Habitual Sleep Measures are Associated with Overall Body Fat,and not Specifically with Visceral Fat,in Men and Women

Objective

This study aimed to investigate the associations of sleep duration and sleep quality with visceral adipose tissue (VAT) in middle‐aged individuals.

Methods

In this cross‐sectional analysis of baseline measurements of the Netherlands Epidemiology of Obesity (NEO) study, participants underwent anthropometry and completed the Pittsburgh Sleep Quality Index (PSQI) for assessing short sleep duration (as sex‐specific age‐adjusted percentiles) and poor quality (PSQI > 5). VAT was assessed by magnetic resonance imaging in a random subgroup. We performed linear regression analyses to examine associations of short sleep and poor sleep with measures of body fat, adjusted for confounding, including total body fat in models with VAT.

Results

A total of 5,094 participants (52% women; mean age of 56 [SD 6] years), 1,947 of whom had VAT measurements, were analyzed. The difference in VAT between poor sleep (PSQI > 5) and good sleep (PSQI ≤ 5) was 7.2cm² (95% CI: 1.2‐13.8) in women and 16.1cm² (95% CI: 6.2‐26.0) in men. These differences attenuated toward the null after the adjustment for total body fat. Similar patterns of associations were observed for short sleep (lowest 10% compared with median 60%).

Conclusions

Our results suggest that measures of sleep are not specifically associated with a higher amount of VAT.     

Environmental Sequencing Fills the Gap Between Parasitic Haplosporidians and Free‐living Giant Amoebae

Class Ascetosporea (Rhizaria; Endomyxa) comprises many parasites of invertebrates. Within this group, recent group‐specific environmental DNA (eDNA) studies have contributed to the establishment of the new order Mikrocytida, a new phylogeny and characterization of Paramyxida, and illuminated the diversity and distribution of haplosporidians. Here, we use general and lineage‐specific PCR primers to investigate the phylogenetic “gap” between haplosporidians and their closest known free‐living relatives, the testate amoeba Gromia and reticulate amoeba Filoreta. Within this gap are Paradinium spp. parasites of copepods, which we show to be highly diverse and widely distributed in planktonic and benthic samples. We reveal a robustly supported radiation of parasites, ENDO‐3, comprised of Paradinium and three further clades (ENDO‐3a, ENDO‐3b and SPP). A further environmental group, ENDO‐2, perhaps comprising several clades, branches between this radiation and the free‐living amoebae. Early diverging haplosporidians were also amplified, often associated with bivalves or deep‐sea samples. The general primer approach amplified an overlapping set of novel lineages within ENDO‐3 and Haplosporida, whereas the group‐specific primer strategy, targeted to amplify from the earliest known divergent haplosporidians to Gromia, generated greater sequence diversity across part of this phylogenetic range.     

The circumscription of Karimbolea Descoings (Asclepiadaceae)

The hitherto monotypic and isolated Madagascan genus Karimbolea is redefined. One species is added to the genus and the new combination K. macrantha (Jum. & H. Perrier) Liede & Meve is provided. The unusual erect orientation of the pollinia in the type species, K. verrucosa, is interpreted as secondary shift from a pendulous position (tribe Asclepiadeae) rather than an indication for affinities with tribes characterized by erect pollinia (Stapelieae, Marsdenieae).     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.     

Decreasing Period-Length of the Endogenous Circadian Rhythm of Oxygen Evolution in Acetabularia and Its Possible Relation to Aging

Endogenous circadian rhythms observed under constant conditions normally show period length variations. However, a general trend is difficult to identify when cells or organisms are entrained with the usual 24-h-period light/dark cycles. Therefore, these variations in time have been considered as fluctuations. In order to gain more insight into this phenomenon, individual Acetabularia cells were exposed to light/dark cycles of 16 h (LD 8:8) and 33.6 h (LD 16.8:16.8), respectively, i.e., periods which lie distinctly outside the range of the normal circadian entrainment. Employing a high-resolution procedure for data analysis, decreasing period lengths could consistently be detected when cells were kept under constant conditions for several weeks. Possible causes of this decrease are discussed.     

Phylogenetics of tribe Sabiceeae (Ixoroideae,Rubiaceae) revisited,with a new subgeneric classification for Sabicea

Tribe Sabiceeae (Ixoroideae, Rubiaceae) has undergone recent taxonomical changes with the incorporation of the related genera Ecpoma, Pseudosabicea and Stipularia into the type genus Sabicea. We use phylogenetic analysis and morphological data to verify the relationships among members of the tribe, including the most comprehensive taxon sampling of the tribe to date with 74 of 145 species. Sequence data from the nuclear internal transcribed spacer (ITS) and three plastid markers (petD, rps16, trnT–F) were used to infer relationships among the members of the tribe. Individual analyses using maximum likelihood, parsimony and Bayesian approaches reveal several supported clades: the former genus Stipularia is resolved as a monophyletic unit, but Ecpoma is monophyletic only if Sabicea urbaniana and Sabicea xanthotricha are included (corresponding to Sabicea subgenus Stipulariopsis sensu Wernham). Pseudosabicea is biphyletic, with one clade corresponding to section Anisophyllae of Hallé (1964) and the other one to the other sections (Floribundae and Sphaericae) of the genus. Eleven morphological characteristics were recorded for all species studied and seven have been mapped onto the phylogenetic tree to study their evolution in the group and assess their value for the classification of Sabicea s.l. Finally, our study shows that a combination of diagnostic characteristics should be used to differentiate each group and we propose to recognise four subgenera in Sabicea.     

Euthanasia in Belgium: trends in reported cases between 2003 and 2013

Background:In 2002, the Belgian Act on Euthanasia came into effect, regulating the intentional ending of life by a physician at the patient’s explicit request. We undertook this study to describe trends in officially reported euthanasia cases in Belgium with regard to patients’ sociodemographic and clinical profiles, as well as decision-making and performance characteristics.Methods:We used the database of all euthanasia cases reported to the Federal Control and Evaluation Committee on Euthanasia in Belgium between Jan. 1, 2003, and Dec. 31, 2013 (n = 8752). The committee collected these data with a standardized registration form. We analyzed trends in patient, decision-making and performance characteristics using a χ² technique. We also compared and analyzed trends for cases reported in Dutch and in French.Results:The number of reported euthanasia cases increased every year, from 235 (0.2% of all deaths) in 2003 to 1807 (1.7% of all deaths) in 2013. The rate of euthanasia increased significantly among those aged 80 years or older, those who died in a nursing home, those with a disease other than cancer and those not expected to die in the near future (p < 0.001 for all increases). Reported cases in 2013 most often concerned those with cancer (68.7%) and those under 80 years (65.0%). Palliative care teams were increasingly often consulted about euthanasia requests, beyond the legal requirements to do so (p < 0.001). Among cases reported in Dutch, the proportion in which the person was expected to die in the foreseeable future decreased from 93.9% in 2003 to 84.1% in 2013, and palliative care teams were increasingly consulted about the euthanasia request (from 34.0% in 2003 to 42.6% in 2013). These trends were not significant for cases reported in French.Interpretation:Since legalization of euthanasia in Belgium, the number of reported cases has increased each year. Most of those receiving euthanasia were younger than 80 years and were dying of cancer. Given the increases observed among non–terminally ill and older patients, this analysis shows the importance of detailed monitoring of developments in euthanasia practice.In 2002, Belgium legalized euthanasia, defined as the intentional ending of life by a physician at the patient’s explicit request.^1,2 For a patient to be eligible for euthanasia, certain formal criteria for due care must be met.¹ These include a voluntary, well-considered, repeated and written request, expressed by a person with full mental capacity who is fully informed about his or her medical condition and the remaining therapeutic possibilities.¹ The person must be in a state of constant and unbearable physical or mental suffering that cannot be alleviated. Due care criteria for the procedure include an a priori consultation with a second independent physician, consultation with a third physician in cases where death is not expected in the foreseeable future and a posteriori reporting of the case for evaluation purposes.¹To safeguard due process and legal compliance and to enable societal control and evaluation, a mandatory notification procedure was built into the legislation.³ Physicians are required to report each case of euthanasia to the multidisciplinary Belgian Federal Control and Evaluation Committee on Euthanasia by completing and submitting a registration form within 4 working days after a death by euthanasia.^1,3 The evaluation committee reviews the form and determines whether euthanasia was performed in accordance with the legal requirements. Initially, only anonymous information is reviewed; where there is doubt about legality, the committee can revoke anonymity by majority decision and can ask the reporting physician for additional information. If the committee is of the opinion, based on a two-thirds majority, that the legal requirements were not fulfilled, the case is sent to the public prosecutor.^1,3 Although not mentioned in the Belgian law, physician-assisted suicide is treated as a form of euthanasia by the committee.⁴To facilitate societal control, the Federal Control and Evaluation Committee on Euthanasia is legally required to issue biennial reports of all reported cases,^1,3–9 providing basic statistics, an evaluation of the law and further recommendations. However, these statistics do not provide an overview of long-term trends. A more complete and thorough evaluation of case characteristics and analysis of trends is needed. In this way, adherence to the legal criteria can be evaluated, and developments in euthanasia practice that might raise concerns can be identified and addressed.Belgium has 2 main language communities: those who speak Dutch (roughly 60% of the population), who mainly live in Flanders, and those who speak French (about 40%), who mainly live in Wallonia. The Brussels-Capital Region is officially bilingual, but predominantly French-speaking. Several empirical studies have found differences in end-of-life practices, knowledge and attitudes between the regions and language communities, showing that Dutch-speaking physicians more often receive and grant euthanasia requests and are more inclined to adhere to legal safeguards.^10–14 The reports issued by the Federal Control and Evaluation Committee on Euthanasia show a striking disparity in euthanasia reporting between the 2 language communities.^4–9 Trends in the characteristics of reported cases and differences among them have not yet been studied.The committee’s reports have shown a continuing increase in the number of euthanasia cases.^4–9 The primary objective of this study was to examine changes in the number and incidence of euthanasia cases and the proportion of euthanasia cases relative to all deaths in Belgium up to and including 2013. The secondary objectives were to determine and report the sociodemographic and clinical characteristics of patients, the decision-making and performance characteristics of reported cases and the differences in trends in characteristics between cases reported in Dutch and cases reported in French.     

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase

Background

Infectious diseases pose a severe worldwide threat to human and livestock health. While early diagnosis could enable prompt preventive interventions, the majority of diseases are found in rural settings where basic laboratory facilities are scarce. Under such field conditions, point-of-care immunoassays provide an appropriate solution for rapid and reliable diagnosis. The limiting steps in the development of the assay are the identification of a suitable target antigen and the selection of appropriate high affinity capture and detection antibodies. To meet these challenges, we describe the development of a Nanobody (Nb)-based antigen detection assay generated from a Nb library directed against the soluble proteome of an infectious agent. In this study, Trypanosoma congolense was chosen as a model system.

Methodology/Principal Findings

An alpaca was vaccinated with whole-parasite soluble proteome to generate a Nb library from which the most potent T. congolense specific Nb sandwich immunoassay (Nb474H-Nb474B) was selected. First, the Nb474-homologous sandwich ELISA (Nb474-ELISA) was shown to detect experimental infections with high Positive Predictive Value (98%), Sensitivity (87%) and Specificity (94%). Second, it was demonstrated under experimental conditions that the assay serves as test-of-cure after Berenil treatment. Finally, this assay allowed target antigen identification. The latter was independently purified through immuno-capturing from (i) T. congolense soluble proteome, (ii) T. congolense secretome preparation and (iii) sera of T. congolense infected mice. Subsequent mass spectrometry analysis identified the target as T. congolense glycosomal aldolase.

Conclusions/Significance

The results show that glycosomal aldolase is a candidate biomarker for active T. congolense infections. In addition, and by proof-of-principle, the data demonstrate that the Nb strategy devised here offers a unique approach to both diagnostic development and target discovery that could be widely applied to other infectious diseases.     

Cytokeratins and cytokeratin filaments in subpopulations of cultured human and rodent cells of nonepithelial origin: modes and patterns of formation

Using immunofluorescence microscopy, we observed that in several established cell culture lines derived from different nonepithelial tissues and species, cells spontaneously emerge, usually at low frequencies, which contain cytoplasmic structures decorated by antibodies specific for cytokeratins 8 and 18. This phenomenon was further examined at both the protein (gel electrophoreses of cytoskeletal proteins, followed by immunoblotting) and the RNA (Northern blots, "nuclear run-on" analysis, in situ hybridization) level. Positive cell lines included simian virus (SV40)-transformed human fibroblasts (HF-SV80, WI-38 VA13), human astrocytic glioma cells (U333 CG/343MG), rat (RVF-SMC) and hamster (BHK-21/13) cells derived from vascular smooth muscle and murine sarcoma MS-180 cells. In two cell lines (HF-SV80 and BHK-21/13), the frequency of the cytokeratin-containing cells and of the cytokeratin fibril arrays per cell was drastically increased upon treatment with 5-azacytidine. The structural appearance of the cytokeratins was variable in the different cell lines but could also differ among cells of the same culture: While small granular or comma-shaped structures or bizarrely shaped filament arrays prevailed in WI-38, RVF and normally grown BHK-21 cells, most of the other lines revealed extended normal-looking, fibrillar arrays. In one line (MS-180), the appearance of cytokeratins was associated with a morphological change, as it was only found in a subpopulation of cells that had lost their typical elongated and spindle-shaped phenotype and assumed a rounded ("coccoid") shape. Our results show that the expression of the genes encoding cytokeratins 8 and 18 is not necessarily restricted to programs of epithelial differentiation and that factors stochastically effective appear in cultured cell lines that allow the synthesis of these cytoskeletal components. Mechanisms possibly involved in this spontaneous and selective advent of cytokeratins 8 and 18 and implications for tumor diagnosis are discussed.