MNN5 encodes an iron-regulated alpha-1,2-mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans

The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans alpha-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both alpha-1,2- and alpha-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Delta mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Delta mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Delta mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.     

The Economics of Dementia-Care Mapping in Nursing Homes: A Cluster-Randomised Controlled Trial

Background

Dementia-care mapping (DCM) is a cyclic intervention aiming at reducing neuropsychiatric symptoms in people with dementia in nursing homes. Alongside an 18-month cluster-randomized controlled trial in which we studied the effectiveness of DCM on residents and staff outcomes, we investigated differences in costs of care between DCM and usual care in nursing homes.

Methods

Dementia special care units were randomly assigned to DCM or usual care. Nurses from the intervention care homes received DCM training, a DCM organizational briefing day and conducted the 4-months DCM-intervention twice during the study. A single DCM cycle consists of observation, feedback to the staff, and action plans for the residents. We measured costs related to health care consumption, falls and psychotropic drug use at the resident level and absenteeism at the staff level. Data were extracted from resident files and the nursing home records. Prizes were determined using the Dutch manual of health care cost and the cost prices delivered by a pharmacy and a nursing home. Total costs were evaluated by means of linear mixed-effect models for longitudinal data, with the unit as a random effect to correct for dependencies within units.

Results

34 units from 11 nursing homes, including 318 residents and 376 nursing staff members participated in the cost analyses. Analyses showed no difference in total costs. However certain changes within costs could be noticed. The intervention group showed lower costs associated with outpatient hospital appointments over time (p = 0^.05) than the control group. In both groups, the number of falls, costs associated with the elderly-care physician and nurse practitioner increased equally during the study (p<0^.02).

Conclusions

DCM is a cost-neutral intervention. It effectively reduces outpatient hospital appointments compared to usual care. Other considerations than costs, such as nursing homes’ preferences, may determine whether they adopt the DCM method.

Trial Registration

Dutch Trials Registry NTR2314     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.     

Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification

Introduction

HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.

Methods

Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.

Results

Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.

Conclusions

The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Commentary: The carboxyl-terminal Crk SH3 domain: Regulatory strategies and new perspectives

Since their discovery as cellular counterparts of viral oncogenes more than two decades ago, enormous progress has been made in unraveling the complex regulatory pathways of signal transduction initiated by the Crk family of proteins. New structural and biochemical studies have uncovered novel insights into both negative and positive regulation of Crk mediated by its atypical carboxyl-terminal SH3 domain (SH3C). Moreover, SH3C is tyrosine phosphorylated by receptor tyrosine kinases and non-receptor tyrosine kinases, thereby permitting assemblages of other SH2/PTB domain containing proteins. Such non-canonical signaling by the Crk SH3C reveals new regulatory strategies for adaptor proteins.     

Assessing the ecological risks from the persistence and spread of feral populations of insect-resistant transgenic maize

One source of potential harm from the cultivation of transgenic crops is their dispersal, persistence and spread in non-agricultural land. Ecological damage may result from such spread if the abundance of valued species is reduced. The ability of a plant to spread in non-agricultural habitats is called its invasiveness potential. The risks posed by the invasiveness potential of transgenic crops are assessed by comparing in agronomic field trials the phenotypes of the crops with the phenotypes of genetically similar non-transgenic crops known to have low invasiveness potential. If the transgenic and non-transgenic crops are similar in traits believed to control invasiveness potential, it may be concluded that the transgenic crop has low invasiveness potential and poses negligible ecological risk via persistence and spread in non-agricultural habitats. If the phenotype of the transgenic crop is outside the range of the non-transgenic comparators for the traits controlling invasiveness potential, or if the comparative approach is regarded as inadequate for reasons of risk perception or risk communication, experiments that simulate the dispersal of the crop into non-agricultural habitats may be necessary. We describe such an experiment for several commercial insect-resistant transgenic maize events in conditions similar to those found in maize-growing regions of Mexico. As expected from comparative risk assessments, the transgenic maize was found to behave similarly to non-transgenic maize and to be non-invasive. The value of this experiment in assessing and communicating the negligible ecological risk posed by the low invasiveness potential of insect-resistant transgenic maize in Mexico is discussed.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Ethanol administration and the relationship of malonyl-coenzyme A concentrations to the rate of fatty acid synthesis in rat liver

1. The effect of ethanol on liver fatty acid synthesis was studied in vivo in 24h-starved and ;meal-fed' rats (i.e. fed for 3h per day and not ad libitum). 2. In the fed animal (3)H(2)O was incorporated into fat at a rate of 0.46mumol of C(2) units/min per g wet wt. of liver. Administration of either ethanol (3.2g/kg) or equicaloric amounts of glucose had no effect on the rate of (3)H(2)O incorporation into lipid. 3. In the 24h-starved animal, administration of the same dose of ethanol produced an increase in the rate of (3)H(2)O incorporation from 0.06 to 0.12mumol of C(2) units/min per g fresh wt. after 3h whereas [malonyl-CoA] increased from 0.006 to 0.009mumol/g. Glucose given in amounts equicaloric to ethanol was significantly more lipogenic, increasing both the (3)H(2)O incorporation from 0.06 to 0.20mumol of C(2) units/min per g and the malonyl-CoA content from 0.006 to 0.013 mumol/g wet wt. at 3h. 4. The decrease in the redox state of free cytoplasm NAD or NADP couples or the changes in content of citrate, glucose 6-phosphate and pyruvate of liver after ethanol administration had no measurable effect on the rate of fatty acid synthesis in vivo. 5. Under the conditions of the experiments there was no significant difference, among any of the groups, in the activity of liver fatty acid synthetase measured in vitro. A double-reciprocal plot of the rate of (3)H(2)O incorporation and the total tissue malonyl-CoA concentrations showed a striking relationship. It has been concluded that the rate of fatty acid synthesis in vivo is determined principally by the V(max.) of fatty acid synthetase and the concentration of free malonyl-CoA. 6. It has also been concluded that under the conditions of the present study, the synthesis of fatty acids de novo is unlikely to be an important factor in the increased liver lipid content associated with ethanol administration.     

Reproduction of the owl monkey (Aotus spp.) in captivity

Abstract: The reproduction performance of captive owl monkeys, a breed used extensively in biomedical research, was observed at the Battelle Primate Facility (BPF). The colony grew through captive breeding, imports from the Peruvian Primatological Project, and others to a peak size of 730. It included seven karyotypes of Aotus sp. Results showed that owl monkeys can breed successfully in a laboratory in numbers sufficient to sustain modest research programs. Reproductive success increases when pairs are compatible, of the same karyotype, and stabilized; however, mated pairs of different karyotype are also productive. Under conditions of controlled lighting and heating, owl monkeys at BPF showed no birth peak nor birth season.