MNC number in the SOP. The minimum target MNC number requested in the SOPs varied from 1 10 to 5 10. Finally, only one of six (16.7%) study publications had a physician author who was clearly affiliated with a TM department or division, but none were found that were trained in TM. Conclusions: Only 50% of approved CAR-T products had specific ACP in their pivotal study protocols, and these parameters likely guided collection SOPs. Target MNC number may be the only required ACP for CAR-T products, providing an opportunity for standardization. The limited TM physician involvement in the protocols and publications may have contributed to the wide variation of ACPs and increased involvement may accelerate standardization.

Lipid droplets (LDs) have emerged not just as storage sites for lipids but as central regulators of metabolism and organelle quality control. These critical functions are achieved, in part, at membrane contact sites (MCS) between LDs and other organelles. MCS are sites of transfer of cellular constituents to or from LDs for energy mobilization in response to nutrient limitations, as well as LD biogenesis, expansion and autophagy. Here, we describe recent findings on the mechanisms underlying the formation and function of MCS between LDs and mitochondria, ER and lysosomes/vacuoles and the role of the cytoskeleton in promoting LD MCS through its function in LD movement and distribution in response to environmental cues.

Although budding yeast, Saccharomyces cerevisiae, is widely used as a model organism in biological research, studying cell biology in yeast was hindered due to its small size, rounded morphology, and cell wall. However, with improved techniques, researchers can acquire high-resolution images and carry out rapid multidimensional analysis of a yeast cell. As a result, imaging in yeast has emerged as an important tool to study cytoskeletal organization, function, and dynamics. This chapter describes techniques and approaches for visualizing the actin cytoskeleton in live yeast cells.

The processes underlying formation and growth of unfolded protein inclusions are relevant to neurodegenerative diseases but poorly characterized in living cells. In S. cerevisiae , inclusions formed by mutant huntingtin (mHtt) have some characteristics of biomolecular condensates but the physical nature and growth mechanisms of inclusion bodies remain unclear. We have probed the relationship between concentration and inclusion growth in vivo and find that growth of mHtt inclusions in living cells is triggered at a cytoplasmic threshold concentration, while reduction in cytoplasmic mHtt causes inclusions to shrink. The growth rate is consistent with incorporation of new material through collision and coalescence. A small remnant of the inclusion is relatively long-lasting, suggesting that it contains a core that is structurally distinct, and which may serve to nucleate it. These observations support a model in which aggregative particles are incorporated by random collision into a phase-separated condensate composed of a particle-rich mixture.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (StiGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N-6-methyladenosine (m(6)A) RNA modification drives recognition and 3' end processing of SgGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m(6)A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe StiGLT. Direct suppression of m(6)A modification of StiGLT or of m(6)A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m(6)A modification, and m(6)A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

Inclusions of disordered protein are a characteristic feature of most neurodegenerative diseases, including Huntington’s disease. Huntington’s disease is caused by expansion of a polyglutamine tract in the huntingtin protein; mutant huntingtin protein (mHtt) is unstable and accumulates in large intracellular inclusions both in affected individuals and when expressed in eukaryotic cells. Using mHtt-GFP expressed in Saccharomyces cerevisiae , we find that mHtt-GFP inclusions are dynamic, mobile, gel-like structures that concentrate mHtt together with the disaggregase Hsp104. Although inclusions may associate with the vacuolar membrane, the association is reversible and we find that inclusions of mHtt in S. cerevisiae are not taken up by the vacuole or other organelles. Instead, a pulse-chase study using photoconverted mHtt-mEos2 revealed that mHtt is directly and continuously removed from the inclusion body. In addition to mobile inclusions, we also imaged and tracked the movements of small particles of mHtt-GFP and determine that they move randomly. These observations suggest that inclusions may grow through the collision and coalescence of small aggregative particles.

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial-endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome.SIGNIFICANCE STATEMENT Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial-endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.

Mitochondria are highly dynamic organelles that undergo directed movement and anchorage, which in turn are critical for calcium buffering and energy mobilization at specific regions within cells or at sites of contact with other organelles. Physical and functional interactions between mitochondria and other organelles also impact processes, including phospholipid biogenesis and calcium homeostasis. Indeed, mitochondrial motility, localization, and interaction with other organelles are compromised in many neurodegenerative diseases. Here, we describe methods to visualize and carry out quantitative analysis of mitochondrial movement in two genetically-manipulatable, widely-used model systems: Drosophila neurons and the budding yeast, Saccharomyces cerevisiae. We also describe approaches for multi-color imaging in living yeast cells that may be used to visualize colocalization of proteins within mitochondria, as well as interactions of mitochondria with other organelles.

The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair.

Tethers that link mitochondria to other organelles are critical for lipid and calcium transport as well as mitochondrial genome replication and fission of the organelle. Here, we review recent advances in the characterization of interorganellar mitochondrial tethers in the budding yeast, Saccharomyces cerevisiae. We specifically focus on evidence for a role for mitochondrial tethers that anchor mitochondria to specific regions within yeast cells. These tethering events contribute to two processes that are critical for normal replicative lifespan: inheritance of fitter mitochondria by daughter cells, and retention of a small pool of higher-functioning mitochondria in mother cells. Since asymmetric inheritance of mitochondria also occurs in human mammary stem-like cells, it is possible that mechanisms underlying mitochondrial segregation in yeast also operate in other cell types.

End-stage lung disease is the third leading cause of death worldwide, accounting for 400,000 deaths per year in the United States alone. To reduce the morbidity and mortality associated with lung disease, new therapeutic strategies aimed at promoting lung repair and increasing the number of donor lungs available for transplantation are being explored. Because of the extreme complexity of this organ, previous attempts at bioengineering functional lungs from fully decellularized or synthetic scaffolds lacking functional vasculature have been largely unsuccessful. An intact vascular network is critical not only for maintaining the blood-gas barrier and allowing for proper graft function but also for supporting the regenerative cells. We therefore developed an airway-specific approach to removing the pulmonary epithelium, while maintaining the viability and function of the vascular endothelium, using a rat model. The resulting vascularized lung grafts supported the attachment and growth of human adult pulmonary cells and stem cell-derived lung-specified epithelial cells. We propose that de-epithelialization of the lung with preservation of intact vasculature could facilitate cell therapy of pulmonary epithelium and enable bioengineering of functional lungs for transplantation.

Myeloid-biased hematopoietic stem cells (MBHSCs) play critical roles in recovery from injury, but little is known about how they are regulated within the bone marrow niche. Here we describe an auto-/paracrine physiologic circuit that controls quiescence of MB-HSCs and hematopoietic progenitors marked by histidine decarboxylase (Hdc). Committed Hdc(+) myeloid cells lie in close anatomical proximity to MB-HSCs and produce histamine, which activates the H-2 receptor on MB-HSCs to promote their quiescence and self-renewal. Depleting histamine- producing cells enforces cell cycle entry, induces loss of serial transplant capacity, and sensitizes animals to chemotherapeutic injury. Increasing demand for myeloid cells via lipopolysaccharide (LPS) treatment specifically recruits MB-HSCs and progenitors into the cell cycle; cycling MB-HSCs fail to revert into quiescence in the absence of histamine feedback, leading to their depletion, while an H-2 agonist protects MB-HSCs from depletion after sepsis. Thus, histamine couples lineage-specific physiological demands to intrinsically primed MBHSCs to enforce homeostasis.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.