Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1)

PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5(-/-) mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5(-/-) mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.     

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.     

Focus: Preventive Medicine: Undiagnosed Hypertension and Undiagnosed Type 2 Diabetes among Overweight and Obese Marshallese Participants in a Diabetes Prevention Program

Hypertension and type 2 diabetes (T2D) are major public health issues that disproportionately affect minority communities, including Native Hawaiians and Pacific Islanders (NHPI). Minority communities are also more likely to have undiagnosed hypertension and T2D. Marshallese Pacific Islanders have been shown to have high proportions of diagnosed and undiagnosed hypertension and T2D. Using survey and biometric data collected from 378 overweight/obese Marshallese Pacific Islander adults, this study documents the prevalence of hypertension and T2D, as well as the prevalence of undiagnosed hypertension and T2D. The study also examines associations between undiagnosed hypertension and undiagnosed T2D and age group, sex, health care access (defined by foregone care due to cost and health insurance status), and body mass index (BMI). Among participants with blood pressure readings indicative of hypertension, 68.4% were undiagnosed, and among participants with HbA1c indicative of T2D, 31.6% were undiagnosed. A quarter of participants (24.5%) had blood pressure and HbA1c measures indicative of both undiagnosed hypertension and undiagnosed T2D. Undiagnosed hypertension was significantly associated with age group (p’s<0.0001) and sex (p=0.028). Undiagnosed T2D was significantly associated with age group (p’s<0.05), forgone care due to cost (p=0.018), health insurance status (p=0.035), and BMI (p=0.001). Participants in this study had high proportions of undiagnosed hypertension and undiagnosed T2D. These findings will be immediately useful for those working to address hypertension and T2D disparities among Marshallese and other NHPI populations.     

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.     

Uncovering hidden genetic variation in photosynthesis of field‐grown maize under ozone pollution

Ozone is the most damaging air pollutant to crops, currently reducing Midwest US maize production by up to 10%, yet there has been very little effort to adapt germplasm for ozone tolerance. Ozone enters plants through stomata, reacts to form reactive oxygen species in the apoplast and ultimately decreases photosynthetic C gain. In this study, 10 diverse inbred parents were crossed in a half‐diallel design to create 45 F₁ hybrids, which were tested for ozone response in the field using free air concentration enrichment (FACE). Ozone stress increased the heritability of photosynthetic traits and altered genetic correlations among traits. Hybrids from parents Hp301 and NC338 showed greater sensitivity to ozone stress, and disrupted relationships among photosynthetic traits. The physiological responses underlying sensitivity to ozone differed in hybrids from the two parents, suggesting multiple mechanisms of response to oxidative stress. FACE technology was essential to this evaluation because genetic variation in photosynthesis under elevated ozone was not predictable based on performance at ambient ozone. These findings suggest that selection under elevated ozone is needed to identify deleterious alleles in the world's largest commodity crop.     

Cost-Effectiveness Analysis of Six Strategies to Treat Recurrent Clostridium difficile Infection

Objective

To assess the cost-effectiveness of six treatment strategies for patients diagnosed with recurrent Clostridium difficile infection (CDI) in Canada: 1. oral metronidazole; 2. oral vancomycin; 3.oral fidaxomicin; 4. fecal transplantation by enema; 5. fecal transplantation by nasogastric tube; and 6. fecal transplantation by colonoscopy.

Perspective

Public insurer for all hospital and physician services.

Setting

Ontario, Canada.

Methods

A decision analytic model was used to model costs and lifetime health effects of each strategy for a typical patient experiencing up to three recurrences, over 18 weeks. Recurrence data and utilities were obtained from published sources. Cost data was obtained from published sources and hospitals in Toronto, Canada. The willingness-to-pay threshold was $50,000/QALY gained.

Results

Fecal transplantation by colonoscopy dominated all other strategies in the base case, as it was less costly and more effective than all alternatives. After accounting for uncertainty in all model parameters, there was an 87% probability that fecal transplantation by colonoscopy was the most beneficial strategy. If colonoscopy was not available, fecal transplantation by enema was cost-effective at $1,708 per QALY gained, compared to metronidazole. In addition, fecal transplantation by enema was the preferred strategy if the probability of recurrence following this strategy was below 8.7%. If fecal transplantation by any means was unavailable, fidaxomicin was cost-effective at an additional cost of $25,968 per QALY gained, compared to metronidazole.

Conclusion

Fecal transplantation by colonoscopy (or enema, if colonoscopy is unavailable) is cost-effective for treating recurrent CDI in Canada. Where fecal transplantation is not available, fidaxomicin is also cost-effective.     

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.     

A Golgi-localized Mannosidase (MAN1B1) Plays a Non-enzymatic Gatekeeper Role in Protein Biosynthetic Quality Control

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes.