760 research outputs found

    Video Magnification for Structural Analysis Testing

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    The goal of this thesis is to allow a user to see minute motion of an object at different frequencies, using a computer program, to aid in vibration testing analysis without the use of complex setups of accelerometers or expensive laser vibrometers. MIT’s phase-based video motion processing ­was modified to enable modal determination of structures in the field using a cell phone camera. The algorithm was modified by implementing a stabilization algorithm and permitting the magnification filter to operate on multiple frequency ranges to enable visualization of the natural frequencies of structures in the field. To implement multiple frequency ranges a new function was developed to implement the magnification filter at each relevant frequency range within the original video. The stabilization algorithm would allow for a camera to be hand-held instead of requiring a tripod mount. The following methods for stabilization were tested: fixed point video stabilization and image registration. Neither method removed the global motion from the hand-held video, even after masking was implemented, which resulted in poor results. Specifically, fixed point did not remove much motion or created sharp motions and image registration introduced a pulsing effect. The best results occurred when the object being observed had contrast from the background, was the largest feature in the video frame, and the video was captured from a tripod at an appropriate angle. The final program can amplify the motion in user selected frequency bands and can be used as an aid in structural analysis testing

    Regulation of gene expression in specific mouse brain cells during neurodegenerative prion disease

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    Why neurodegenerative diseases target specific brain regions is poorly understood. We hypothesize that this selective vulnerability is caused by specific brain cells in these regions having unique strategies and capacities to cope with various disease related protein conformers that ultimately fail. To study how specific cell types in the mouse brain respond to a neurodegenerative disease we used prion infection (RML scrapie strain) as a model of neurodegenerative disease. Prion infection was combined with the RiboTag method to isolate ribosome associated, actively translated mRNA from specific brain cell types. Mice expressing the epitope-tagged ribosomes specifically in astrocytes or subsets of neurons, including glutamatergic, GABAergic, parvalbumin and somatostatin neurons, were injected with brain homogenate from either normal or prion infected mice. Early changes to gene expression were analyzed by next generation sequencing at a stage when clinical signs first become apparent (disease onset) and at a much earlier stage (preclinical time point) in the disease process. Neuropathological changes like microglia activation, astrogliosis, aggregated prions and spongiosis were analyzed by different immunohistochemistry stainings. This work gives clues into which cells are affected earliest and how they respond to the emerging disease. Investigating cell-type-specific mechanisms of selective vulnerability are needed for a better understanding of mechanism in neurodegenerative diseases and developing therapies

    Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis

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    Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call ‘Organ-On-VascularNet’. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting

    Generation and characterization of a mouse model for the achalasia, alacrima and mental retardation (AAMR) syndrome

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    The AAMR syndrome is characterized by mental retardation and gait abnormalities as well as achalasia and alacrima. This disorder is inherited as an autosomal recessive trait and is caused by mutations in the Guanosine-diphosphate-(GDP)-mannose-pyrophosphorylase A (GMPPA) gene. GMPPA encodes the 420 aa protein GMPPA. Its homolog, the GDP-mannose-pyrophosphorylase B (GMPPB), converts mannose-1-phosphate and guanosine triphosphate (GTP) to GDP-mannose, which is an essential substrate for glycosylation. Up to date, 18 patients with inactivating mutations in the GMPPA gene have been reported worldwide. To elucidate the function of GMPPA in more detail we generated a Gmppa knockout (KO) mouse model. Importantly, these mice recapitulate many features of human AAMR syndrome patients, e.g. homozygous Gmppa KO mice show structural brain alterations. Moreover, Gmppa KO mice show a progressive gait disorder with muscle weakness accompanied by centralization of nuclei, alterations of the mean fiber diameter, the distribution of extracellular matrix (ECM) proteins and of the Z-disc related protein α-Actinin. In immunoblot analysis we found hyperglycosylation of proteins, especially hyperglycosylation of alpha-Dystroglycan (α-DG). Hyperglycosylated α-DG shows decreased protein stability and an increased binding to ECM proteins. Suggesting that the observed muscle phenotype is muscle intrinsic, sciatic nerve structure and nerve conduction velocities are normal in Gmppa KO mice. Mechanistically, elevated GDP-mannose levels and a direct interaction of GMPPA with GMPPB support a role of GMPPA as an allosteric feedback inhibitor of GMPPB. Gmppa knockdown studies in myoblasts revealed an increased α-DG turnover and activation of ERK signaling. In mice, a mannose-depleted diet dramatically improved the motor phenotype and almost normalized glycosylation of α-DG and ERK signaling. Thus, we propose that AAMR syndrome caused by GMPPA mutations is at least in part a treatable condition.Beim AAMR-Syndrom leiden Betroffene unter einer geistigen Behinderung, Gangstörungen sowie Achalasie und Alakrimie. Diese autosomal-rezessiv vererbbare Erkrankung wird durch Mutationen im Guanosin-Diphosphat-(GDP)-Mannose-Pyrophosphorylase A (GMPPA) Gen hervorgerufen, welches das 420 AminosĂ€uren lange Protein GMPPA kodiert. Das Homolog von GMPPA, die GDP-Mannose-Pyrophosphorylase B (GMPPB), konvertiert Mannose-1-Phosphat und Guanosin-Triphosphat (GTP) zu GDP-Mannose, welches ein essentielles Substrat der Glykosylierungskette darstellt. Bis heute sind nur 18 Patienten mit GMPPA-Mutationen beschrieben worden. Um die Funktion von GMPPA nĂ€her zu beleuchten, haben wir ein Gmppa-Knockout-(KO)-Mausmodell generiert. Diese MĂ€use zeigen ein Krankheitsbild das viele Aspekte der humanen Patienten wiederspiegelt, so zeigen homozygote Gmppa-KO-MĂ€use kognitive Defekte sowie strukturelle VerĂ€nderungen des Gehirns. Des Weiteren zeigen Gmppa-KO-MĂ€use eine progressive Gangstörung mit zunehmender MuskelschwĂ€che sowie VerĂ€nderungen extrazellulĂ€rer Matrix-(ECM) und Z-Disk-Proteine. Wir konnten an Gewebe-Lysaten zeigen, dass Proteine von Gmppa-KO-MĂ€usen, insbesondere auch alpha-Dystroglykan (α-DG), hyperglykosyliert sind. Die Hyperglykosylierung von α-DG fĂŒhrt zu einer verminderten ProteinstabilitĂ€t und einer erhöhten Bindung an ECM-Proteine. Da in Gmppa-KO-MĂ€usen der Ischiasnerv morphologisch unverĂ€ndert war und die elektrophysiologischen Eigenschaften unauffĂ€llig waren, gehen wir von einem muskelintrinsischen Defekt aus. Da die GDP-Mannose-Spiegel im Gewebe erhöht waren und eine direkte Interaktion von GMPPA mit GMPPB gezeigt werden konnte, vermuten wir, dass GMPPA als allosterischer Inhibitor von GMPPB fungiert. Gmppa-knockdown-Studien in Myoblasten lassen auf eine Aktivierung des ERK-Signalweges schließen. Wir konnten durch Gabe einer Mannose-freien DiĂ€t eine erhebliche Verbesserung der motorischen AuffĂ€lligkeiten sowie eine fast vollstĂ€ndige Normalisierung der Glykosylierung und der ERK-Aktivierung erreichen. Möglicherweise ist eine Mannose-freie DiĂ€t auch eine therapeutische Option fĂŒr Patienten

    Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche

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    Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-alpha (LXR-alpha). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-alpha via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity

    Advanced sensors technology survey

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    This project assesses the state-of-the-art in advanced or 'smart' sensors technology for NASA Life Sciences research applications with an emphasis on those sensors with potential applications on the space station freedom (SSF). The objectives are: (1) to conduct literature reviews on relevant advanced sensor technology; (2) to interview various scientists and engineers in industry, academia, and government who are knowledgeable on this topic; (3) to provide viewpoints and opinions regarding the potential applications of this technology on the SSF; and (4) to provide summary charts of relevant technologies and centers where these technologies are being developed

    An injury survey and biomechanical analysis of strength and conditioning exercises and maximal hiking test (HM180) in junior sailors

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    In Olympic sailing, the hiking position is adopted by sailors to counteract forces of the wind acting on the sail and. improve boat speed. Hiking is widely regarded as the main physical challenge faced by single-handed dinghy sailors and senior dinghy sailors are known to have high rates of low back and knee injury. However, the extent of these injuries in junior sailors is yet to be reported. Although strength and conditioning exercises have been prescribed to enhance performance and prevent injury in sailors, little is known about these exercises in comparison to the demands placed on the sailor\u27s musculature whilst hiking maximally. Therefore, the first aim of this study was to determine the incidence of back and knee pain in a group of 29 male and female sailors from the Singaporean National Byte Class training squad (n=12) and the Singapore High Participation Group (n=17). Utilising this group of participants, the second aim of the study was to compare the levels of muscle (EMG) activation in four muscle bilaterally (rectus abdominus, superficial lumbar multifidus, vastus lateralis, and biceps femoris) in selected strength and conditioning exercises (leg extension, back squat, back extension, 30 second isometric hiking hold) and a three-minute maximal hiking test performed on a hiking simulator (called the HM180). In the first part of this study, there was a low incidence of low back pain and knee pain (14.8% and 31% respectively). Results from the second part of this study indicated that both the leg extension and back squat are capable of providing an overload stimulus for the HM180 test. However, higher than expected activation of the lumbar multifidus during the back squat exercise suggests that the leg extension exercise is an appropriate exercise for development. of quadriceps strength whilst squatting technique is refined. When comparing the level of muscle activation in the strength and conditioning exercises to the HM180 test, it was evident that the level of muscle activation was greater for; 1) the superficial lumbar multifidus in the back extension exercise, 2) the rectus abominus in the hiking hold exercise, and 3) the vastus lateralis muscle in the back squat and leg extension exercises. Between-ability group and between-gender comparisons for the HM18o test revealed that significant differences existed (p=0.002 and p=0.027 respectively). Findings from this study which examined a developmental sailing cohort has the potential to inform practical decision making in everyday exercise prescription

    Environmental and Neurogenetic Framework of Mate-Choice Relevant Behaviors in Xiphophorus Fishes

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    Mate-choice related behaviors are highly variable and sensitive to a wide array of environmental and social factors. Therefore, the stability of a given behavior can largely depend on the level of environmental variability within a population. My research aims to understand the mechanisms whereby behaviors are influenced by social conditions and other environmental factors. I first describe the level of preference variation within a population of swordtail fish across time and small-scale space. Over three years, I found marked, but highly variable differences in female mating preferences between sampling sites. These results highlight the importance of accounting for small-scale heterogeneity when modelling and measuring the evolution of mating preferences and display traits, and may help explain why empirical measures of sexual selection via mate choice are often very weak. Next, I take advantage of the socially-sensitive olfactory mating preferences of female Xiphophorus birchmanni to elucidate the neurogenetic mechanisms by which these preferences are learned. I compare whole brain and olfactory epithelial gene expression profiles of females that were socially isolated from adults, or exposed to either adult conspecifics or members of the closely related X. malinche. I found that conspecific-exposed females experienced an upregulation of genes with functional roles in immune response and the detection of visual and olfactory cues. Meanwhile, heterospecific-exposed females showed upregulation of genes involved in neurogenesis and synaptic transmission, suggesting a prioritization of processing sensory cues. Lastly, I used this same system to determine the role of cultural transmission -- the intergenerational transfer of information -- in shaping male and female personalities. I found that both males and females learn to develop boldness behaviors similar to those of their exposure models. These culturally-sensitive personalities are likely to have important mate choice and evolutionary implications. Together, these studies describe the complex direct and indirect relationships between the environment and female mate choice

    Implementation of Digital Technologies on Beverage Fermentation

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    In the food and beverage industries, implementing novel methods using digital technologies such as artificial intelligence (AI), sensors, robotics, computer vision, machine learning (ML), and sensory analysis using augmented reality (AR) has become critical to maintaining and increasing the products’ quality traits and international competitiveness, especially within the past five years. Fermented beverages have been one of the most researched industries to implement these technologies to assess product composition and improve production processes and product quality. This Special Issue (SI) is focused on the latest research on the application of digital technologies on beverage fermentation monitoring and the improvement of processing performance, product quality and sensory acceptability

    XLF-Dependent Nonhomologous End Joining of Complex DNA Double-Strand Breaks with Proximal Thymine Glycol and Screening for XRCC4-XLF Interaction Inhibitors

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    DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (\u3e10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. XLFL115D mutant completely eliminates ligation of all five substrates and previous X‑ray crystallography shows that XLF binds to XRCC4 via a “leucine lock” motif wherein L115 of XLF slips into a hydrophobic pocket in XRCC4. This makes the XRCC4-XLF interaction a good target to develop peptide inhibitors in order to radiosensitize breast tumor cells that are dependent on NHEJ to repair their DSBs after ionizing radiation exposure. Using mRNA display, we created a diverse library of 870 billion unique peptide sequences. After seven rounds of in vitro selection, the eluted fusions were cloned and sequenced. The results showed homology of sequences of five main families. We have selected representative peptides from those families (Pep 7.1-7.5), and several were chemically synthesized. However, none of these significantly inhibited XLF-dependent end joining in whole-cell extracts. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. The effectiveness of XLF-XLRCC4 inhibitors in blocking nonhomologous end joining remains to be determined
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