The 3D printing of freestanding PLLA thin layers and improving first layer consistency through the introduction of sacrificial PVA

Fused filament fabrication (FFF) is an inexpensive way of producing objects through a programmed layer-by-layer deposition. For multi-layer, macro-scaled prints, acceptable printing is achieved provided, amongst other factors, first layer adhesion is sufficient to fix a part to the surface during printing. However, in the deposition of structures with a single or few layers, first layer consistency is significantly more important and is an issue that has been previously overlooked. As layer-to-bed adhesion is prioritised in first layer printing, thin layer structures are difficult to remove without damage. The deposition of controllable thin structures has potential in tissue engineering through the use of bioactive filaments and incorporation of microfeatures into complex, patient-specific scaffolds. This paper presents techniques to progress the deposition of thin, reproducible structures. The linear thickness variation of 3D-printed single PVA and PLLA layers is presented as a function of extrusion factor and the programmed vertical distance moved by the nozzle between layers (the layer separation). A sacrificial PVA layer is shown to significantly improve first layer consistency, reducing the onus on fine printer calibration in the deposition of single layers. In this way, the linear variation in printed single PLLA layers with bed deviation is drastically reduced. Further, this technique is used to demonstrate the printing of freestanding thin layers of ~25 µm in thickness.</jats:p

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance

Mosquito Abundance, Bed net Coverage and Other Factors Associated with Variations in Sporozoite Infectivity Rates in Four Villages of Rural Tanzania.

Entomological surveys are of great importance in decision-making processes regarding malaria control strategies because they help to identify associations between vector abundance both species-specific ecology and disease intervention factors associated with malaria transmission. Sporozoite infectivity rates, mosquito host blood meal source, bed net coverage and mosquito abundance were assessed in this study. A longitudinal survey was conducted in four villages in two regions of Tanzania. Malaria vectors were sampled using the CDC light trap and pyrethrum spray catch methods. In each village, ten paired houses were selected for mosquitoes sampling. Sampling was done in fortnight case and study was undertaken for six months in both Kilimanjaro (Northern Tanzania) and Dodoma (Central Tanzania) regions. A total of 6,883 mosquitoes were collected including: 5,628 (81.8%) Anopheles arabiensis, 1,100 (15.9%) Culex quinquefasciatus, 89 (1.4%) Anopheles funestus, and 66 (0.9%) Anopheles gambiae s.s. Of the total mosquitoes collected 3,861 were captured by CDC light trap and 3,022 by the pyrethrum spray catch method. The overall light trap: spray catch ratio was 1.3:1. Mosquito densities per room were 96.5 and 75.5 for light trap and pyrethrum spray catch respectively. Mosquito infectivity rates between villages that have high proportion of bed net owners and those without bed nets was significant (P < 0.001) and there was a significant difference in sporozoite rates between households with and without bed nets in these four villages (P < 0.001). Malaria remains a major problem in the study areas characterized as low transmission sites. Further studies are required to establish the annual entomological inoculation rates and to observe the annual parasitaemia dynamics in these communities. Outdoor mosquitoes collection should also be considered

Sarcomere Formation Occurs by the Assembly of Multiple Latent Protein Complexes

The stereotyped striation of myofibrils is a conserved feature of muscle organization that is critical to its function. Although most components that constitute the basic myofibrils are well-characterized biochemically and are conserved across the animal kingdom, the mechanisms leading to the precise assembly of sarcomeres, the basic units of myofibrils, are poorly understood. To gain insights into this process, we investigated the functional relationships of sarcomeric protein complexes. Specifically, we systematically analyzed, using either RNAi in primary muscle cells or available genetic mutations, the organization of myofibrils in Drosophila muscles that lack one or more sarcomeric proteins. Our study reveals that the thin and thick filaments are mutually dependent on each other for striation. Further, the tension sensor complex comprised of zipper/Zasp/α-actinin is involved in stabilizing the sarcomere but not in its initial formation. Finally, integrins appear essential for the interdigitation of thin and thick filaments that occurs prior to striation. Thus, sarcomere formation occurs by the coordinated assembly of multiple latent protein complexes, as opposed to sequential assembly

Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division

Congenital Plasmodium falciparum infection in neonates in Muheza District, Tanzania

BACKGROUND\ud \ud Although recent reports on congenital malaria suggest that the incidence is increasing, it is difficult to determine whether the clinical disease is due to parasites acquired before delivery or as a result of contamination by maternal blood at birth. Understanding of the method of parasite acquisition is important for estimating the time incidence of congenital malaria and design of preventive measures. The aim of this study was to determine whether the first Plasmodium falciparum malaria disease in infants is due to same parasites present on the placenta at birth.\ud \ud METHODS\ud \ud Babies born to mothers with P. falciparum parasites on the placenta detected by PCR were followed up to two years and observed for malaria episodes. Paired placental and infant peripheral blood samples at first malaria episode within first three months of life were genotyped (msp2) to determine genetic relatedness. Selected amplifications from nested PCR were sequenced and compared between pairs.\ud \ud RESULTS\ud \ud Eighteen (19.1%) out of 95 infants who were followed up developed clinical malaria within the first three months of age. Eight pairs (60%) out of 14 pairs of sequenced placental and cord samples were genetically related while six (40%) were genetically unrelated. One pair (14.3%) out of seven pairs of sequenced placental and infants samples were genetically related. In addition, infants born from primigravidae mothers were more likely to be infected with P. falciparum (P < 0.001) as compared to infants from secundigravidae and multigravidae mothers during the two years of follow up. Infants from multigravidae mothers got the first P. falciparum infection earlier than those from secundigravidae and primigravidae mothers (RR = 1.43).\ud \ud CONCLUSION\ud \ud Plasmodium falciparum malaria parasites present on the placenta as detected by PCR are more likely to result in clinical disease (congenital malaria) in the infant during the first three months of life. However, sequencing data seem to question the validity of this likelihood. Therefore, the relationship between placental parasites and first clinical disease need to be confirmed in larger studies

Glutamate May Be an Efferent Transmitter That Elicits Inhibition in Mouse Taste Buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (∼50%) respond to 100 µM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca2+. In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 µM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors

Amiloride-sensitive channels in type I fungiform taste cells in mouse

<p>Abstract</p> <p>Background</p> <p>Taste buds are the sensory organs of taste perception. Three types of taste cells have been described. Type I cells have voltage-gated outward currents, but lack voltage-gated inward currents. These cells have been presumed to play only a support role in the taste bud. Type II cells have voltage-gated Na⁺and K⁺current, and the receptors and transduction machinery for bitter, sweet, and umami taste stimuli. Type III cells have voltage-gated Na⁺, K⁺, and Ca²⁺currents, and make prominent synapses with afferent nerve fibers. Na⁺salt transduction in part involves amiloride-sensitive epithelial sodium channels (ENaCs). In rodents, these channels are located in taste cells of fungiform papillae on the anterior part of the tongue innervated by the chorda tympani nerve. However, the taste cell type that expresses ENaCs is not known. This study used whole cell recordings of single fungiform taste cells of transgenic mice expressing GFP in Type II taste cells to identify the taste cells responding to amiloride. We also used immunocytochemistry to further define and compare cell types in fungiform and circumvallate taste buds of these mice.</p> <p>Results</p> <p>Taste cell types were identified by their response to depolarizing voltage steps and their presence or absence of GFP fluorescence. TRPM5-GFP taste cells expressed large voltage-gated Na⁺and K⁺currents, but lacked voltage-gated Ca²⁺currents, as expected from previous studies. Approximately half of the unlabeled cells had similar membrane properties, suggesting they comprise a separate population of Type II cells. The other half expressed voltage-gated outward currents only, typical of Type I cells. A single taste cell had voltage-gated Ca²⁺current characteristic of Type III cells. Responses to amiloride occurred only in cells that lacked voltage-gated inward currents. Immunocytochemistry showed that fungiform taste buds have significantly fewer Type II cells expressing PLC signalling components, and significantly fewer Type III cells than circumvallate taste buds.</p> <p>Conclusion</p> <p>The principal finding is that amiloride-sensitive Na⁺channels appear to be expressed in cells that lack voltage-gated inward currents, likely the Type I taste cells. These cells were previously assumed to provide only a support function in the taste bud.</p

Enhanced Hippocampal Long-Term Potentiation and Fear Memory in Btbd9 Mutant Mice

Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS