Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.

Mitochondrial point mutations do not limit the natural lifespan of mice.

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single-base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice.

Mitochondrial DNA polymerase editing mutation, PolgD257A, reduces the diabetic phenotype of Akita male mice by suppressing appetite.

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice.

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.

A stepwise illustration of the retrosigmoid approach for resection of a cerebellopontine meningioma.

The retrosigmoid approach is a work-horse approach to the cerebellopontine angle (CPA), providing access from the foremen magnum to the tentorium. Indications for this approach are variable such as resection of meningiomas, acoustic neuromas and epidermoid tumors, treatment of vascular lesions of vertebrobasilar system, vascular decompression of cranial nerves (V, VII, IX, X), cranial nerve neurectomies, and intrinsic lesions of the cerebellum and brainstem. In this video, we demonstrate the use of retrosigmoid craniotomy for resection of a large CPA meningioma, delineating all steps including positioning, mapping. The video can be found here: http://youtu.be/kISkYS16Brk .

Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption.

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

The role of mitochondrial DNA mutations in mammalian aging.

Mitochondrial DNA (mtDNA) accumulates both base-substitution mutations and deletions with aging in several tissues in mammals. Here, we examine the evidence supporting a causative role for mtDNA mutations in mammalian aging. We describe and compare human diseases and mouse models associated with mitochondrial genome instability. We also discuss potential mechanisms for the generation of these mutations and the means by which they may mediate their pathological consequences. Strategies for slowing the accumulation and attenuating the effects of mtDNA mutations are discussed.

Gene Editing in Dimorphic Fungi Using CRISPR/Cas9.

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.

Prematurely aging mitochondrial DNA mutator mice display subchondral osteopenia and chondrocyte hypertrophy without further osteoarthritis features.

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.

Establishment of Quantitative PCR Assays for Active Long Interspersed Nuclear Element-1 Subfamilies in Mice and Applications to the Analysis of Aging-Associated Retrotransposition.

The retrotransposon long interspersed nuclear element-1 (LINE-1) can autonomously increase its copy number within a host genome through the retrotransposition process. LINE-1 is active in the germline and in neural progenitor cells, and its somatic retrotransposition activity has a broad impact on neural development and susceptibility to neuropsychiatric disorders. The method to quantify the genomic copy number of LINE-1 would be important in unraveling the role of retrotransposition, especially in the brain. However, because of the species-specific evolution of LINE-1 sequences, methods for quantifying the copy number should be independently developed. Here, we developed a quantitative PCR (qPCR) assay to measure the copy number of active LINE-1 subfamilies in mice. Using the assay, we investigated aging-associated alterations of LINE-1 copy number in several brain regions in wild-type mice and Polg+/D257A mice as a model for accelerated aging. We found that aged Polg+/D257A mice showed higher levels of the type GfII LINE-1 in the basal ganglia than the wild-type mice did, highlighting the importance of assays that focus on an individual active LINE-1 subfamily.

Antigen discovery unveils resident memory and migratory cell roles in antifungal resistance.

Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue-resident memory (Trm) CD4+ T cells in the lung parenchyma. In contrast to expectations, CD69+, CXCR3+, CD103- Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer+ CD4+ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Operating microscopes: past, present, and future.

The operating microscope is a fixture of modern surgical facilities, and it is a critically important factor in the success of many of the most complex and difficult surgical interventions used in medicine today. The rise of this key surgical tool reflects advances in understanding the principles of optics and vision that have occurred over centuries. The development of reading spectacles in the late 13th century led to the construction of early compound microscopes in the 16th and 17th centuries by Lippershey, Janssen, Galileo, Hooke, and others. Perhaps surprisingly, Leeuwenhoek's simple microscopes of this era offered improved performance over his contemporaries' designs. The intervening years saw improvements that reduced the spherical and chromatic aberrations present in compound microscopes. By the late 19th century, Carl Zeiss and Ernst Abbe ushered the compound microscope into the beginnings of the modern era of commercial design and production. The introduction of the microscope into the operating room by Nylén in 1921 initiated a revolution in surgical practice that gained momentum throughout the 1950s with multiple refinements, the introduction of the Zeiss OPMI series, and Kurze's application of the microscope to neurosurgery in 1957. Many of the refinements of the last 50 years have greatly improved the handling and practical operation of the surgical microscope, considerations which are equally important to its optical performance. Today's sophisticated operating microscopes allow for advanced real-time angiographic and tumor imaging. In this paper the authors discuss what might be found in the operating rooms of tomorrow.

Evolving insight into the role of mitochondrial DNA mutations in aging.

Mitochondria have occupied a central place in theories on the underlying cellular mechanisms of eukaryotic aging for several decades and much debate has ensued regarding the role of oxidative stress and mitochondrial genomic damage in these processes. Mouse models with greatly enhanced mitochondrial mutagenesis have produced dramatic aging-like phenotypes but recent results have led some to reassess whether such models are relevant to naturally occurring aging mechanisms. Here, we discuss the evolving insight that may be gained from these models regarding the contribution of mitochondrial DNA mutations to aging.

Mitochondrial DNA mutations and apoptosis in mammalian aging.

Mutations in mitochondrial DNA (mtDNA) accumulate during aging, but their significance to longevity and age-associated disease has been uncertain. Recently, in support of the hypothesis that mtDNA integrity is important, we have shown that age-associated diseases arise more rapidly in mice where mtDNA mutations and increased levels of apoptosis occur at higher rates than normal due to expression of an error-prone mtDNA polymerase. Further studies in this model may provide deeper insights into the relationship between mitochondria, aging, and susceptibility to age-associated diseases, such as cancer.

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis.

Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivoIMPORTANCEBlastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.

Evaluation of sex differences on mitochondrial bioenergetics and apoptosis in mice.

It has been postulated that the differences in longevity observed between organisms of different sexes within a species can be attributed to differences in oxidative stress. It is generally accepted that differences are due to the higher female estrogen levels. However, in some species males live the same or longer despite their lower estrogen values. Therefore, in the present study, we analyze key parameters of mitochondrial bioenergetics, oxidative stress and apoptosis in the B6 (C57Bl/6J) mouse strain. There are no differences in longevity between males and females in this mouse strain, although estrogen levels are higher in females. We did not find any differences in heart, skeletal muscle and liver mitochondrial oxygen consumption (State 3 and State 4) and ATP content between male and female mice. Moreover, mitochondrial H(2)O(2) generation and oxidative stress levels determined by cytosolic protein carbonyls and concentration of 8-hydroxy-2'-deoxyguanosine in mitochondrial DNA were similar in both sexes. In addition, markers of apoptosis (caspase-3, caspase-9 and mono- and oligonucleosomes: the apoptosis index) were not different between male and female mice. These data show that there are no differences in mitochondrial bioenergetics, oxidative stress and apoptosis due to gender in this mouse strain according with the lack of differences in longevity. These results support the Mitochondrial Free Radical Theory of Aging, and indicate that oxidative stress generation independent of estrogen levels determines aging rate.

Effects of calorie restriction on the lifespan and healthspan of POLG mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.

Monitoring Ischemic Cerebral Injury in Spontaneously Hypertensive Rats by Diffusion Tensor Imaging.

AIM: We have applied diffusion tensor imaging (DTI) to interrogate microstructural changes in white matter integrity in a widely used middle cerebral artery occlusion (MCAO) model of cerebral ischemia. MATERIAL AND METHODS: We performed ex vivo DTI 35 days after 60 minutes transient focal ischemia in male spontaneously hypertensive rats and generated fractional anisotropy (FA), mean, axial and radial diffusivity maps. Regions of interest corresponding to external capsule (EC), corpus callosum (CC) and internal capsule (IC) were compared among sham and stroked rats. We compared tractographic projections of white matter fiber patterns and examined white matter integrity by Luxol fast blue histological analysis. We also determined infarct lesion volumes at 24 hours post-ischemia by T2-weighted magnetic resonance imaging (MRI) or at 35 days by histological staining with cresyl violet. RESULTS: We found alterations in EC and IC, but not CC, as represented by decreased FA and increased mean, axial and radial diffusivities. The size of the ischemic lesion detected subacutely by T2-weighted MRI or at 35 days by histological staining correlated with the decline in FA in the affected structures. Tractography revealed disruption of fiber trajectories through the EC and reorientation of fibers within the caudate/putamen of rats subjected to MCAO. Similarly, loss of white matter integrity in the EC and increased white matter density in the caudate/putamen along the infarct border zone was evidenced by Luxol fast blue staining. CONCLUSION: Diffusion tensor imaging therefore allows for monitoring of white matter injury and reorganization in hypertensive rats.