LPA5-Dependent signaling regulates regeneration of the intestinal epithelium following irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.NEW & NOTEWORTHY We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

Diving into CDC pregnancy data in the United States: longitudinal study and interactive application.

Objective: Preterm birth (PTB) is a major determinant of neonatal mortality, morbidity, and childhood disability. In this article, we present a longitudinal analysis of the risk factors associated with PTB and how they have varied over the years: starting from 1968 when the CDC first started, reporting the natality data, up until 2021. Along with this article, we are also releasing an RShiny web application that will allow for easy consumption of this voluminous dataset by the research community. Further, we hope this tool can aid clinicians in the understanding and prevention of PTB. Materials and Methods: This study used the CDC Natality data from 1968 to 2021 to analyze trends in PTB outcomes across the lens of various features, including race, maternal age, education, and interval length between pregnancies. Our interactive RShiny web application, CDC NatView, allows users to explore interactions between maternal risk factors and maternal morbidity conditions and the aforementioned features. Results: Our study demonstrates how CDC data can be leveraged to conduct a longitudinal analysis of natality trends in the United States. Our key findings reveal an upward trend in late PTBs, which is concerning. Moreover, a significant disparity exists between African American and White populations in terms of PTB. These disparities persist in other areas, such as education, body-mass index, and access to prenatal care later in pregnancy. Discussion: Another notable finding is the increase in maternal age over time. Additionally, we confirm that short interpregnancy intervals (IPIs) are a risk factor for PTBs. To facilitate the exploration of pregnancy risk factors, infections, and maternal morbidity, we developed an open-source RShiny tool called CDC NatView. This software offers a user-friendly interface to interact with and visualize the CDC natality data, which constitutes an invaluable resource. Conclusion: In conclusion, our study has shed light on the rise of late PTBs and the persistent disparities in PTB rates between African American and White populations in the US. The increase in maternal age and the confirmation of a short IPI as a risk factor for PTB are noteworthy findings. Our open-source tool, CDC NatView, can be a valuable resource for further exploration of the CDC natality data to enhance our understanding of pregnancy risk factors and the interaction of PTB outcomes and maternal morbidities.

Regulation of the intestinal Na+/H+ exchanger NHE3 by AMP-activated kinase is dependent on phosphorylation of NHE3 at S555 and S563.

Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.

Cryptococcus neoformans requires the TVF1 gene for thermotolerance and virulence.

Cryptococcus neoformans is the primary causative agent of cryptococcosis. Since C. neoformans thrives in environments and its optimal growth temperature is 25-30°C, it needs to adapt to heat stress in order to cause infection in mammalian hosts. In this study, we aimed to investigate the role of an uncharacterized gene, CNAG_03308. Although the CNAG_03308 deletion strain grew as well as the parent strain KN99, it produced yeast cells with abnormal morphology at 37°C and failed to propagate at 39°C. Furthermore, the deletion strain exhibited slower growth at 37°C in the presence of congo red, which is a cell wall stressor. When cultured at 39°C, the deletion strain showed strong staining with fluorescent probes for cell wall chitin and chitosan, including FITC-labeled wheat germ agglutinin, Eosin Y, and calcofluor white. The transmission electron microscopy of the deletion strain revealed a thickened inner layer of the cell wall containing chitin and chitosan under heat stress. This cell-surface altered deletion strain induced dendritic cells to secrete more interleukin (IL)-6 and IL-23 than the control strains under heat stress. In a murine infection study, C57BL/6 mice infected with the deletion strain exhibited lower mortality and lower fungal burden in the lungs and brain compared to those infected with the control strains. Based on these findings, we concluded that CNAG_03308 gene is necessary for C. neoformans to adapt to heat stress both in vitro and in the host environment. Therefore, we designated the CNAG_03308 gene as TVF1, which stands for thermotolerance and virulence-related factor 1.

Cryptococcus neoformans is a fungal pathogen causing cryptococcosis, which requires thermotolerance to proliferate in the host environment. In the present study, we identified a novel gene, TVF1 (CNAG_03308), required for thermotolerance and virulence by reverse genetics approach.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Refining the serum miR-371a-3p test for viable germ cell tumor detection.

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (GCT) pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28-35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to (a) utilize threshold-based approaches using raw Cq values, (b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and (c) to re-run any sample with an indeterminate result.

X-linked hydrocephalus genes: Their proximity to telomeres and high A + T content compared to Parkinson's disease.

Proximity to telomeres (i) and high adenine and thymine (A + T) content (ii) are two factors associated with high mutation rates in human chromosomes. We have previously shown that >100 human genes when mutated to cause congenital hydrocephalus (CH) meet either factor (i) or (ii) at 91% matching, while two factors are poorly satisfied in human genes associated with familial Parkinson's disease (fPD) at 59%. Using the sets of mouse, rat, and human chromosomes, we found that 7 genes associated with CH were located on the X chromosome of mice, rats, and humans. However, genes associated with fPD were in different autosomes depending on species. While the contribution of proximity to telomeres in the autosome was comparable in CH and fPD, high A + T content played a pivotal contribution in X-linked CH (43% in all three species) than in fPD (6% in rodents or 13% in humans). Low A + T content found in fPD cases suggests that PARK family genes harbor roughly 3 times higher chances of methylations in CpG sites or epigenetic changes than X-linked genes.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

MDA5 signaling induces type 1 IFN- and IL-1-dependent lung vascular permeability which protects mice from opportunistic fungal infection.

Lungs balance threat from primary viral infection, secondary infection, and inflammatory damage. Severe pulmonary inflammation induces vascular permeability, edema, and organ dysfunction. We previously demonstrated that poly(I:C) (pICLC) induced type 1 interferon (t1IFN) protected mice from Cryptococcus gattii (Cg) via local iron restriction. Here we show pICLC increased serum protein and intravenously injected FITC-dextran in the lung airspace suggesting pICLC induces vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target.

Compensatory Upregulation of LPA2 and Activation of the PI3K-Akt Pathway Prevent LPA5-Dependent Loss of Intestinal Epithelial Cells in Intestinal Organoids.

Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of Lpar5 (Lpar5iKO) in mice ablates the intestinal epithelium and increases morbidity. In contrast, constitutive Lpar5 deletion (Lpar5cKO) does not cause a defect in intestinal crypt regeneration. In this study, we investigated whether another LPA receptor (LPAR) compensates for constitutive loss of LPA5 function to allow regeneration of intestinal epithelium. In Lpar5cKO intestinal epithelial cells (IECs), Lpar2 was upregulated and blocking LPA2 function reduced proliferation and increased apoptosis of Lpar5cKO IECs. Similar to Lpar5cKO mice, the absence of Lpar2 (Lpar2-/-) resulted in upregulation of Lpar5 in IECs, indicating that LPA2 and LPA5 reciprocally compensate for the loss of each other. Blocking LPA2 in Lpar5cKO enteroids reduced phosphorylation of Akt, indicating that LPA2 maintains the growth of Lpar5cKO enteroids through activation of the PI3K-Akt pathway. The present study provides evidence that loss of an LPAR can be compensated by another LPAR. This ability to compensate needs to be considered in studies aimed to define receptor functions or test the efficacy of a LPAR-targeting drug using genetically engineered animal models.

Metformin Inhibits Na+/H+ Exchanger NHE3 Resulting in Intestinal Water Loss.

Glycemic control is the key to the management of type 2 diabetes. Metformin is an effective, widely used drug for controlling plasma glucose levels in diabetes, but it is often the culprit of gastrointestinal adverse effects such as abdominal pain, nausea, indigestion, vomiting, and diarrhea. Diarrhea is a complex disease and altered intestinal transport of electrolytes and fluid is a common cause of diarrhea. Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and our previous study has demonstrated that decreased NHE3 contributes to diarrhea associated with type 1 diabetes. The goal of this study is to investigate whether metformin regulates NHE3 and inhibition of NHE3 contributes to metformin-induced diarrhea. We first determined whether metformin alters intestinal water loss, the hallmark of diarrhea, in type 2 diabetic db/db mice. We found that metformin decreased intestinal water absorption mediated by NHE3. Metformin increased fecal water content although mice did not develop watery diarrhea. To determine the mechanism of metformin-mediated regulation of NHE3, we used intestinal epithelial cells. Metformin inhibited NHE3 activity and the effect of metformin on NHE3 was mimicked by a 5'-AMP-activated protein kinase (AMPK) activator and blocked by pharmacological inhibition of AMPK. Metformin increased phosphorylation and ubiquitination of NHE3, resulting in retrieval of NHE3 from the plasma membrane. Previous studies have demonstrated the role of neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2) in regulation of human NHE3. Silencing of Nedd4-2 mitigated NHE3 inhibition and ubiquitination by metformin. Our findings suggest that metformin-induced diarrhea in type 2 diabetes is in part caused by reduced Na+ and water absorption that is associated with NHE3 inhibition, probably by AMPK.

Survival of Stem Cells and Progenitors in the Intestine Is Regulated by LPA5-Dependent Signaling.

BACKGROUND & AIMS: Regeneration of the epithelium by stem cells in the intestine is supported by intrinsic and extrinsic factors. Lysophosphatidic acid (LPA), a bioactive lipid mediator, regulates many cellular functions, including cell proliferation, survival, and cytokine secretion. Here, we identify LPA5 receptor as a potent regulator of the survival of stem cells and transit-amplifying cells in the intestine. METHODS: We have used genetic mouse models of conditional deletion of Lpar5, Lpar5f/f;Rosa-CreERT (Lpar5KO), and intestinal epithelial cell-specific Lpar5f/f;AhCre (Lpar5IECKO) mice. Mice were treated with tamoxifen or ß-naphthoflavone to delete Lpar5 expression. Enteroids derived from these mice were used to determine the effect of Lpar5 loss on the apoptosis and proliferation of crypt epithelial cells. RESULTS: Conditional loss of Lpar5 induced ablation of the intestinal mucosa, which increased morbidity of Lpar5KO mice. Epithelial regeneration was compromised with increased apoptosis and decreased proliferation of crypt epithelial cells by Lpar5 loss. Interestingly, intestinal epithelial cell-specific Lpar5 loss did not cause similar phenotypic defects in vivo. Lpar5 loss reduced intestinal stem cell marker gene expression and reduced lineage tracing from Lgr5+ ISCs. Lpar5 loss induced CXCL10 expression which exerts cytotoxic effects on intestinal stem cells and progenitors in the intestinal crypts. By co-culturing Lpar5KO enteroids with wild-type or Lpar5KO splenocytes, we demonstrated that lymphocytes protect the intestinal crypts via a LPA5-dependent suppression of CXCL10. CONCLUSIONS: LPA5 is essential for the regeneration of intestinal epithelium. Our findings reveal a new finding that LPA5 regulates survival of stem cells and transit-amplifying cells in the intestine.

A Modification to the Situation-based Scheme for Sorting Exposures Proposed in ICRP Publication 103.

ABSTRACT: The International Commission on Radiological Protection (ICRP) issued its current recommendations in 2007, introducing an evolution from a previously process-based protection approach (practices and interventions) to a situation-based approach (planned, existing, and emergency). In most countries, radiation protection standards are generally in line with the new recommendations by the ICRP. Unfortunately, in many cases, situations involving sources of natural or artificial origin may be in a state of contradiction. Logical incompleteness and unsoundness in the framework of exposure situations can be found in current ICRP recommendations. There are situations that do not readily fall into any category. To contribute to a more reasonable situation-based approach for the radiological protection community, we recommend here a possible framework of exposure situations, i.e., planned, unplanned, and naturally occurring, that forms one coherent system. We believe this system will provide increased clarity that will help resolve possible sorting problems.

Nedd4-2-dependent Ubiquitination Potentiates the Inhibition of Human NHE3 by Cholera Toxin and Enteropathogenic Escherichia coli.

BACKGROUND & AIMS: Diarrhea is one of the most common illnesses and is often caused by bacterial infection. Recently, we have shown that human Na+/H+ exchanger NHE3 (hNHE3), but not non-human NHE3s, interacts with the E3 ubiquitin ligase Nedd4-2. We hypothesize that this property of hNHE3 contributes to the increased severity of diarrhea in humans. METHODS: We used humanized mice expressing hNHE3 in the intestine (hNHE3int) to compare the contribution of hNHE3 and mouse NHE3 to diarrhea induced by cholera toxin (CTX) and enteropathogenic Escherichia coli (EPEC). We measured Na+/H+ exchange activity and fluid absorption. The role of Nedd4-2 on hNHE3 activity and ubiquitination was determined by knockdown in Caco-2bbe cells. The effects of protein kinase A (PKA), the primary mediator of CTX-induced diarrhea, on Nedd4-2 and hNHE3 phosphorylation and their interaction were determined. RESULTS: The effects of CTX and EPEC were greater in hNHE3int mice than in control wild-type (WT) mice, resulting in greater inhibition of NHE3 activity and increased fluid accumulation in the intestine, the hallmark of diarrhea. Activation of PKA increased ubiquitination of hNHE3 and enhanced interaction of Nedd4-2 with hNHE3 via phosphorylation of Nedd4-2 at S342. S342A mutation mitigated the Nedd4-2-hNHE3 interaction and blocked PKA-induced inhibition of hNHE3. Unlike non-human NHE3s, inhibition of hNHE3 by PKA is independent of NHE3 phosphorylation, suggesting a distinct mechanism of hNHE3 regulation. CONCLUSIONS: The effects of CTX and EPEC on hNHE3 are amplified, and the unique properties of hNHE3 may contribute to diarrheal symptoms occurring in humans.

Novel angular naphthopyrone formation by Arp1p dehydratase involved in Aspergillus fumigatus melanin biosynthesis.

Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Micromagnetic simulation of microstructure effect for binary-main-phase Nd-Ce-Fe-B magnets.

We investigate the magnetic properties of a chemically heterogeneous binary-main-phase (BMP) Nd-Ce-Fe-B magnet with a core-shell structure via micromagnetic simulation. It is found that the coercivity strongly depends on the shell thickness. The BMP magnet's coercivity initially increases and then decreases with increasing Nd-rich shell thickness, and so there is the optimal shell thickness which shows the maximum coercivity for any given Ce concentration. The simulation shows the significant difference in coercivity and maximum energy product between the BMP and single-main-phase magnets. Notably, the magnetization reversal mechanism of the BMP magnet is revealed in the simulation. Local reversals in the BMP magnet first occur in the Ce-rich shells, followed by the Nd-rich cores. Then, the magnetization in Ce-rich core/Nd-rich shell typed grains is switched after reversed magnetization of all the Nd-rich core/Ce-rich shell typed grains. The BMP magnet represents a further increased coercivity for a larger GB thickness, which can be well explained by a maximum stray field.

Moderate levels of 5-fluorocytosine cause the emergence of high frequency resistance in cryptococci.

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.