Plasmodium DEH is ER-localized and crucial for oocyst mitotic division during malaria transmission.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage, and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase-DEH-is a key component of very long chain fatty acid synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics, and ultrastructure analysis using the mouse malaria model Plasmodium berghei DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δdeh mutants showed a small but significant reduction in oocyst size compared with WT controls from day 10 postinfection onwards, and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δdeh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER, and plays a crucial role in sporogony.

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission.

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal '9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission.

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

First year post-stroke healthcare costs and fall-status among those discharged to the community.

INTRODUCTION: Falls are common post-stroke events but their relationship with healthcare costs is unclear. The aim of this study was to examine the relationship between healthcare costs in the first year after stroke and falls among survivors discharged to the community. PATIENTS AND METHODS: Survivors of acute stroke with planned home discharges from five large hospitals in Ireland were recruited. Falls and healthcare utilisation data were recorded using inpatient records, monthly calendars and post-discharge interviews. Cost of stroke was estimated for each participant from hospital admission for one year. The association of fall-status with overall cost was tested with multivariable linear regression analysis adjusting for pre-stroke function, stroke severity, age and living situation. RESULTS: A total of 109 stroke survivors with complete follow-up data (mean age = 68.5 years (SD = 13.5 years)) were included. Fifty-three participants (49%) fell following stroke, of whom 28 (26%) had recurrent falls. Estimated mean total healthcare cost was €20,244 (SD=€23,456). The experience of one fall and recurrent falls was independently associated with higher costs of care (p = 0.02 and p < 0.01, respectively). DISCUSSION: The observed relationship between falls and cost is likely to be underestimated as aids and adaptions, productivity losses, and nursing home care were not included. CONCLUSION: This study points at differences across fall-status in several healthcare costs categories, namely the index admission, secondary/tertiary care (including inpatient re-admissions) and allied healthcare. Future research could compare the cost-effectiveness of inpatient versus community-based fall-prevention after stroke. Further studies are also required to inform post-stroke bone-health management and fracture-risk reduction.

Falls-Related EvEnts in the first year after StrokE in Ireland: Results of the multi-centre prospective FREESE cohort study.

INTRODUCTION: Falls are common post-stroke adverse events. This study aimed to describe the first-year falls incidence, circumstances and consequences among persons discharged home after stroke in Ireland, and to examine the association between potential risk factors and recurrent falls. PATIENTS AND METHODS: Patients with acute stroke and planned home-discharge were recruited consecutively from five hospitals. Variables recorded pre-discharge included: age, stroke severity, co-morbidities, fall history, prescribed medications, hemi-neglect, cognition and functional independence (Barthel index). Falls were recorded with monthly diaries, and 6 and 12-month interviews. The association of pre-discharge factors with recurrent falls (>1 fall) was examined using univariable logistic regression. RESULTS: A total of 128 participants (mean age = 68.6, SD = 13.3) were recruited; 110 completed the 12-month follow-up. The first-year falls incidence was 44.5% (95% CI = 35.1-53.6) with 25.6% falling repeatedly (95% CI = 18.5-34.4). Fallers experienced 1-18 falls (median = 2) and five reported fractures; 47% of fallers experienced at least one fall outdoors. Only 10% of recurrent fallers had bone health medication prescribed at discharge. Lower Barthel index scores (<75/100, RR = 4.38, 1.64-11.72) and psychotropic medication prescription (RR = 2.10, 1.13-3.91) were associated with recurrent falls. DISCUSSION: This study presents prospectively collected information about falls circumstances. It was not powered for multivariable analysis of risk factors. CONCLUSION: One-quarter of stroke survivors discharged to the community fall repeatedly and mostly indoors in the first year. Specific attention may be required for individuals with poor functional independence or those on psychotropic medication. Future falls-management research in this population should explore falls in younger individuals, outdoor as well as indoor falls and post-stroke bone health status.

[Bone and adipose tissue formation]. / Knochen- und Fettgewebebildung.

Leptin has been described to have a crucial role in bone homeostasis by systemic as well as local action. Systemically, leptin seems to inhibit bone formation controlled by a feedback loop including osteocalcin and insulin. Even though the action seems to be bone site specific, as well as gender- and time-dependent, the results showing the interaction of these three factors are in part still inconsistent. In this article the complex effects of leptin, insulin, and osteocalcin on bone and fat metabolism are summarized.

Placental Size Is Associated Differentially With Postnatal Bone Size and Density.

We investigated relationships between placental size and offspring adolescent bone indices using a population-based, mother-offspring cohort. The Avon Longitudinal Study of Parents and Children (ALSPAC) recruited pregnant women from the southwest of England between 1991 and 1993. There were 12,942 singleton babies born at term who survived at least the first 12 months. From these, 8933 placentas were preserved in formaldehyde, with maternal permission for their use in research studies. At the approximate age of 15.5 years, the children underwent a dual-energy X-ray absorptiometry (DXA) scan (measurements taken of the whole body minus head bone area [BA], bone mineral content [BMC], and areal bone mineral density [aBMD]). A peripheral quantitative computed tomography (pQCT) scan (Stratec XCT2000L; Stratec, Pforzheim, Germany) at the 50% tibial site was performed at this visit and at approximately age 17.7 years. In 2010 a sample of 1680 placentas were measured and photographed. To enable comparison of effect size across different variables, predictor and outcome variables were standardized to Z-scores and therefore results may be interpreted as partial correlation coefficients. Complete placental, DXA, and pQCT data were available for 518 children at age 15.5 years. After adjustment for gender, gestational age at birth, and age at time of pQCT, the placental area was positively associated with endosteal circumference (ß [95% CI]: 0.21 [0.13, 0.30], p < 0.001), periosteal circumference (ß [95% CI]: 0.19 [0.10, 0.27], p < 0.001), and cortical area (ß [95% CI]: 0.10 [0.01, 0.18], p = 0.03), and was negatively associated with cortical density (ß [95% CI]: -0.11 [-0.20, -0.03], p = 0.01) at age 15.5 years. Similar relationships were observed for placental volume, and after adjustment for additional maternal and offspring covariates. These results suggest that previously observed associations between placental size and offspring bone development persist into older childhood, even during puberty, and that placental size is differentially related to bone size and volumetric density. © 2016 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

Rsk2, the Kinase Mutated in Coffin-Lowry Syndrome, Controls Cementum Formation.

The ribosomal S6 kinase RSK2 is essential for osteoblast function, and inactivating mutations of RSK2 cause osteopenia in humans with Coffin-Lowry syndrome (CLS). Alveolar bone loss and premature tooth exfoliation are also consistently reported symptoms in CLS patients; however, the pathophysiologic mechanisms are unclear. Therefore, aiming to identify the functional relevance of Rsk2 for tooth development, we analyzed Rsk2-deficient mice. Here, we show that Rsk2 is a critical regulator of cementoblast function. Immunohistochemistry, histology, micro-computed tomography imaging, quantitative backscattered electron imaging, and in vitro assays revealed that Rsk2 is activated in cementoblasts and is necessary for proper acellular cementum formation. Cementum hypoplasia that is observed in Rsk2-deficient mice causes detachment and disorganization of the periodontal ligament and was associated with significant alveolar bone loss with age. Moreover, Rsk2-deficient mice display hypomineralization of cellular cementum with accumulation of nonmineralized cementoid. In agreement, treatment of the cementoblast cell line OCCM-30 with a Rsk inhibitor reduces formation of mineralization nodules and decreases the expression of cementum markers. Western blot analyses based on antibodies against Rsk1, Rsk2, and an activated form of the 2 kinases confirmed that Rsk2 is expressed and activated in differentiating OCCM-30 cells. To discriminate between periodontal bone loss and systemic bone loss, we additionally crossed Rsk2-deficient mice with transgenic mice overexpressing the osteoanabolic transcription factor Fra1. Fra1 overexpression clearly increases systemic bone volume in Rsk2-deficient mice but does not protect from alveolar bone loss. Our results indicate that cell autonomous cementum defects are causing early tooth loss in CLS patients. Moreover, we identify Rsk2 as a nonredundant regulator of cementum homeostasis, alveolar bone maintenance, and periodontal health, with all these features being independent of Rsk2 function in systemic bone formation.

An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

The electrical stimulation of carbon nanotubes to provide a cardiomimetic cue to MSCs.

Once damaged, cardiac muscle has little intrinsic repair capability due to the poor regeneration potential of remaining cardiomyocytes. One method of overcoming this issue is to deliver functional cells to the injured myocardium to promote repair. To address this limitation we sought to test the hypothesis that electroactive carbon nanotubes (CNT) could be employed to direct mesenchymal stem cell (MSC) differentiation towards a cardiomyocyte lineage. Using a two-pronged approach, MSCs exposed to medium containing CNT and MSCs seeded on CNT based polylactic acid scaffolds were electrically stimulated in an electrophysiological bioreactor. After electrical stimulation the cells reoriented perpendicular to the direction of the current and adopted an elongated morphology. Using qPCR, an upregulation in a range of cardiac markers was detected, the greatest of which was observed for cardiac myosin heavy chain (CMHC), where a 40-fold increase was observed for the electrically stimulated cells after 14 days, and a 12-fold increase was observed for the electrically stimulated cells seeded on the PLA scaffolds after 10 days. Differentiation towards a cardioprogenitor cell was more evident from the western blot analysis, where upregulation of Nkx2.5, GATA-4, cardiac troponin t (CTT) and connexin43 (C43) was seen to occur. This was echoed in immunofluorescent staining, where increased levels of CTT, CMHC and C43 protein expression were observed after electrical stimulation for both cells and cell-seeded scaffolds. More interestingly, there was evidence of increased cross talk between the cells as shown by the pattern of C43 staining after electrical stimulation. These results establish a paradigm for nanoscale biomimetic cues that can be readily translated to other electroactive tissue repair applications.

In vitro characterization of an electroactive carbon-nanotube-based nanofiber scaffold for tissue engineering.

In an effort to reduce organ replacement and enhance tissue repair, there has been a tremendous effort to create biomechanically optimized scaffolds for tissue engineering applications. In contrast, the development and characterization of electroactive scaffolds has attracted little attention. Consequently, the creation and characterization of a carbon nanotube based poly(lactic acid) nanofiber scaffold is described herein. After 28 d in physiological solution at 37 °C, a change in the mass, chemical properties and polymer morphology is seen, while the mechanical properties and physical integrity are unaltered. No adverse cytotoxic affects are seen when mesenchymal stem cells are cultured in the presence of the scaffold. Taken together, these data auger well for electroactive tissue engineering.

Childhood body mass index and adult pro-inflammatory and pro-thrombotic risk factors: data from the New Delhi birth cohort.

OBJECTIVE: Weight gain and growth in early life may influence adult pro-inflammatory and pro-thrombotic cardiovascular risk factors. METHODS: Follow-up of a birth cohort in New Delhi, India, whose weight and height were measured every 6 months until age 21 years. Body mass index (BMI) at birth, during infancy (2 years), childhood (11 years) and adulthood (26-32 years) and BMI gain between these ages were analysed in 886 men and 640 women with respect to adult fibrinogen, high-sensitivity C-reactive protein (hsCRP) and plasminogen activator inhibitor-1 (PAI-1) concentrations. RESULTS: All the pro-inflammatory/pro-thrombotic risk factors were higher in participants with higher adiposity. In women, BMI at birth and age 2 years was inversely related to fibrinogen (P = 0.002 and 0.05) and, after adjusting for adult adiposity, to hsCRP (P = 0.02 and 0.009). After adjusting for adult adiposity, BMI at 2 years was inversely related to hsCRP and PAI-1 concentrations (P < 0.001 and 0.02) in men. BMI gain between 2 and 11 years and/or 11 years to adulthood was positively associated with fibrinogen and hsCRP in women and with hsCRP and PAI-1 in men. CONCLUSIONS: Thinness at birth or during infancy, and accelerated BMI gain during childhood/adolescence are associated with a pro-inflammatory/pro-thrombotic state in adult life. An altered inflammatory state could be one link between small newborn/infant size and adult cardiovascular disease. Associations between pro-inflammatory markers and childhood/adolescent BMI gain are probably mediated through adult adiposity.

Blockade of Dickkopf (DKK)-1 induces fusion of sacroiliac joints.

OBJECTIVE: To study whether Dickkopf (DKK)-1, an inhibitor of wingless (Wnt) signalling, is involved in the fusion of sacroiliac joints. METHODS: Mice transgenic for tumour necrosis factor (TNFtg mice), which develop bilateral sacroiliitis, were treated with vehicle, anti-TNF antibody or anti-DKK1 antibody. Sacroiliac joints were analysed for histological signs of inflammation, bone erosion, osteoclast formation and ankylosis. Moreover, expression of collagen type X, beta-catenin and DKK-1 was assessed by immunohistochemistry. RESULTS: There were no signs of spontaneous ankylosis of the sacroiliac joints in TNFtg mice. TNF blockade effectively reduced inflammation, bone erosion and osteoclast numbers in the sacroiliac joints, but did not lead to ankylosis. Blockade of DKK1 had no effect on inflammatory signs of sacroiliitis, but significantly reduced bone erosions and osteoclast counts. Moreover, DKK1 blockade promoted expression of collagen type X, the formation of hypertrophic chondrocytes and ankylosis of sacroiliac joints. CONCLUSION: DKK1 influences inflammatory remodelling of sacroiliac joints by prevention of joint ankylosis. This may indicate an important role of the Wnt signalling pathway in the structural bone changes of axial joint disease. Although this model does not reflect the entire spectrum of ankylosing spondylitis in humans, it helps to explain the pathophysiological processes of sacroiliac joint ankylosis, which is a hallmark of the spondyloarthritides.

[Mesenchymal stem cells in arthritis]. / Mesenchymale Stammzellen bei Arthritis.

While one of the major achievements of the 20th century was prolonging life expectancy in developed countries, the main challenge of the 21st century is to improve the quality of life of the aging population. Aging is associated with a progressive reduction of organ system function. Therefore, regenerative medicine will be one of the major developing fields of medicine. This new medical field does not only apply to aging but also to all degenerative diseases, such as arthritis and degenerative joint disease, which lead to progressive degeneration of mesenchymal tissues such as bone and cartilage. The discovery of pluripotent mesenchymal stem cells (MSCs) offers a promising alternative to surgery for non-invasive regenerative therapies of mesenchymal tissues. This review focuses on the characterization and potential application of MSCs in the regeneration of damaged joints.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Body mass index during childhood and adult body composition in men and women aged 56-70 y.

BACKGROUND: The relation between the change in body mass index (BMI) through childhood and body composition in adult life is important because body composition is known to affect adult health. OBJECTIVE: The objective was to examine how the change in BMI throughout childhood is related to adult lean and fat mass. DESIGN: We examined how the change in BMI in childhood was related to adult body composition in 885 men and 1032 women born during 1934-1944, whose weights and heights during childhood were recorded serially. Adult lean and fat mass were measured by bioelectrical impedance with an 8-polar tactile electrode system. RESULTS: In these 56-70-y-old men and women, adult lean body mass index (lean mass/height(2); in kg/m(2)) was positively associated with BMI at birth (0.24 and 0.20 higher for each 1-SD increase in BMI at birth, respectively) and with more rapid gain in BMI from birth to 1 y (0.17 and 0.22), 1-2 y (0.21 and 0.20), 2-7 y (0.44 and 0.46), and 7-11 y (0.32 and 0.26) of age. Fat mass index (fat mass/height(2)) was positively associated with more rapid increases in BMI between 2 and 11 y of age. CONCLUSIONS: Rapid gain in BMI before the age of 2 y increased adult lean body mass without excess fat accumulation, whereas rapid gain in BMI in later childhood, despite the concurrent rise in lean mass, resulted in relatively larger increases in fat mass.