Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.     

Design of reference populations for genomic selection in crossbreeding programs

Background

In crossbreeding programs, genomic selection offers the opportunity to make efficient use of information on crossbred (CB) individuals in the selection of purebred (PB) candidates. In such programs, reference populations often contain genotyped PB animals, although the breeding objective is usually more focused on CB performance. The question is what would be the benefit of including a larger proportion of CB individuals in the reference population.

Methods

In a deterministic simulation study, we evaluated the benefit of including various proportions of CB animals in a reference population for genomic selection of PB animals in a crossbreeding program. We used a pig breeding scheme with selection for a moderately heritable trait and a size of 6000 for the reference population.

Results

Applying genomic selection to improve the performance of CB individuals, with a genetic correlation between PB and CB performance (r_PC) of 0.7, selection accuracy of PB candidates increased from 0.49 to 0.52 if the reference population consisted of PB individuals, it increased to 0.55 if the reference population consisted of the same number of CB individuals, and to 0.60 if the size of the CB reference population was twice that of the reference population for each PB line. The advantage of using CB rather than PB individuals increased linearly with the proportion of CB individuals in the reference population. This advantage disappeared quickly if r_PC was higher or if the breeding objective put some emphasis on PB performance. The benefit of adding CB individuals to an existing PB reference population was limited for high r_PC.

Conclusions

Using CB rather than PB individuals in a reference population for genomic selection can provide substantial advantages, but only when correlations between PB and CB performances are not high and PB performance is not part of the breeding objective.     

Patterns of Human Papillomavirus DNA and Antibody Positivity in Young Males and Females,Suggesting a Site-Specific Natural Course of Infection

Background

To monitor the impact of human papillomavirus types 16 and 18 vaccine on HPV infection dynamics in the Netherlands, we started an ongoing study in sexually transmitted infection (STI) clinics in 2009. Here, we analyze baseline type-specific HPV DNA and HPV-specific antibody positivity rates.

Methods

We enrolled 3569 men and women, 16–24 years of age, from 14 STI clinics, and estimated genital and anal HPV DNA and antibody positivity rates of 7 main carcinogenic HPV types. Generalized estimating equations regression analyses were applied to determine risk factors for, and associations between, type-specific HPV DNA and antibody positivity.

Results

Genital HPV DNA positivity rates were higher in women than in men; anal HPV DNA was especially high in men who have sex with men (MSM). HPV antibody seropositivity rates were also highest in women and MSM. High-risk sexual behavior was predictive of both HPV DNA and antibody positivity. Despite a strong correlation in serological profiles for multiple HPV types, seropositivity was independently associated with homologous HPV DNA detection.

Conclusions

HPV DNA and antibody positivity rates are higher in women and MSM than in heterosexual men, but their association is similar across gender. This suggests a site-specific natural course of infection.     

Selective Hepatitis B Virus Vaccination Has Reduced Hepatitis B Virus Transmission in The Netherlands

Background & Aims

In the Netherlands, a selective hepatitis B virus (HBV) vaccination programme started in 2002 for men having sex with men, drug users, commercial sex workers and heterosexuals with frequent partner changes. We assessed the programme''s effectiveness to guide policy on HBV prevention.

Methods

We analysed reports of acute HBV infection in the Netherlands between 2004 and 2010 requesting serum from patients for HBV-genome S- and C-region sequencing. We used coalescence analyses to assess genetic diversity of nonimported genotype-A cases over time.

Results

1687 patients with acute HBV infection were reported between 2004 and 2010. The incidence of reported acute HBV infection decreased from 1.8 to 1.2 per 100,000 inhabitants, mostly due to a reduction in the number of cases in men who have sex with men. Men were overrepresented among cases with an unknown route of transmission, especially among genotype A2 cases mainly associated with transmission through male homosexual contact. The genetic diversity of nonimported genotype-A strains obtained from men who have sex with men decreased from 2006 onwards, suggesting HBV incidence in this group decreased.

Conclusions

The selective HBV-vaccination programme for behavioural high-risk groups very likely reduced the incidence of HBV infection in the Netherlands mainly by preventing HBV infections in men who have sex with men. A considerable proportion of cases in men who did not report risk behaviour was probably acquired through homosexual contact. Our findings support continuation of the programme, and adopting similar approaches in other countries where HBV transmission is focused in high-risk adults.     

A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases

Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.     

Exercise prevents leptin-induced increase in blood pressure in Sprague–Dawley rats

Although leptin has been shown to increase blood pressure (BP), it is however unclear if this increase can be prevented by exercise. This study therefore investigated the effect of leptin treatment with concurrent exercise on blood pressure (BP), sodium output, and endothelin-1 (ET-1) levels in normotensive rats. Male Sprague–Dawley rats weighing 250–270 g were divided into four groups consisting of a control group (n?=?6), leptin-treated (n?=?8), non-leptin-treated exercise group (n?=?8), and a leptin-treated exercise group (n?=?8). Leptin was given subcutaneously daily for 14 days (60 μg/kg/day). Animals were exercised on a treadmill for 30 min at a speed of 0.5 m/s and at 5° incline four times per week. Measurement of systolic blood pressure (SBP) and collection of urine samples for estimation of sodium and creatinine was done once a week. Serum samples were collected at the end of the experiment for determination of sodium, creatinine and ET-1. At day 14, mean SBP and serum ET-1 level in the leptin-treated group was significantly higher than that in the control group whereas mean SBP and serum ET-1 level was significantly lower in the leptin-treated exercise group than those in leptin-treated and control groups. Creatinine clearance, urinary sodium excretion, and urine output were not different between the four groups. Regular treadmill exercise prevents leptin-induced increases in SBP in rats, which might in part result from increased urinary sodium excretion and preventing the leptin-induced increases in serum ET-1 concentration.     

Obesity is underestimated using body mass index and waist-hip ratio in long-term adult survivors of childhood cancer

Objective

Obesity, represented by high body mass index (BMI), is a major complication after treatment for childhood cancer. However, it has been shown that high total fat percentage and low lean body mass are more reliable predictors of cardiovascular morbidity. In this study longitudinal changes of BMI and body composition, as well as the value of BMI and waist-hip ratio representing obesity, were evaluated in adult childhood cancer survivors.

Methods

Data from 410 survivors who had visited the late effects clinic twice were analyzed. Median follow-up time was 16 years (interquartile range 11–21) and time between visits was 3.2 years (2.9–3.6). BMI was measured and body composition was assessed by dual X-ray absorptiometry (DXA, Lunar Prodigy; available twice in 182 survivors). Data were compared with healthy Dutch references and calculated as standard deviation scores (SDS). BMI, waist-hip ratio and total fat percentage were evaluated cross-sectionally in 422 survivors, in who at least one DXA scan was assessed.

Results

BMI was significantly higher in women, without significant change over time. In men BMI changed significantly with time (ΔSDS = 0.19, P<0.001). Percentage fat was significantly higher than references in all survivors, with the highest SDS after cranial radiotherapy (CRT) (mean SDS 1.73 in men, 1.48 in women, P<0.001). Only in men, increase in total fat percentage was significantly higher than references (ΔSDS = 0.22, P<0.001). Using total fat percentage as the gold standard, 65% of female and 42% of male survivors were misclassified as non-obese using BMI. Misclassification of obesity using waist-hip ratio was 40% in women and 24% in men.

Conclusions

Sixteen years after treatment for childhood cancer, the increase in BMI and total fat percentage was significantly greater than expected, especially after CRT. This is important as we could show that obesity was grossly underestimated using BMI and waist-hip ratio.     

Time Space Translation: A Hox Mechanism for Vertebrate A-P Patterning

The vertebrate A-P axis is a time axis. The head is made first and more and more posterior levels are made at later and later stages. This is different to the situation in most other animals, for example, in Drosophila. Central to this timing is Hox temporal collinearity (see below). This occurs rarely in the animal kingdom but is characteristic of vertebrates and is used to generate the primary axial Hox pattern using time space translation and to integrate successive derived patterns (see below). This is thus a different situation than in Drosophila, where the primary pattern guiding Hox spatial collinearity is generated externally, by the gap and segmentation genes.