Angioinvasion is a hallmark of mucormycosis. Previously, we identified endothelial cell glucose-regulated protein 78 (GRP78) as a receptor for Mucorales that mediates host cell invasion. Here we determined that spore coat protein homologs (CotH) of Mucorales act as fungal ligands for GRP78. CotH proteins were widely present in Mucorales and absent from noninvasive pathogens. Heterologous expression of CotH3 and CotH2 in Saccharomyces cerevisiae conferred the ability to invade host cells via binding to GRP78. Homology modeling and computational docking studies indicated structurally compatible interactions between GRP78 and both CotH3 and CotH2. A mutant of Rhizopus oryzae, the most common cause of mucormycosis, with reduced CotH expression was impaired for invading and damaging endothelial cells and CHO cells overexpressing GRP78. This strain also exhibited reduced virulence in a diabetic ketoacidotic (DKA) mouse model of mucormycosis. Treatment with anti-CotH Abs abolished the ability of R. oryzae to invade host cells and protected DKA mice from mucormycosis. The presence of CotH in Mucorales explained the specific susceptibility of DKA patients, who have increased GRP78 levels, to mucormycosis. Together, these data indicate that CotH3 and CotH2 function as invasins that interact with host cell GRP78 to mediate pathogenic host-cell interactions and identify CotH as a promising therapeutic target for mucormycosis.

SummaryRhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

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Candida albicans is the most common cause of invasive fungal infections in humans. It is unclear how C. albicans escapes from phagocytic attack and survives in the hostile blood environment during life-threatening systemic infections. Using a conditional overexpression or suppression genetic strategy, we discovered that HYR1 gene reduced phagocytic killing activity of C. albicans in vitro and increased tissue fungal burden in vivo. Concordant with its positive regulation by the transcription factor Bcr1p, autonomous expression of HYR1 complemented the hypersusceptibility to phagocyte-mediated killing of a bcr1 null mutant of C. albicans in vitro. As for C. albicans, heterologous expression of HYR1 in Candida glabrata rendered the organism more resistant to neutrophil killing activity. Vaccination with a recombinant Hyr1p significantly protected mice against hematogenously disseminated candidiasis (P = .001). Finally, anti-rHyr1p polyclonal antibodies enhanced mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro. Thus, Hyr1 is an important virulence factor for C. albicans, mediating resistance to phagocyte killing. Hyr1p is a promising target for vaccine or other immunological or small molecule intervention to improve the outcomes of disseminated candidiasis.

-3D IC technologies can help to improve circuit performance and lower power consumption by reducing wirelength. Also, 3D IC technology can be used to realize heterogeneous system-on-chip design, by integrating different modules together with less interference with each other. In this paper, we propose a novel thermal-aware 3D cell placement approach, named T3Place, based on transforming a 2D placement with good wirelength to a 3D placement, with the objectives of half-perimeter wirelength, through-the-silicon (TS) via number and temperature. T3Place is composed of two steps, transformation from a 2D placement to a 3D placement and the refinement of the resulting 3D placement. We proposed and compared several different transformation techniques, including local stacking transformation (LST), folding-2, folding-4 and window-based stacking/folding transformation, and concluded that (i) LST can generate 3D placements with the least wirelength, (ii) the folding-based transformations result in 3D placements with the fewest TS vias, and (iii) the window-based stacking/folding transformations provide good TS via number and wirelength tradeoffs. For example, with four device layers, LST can reduce the wirelength by over 2 compared to the initial 2D placement, while window-based stacking/folding can provide over 10 variation in terms of the TS via number, thus adaptive to different manufacturing ability for TS via density. Moreover, we proposed a novel relaxed conflict-net (RCN) graph-based layer assignment method to further refine the 3D placements. Compared to LST results, thermal-aware RCN graph-based layer assignment algorithm (r = 10%) can further reduce the maximum on-chip temperature by 37%, with only 6% TS via number increase and 8% wirelength increase.

e Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections. Mucormycoses are uncommon life-threatening fungal infections caused by fungi of the order Mucorales (1-3). These infections almost uniformly afflict the immunocompromised hosts, those with diabetic ketoacidosis (DKA) or other forms of acidosis, and trauma patients (e.g., victims of the recent natural disasters of the Joplin tornado [4] and the Indian tsunami [5]) (6, 7). Due to the rising prevalence of diabetes, cancer, and organ transplantation in aging populations, as well as the recent increase in natural disasters, the number of patients at risk for this deadly infection is significantly rising and is expected to continue to rise (8-10).Fungi belonging to the order Mucorales are distributed into six families, all of which can cause cutaneous and hematogenously disseminated infections (1, 6). Species belonging to the family Mucoraceae are isolated more frequently from patients with mucormycosis than any other family. Among the Mucoraceae, Rhizopus spp. are the most common cause of mucormycosis and are responsible for approximately 70% of all infections and 90% of rhinocerebral cases (6,11,12). However, recently more cases caused by the previously less frequent species of Lichtheimia (formerly Absidia) corymbifera, Apophysomyces elegans, and Mucor species have been reported (4, 6, 13-17). Increasing numbers of cases of mucormycosis have been also reported due to infection with Cunninghamella spp. (18)(19)(20).Despite disfiguring surgical debridement and adjunctive antifungal therapy, the overall mortality of mucormycosis remains approximately 50%. In the absence of surgical removal of the infected focus, antifungal therapy alone is rarely curative, resulting in a 100% mortality rate for patients with hematogenously disseminated disease (1,9,(21)(22)(23)(24). Clearly, new strategies to prevent and treat mucormycosis are urgently needed. Because of the rarity of the disease, controlled clinical trials are hard to conduct. Consequently...

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