Mechanisms of secretory cargo sorting at the trans-Golgi network

Protein sorting, processing and trafficking are central processes that maintain cellular functions, migration and homeostasis, ultimately defining health or disease of the organism. The secretory pathway comprises the compartments where these processes are realized. In this thesis the focus lies in particular on the last station of the Golgi apparatus (GA) and main sorting hub - the trans-Golgi network (TGN). There, proteins that have been modified post-translationally in earlier Golgi compartments, are sorted by their features, such as a transmembrane domain or a tag via certain receptors to intracellular destinations (e.g. endosomes, lysosomes) or for secretion (to apical and basolateral plasma membrane). Importantly, not all soluble secretory proteins are known to be recognized by a certain receptor, however, they are still sorted specifically to their final destinations. One mechanism involving the Ca2+-binding protein Cab45 was reported to facilitate the sorting of some secretory soluble proteins in the TGN (von Blume et al., 2011, 2012; Crevenna et al., 2016; Deng et al., 2018, Hecht et al., 2020): Ca2+-influx by secretory Ca2+- ATPase (SPCA1) in the TGN lumen after activation via actin/cofilin, leads to oligomerization of Cab45, which can bind its cargo (client) proteins and form Cab45-client-complexes that cluster and remain in the TGN lumen. Upon phosphorylation by Golgi kinase Fam20C the clusters are broken down for packaging into SM-rich vesicles destined for secretion. However, how these Cab45-client complexes are recognized for packaging into specific vesicles is unclear. In this thesis one more component is added to the machinery, the transmembrane protein TGN46, found in proximity-labelling approaches (BioID, APEX ; Deng et al., 2018), and shown here to be involved in the sorting of Cab45 client Lysozyme C (LyzC) by possibly indirect interaction with Cab45. Depletion of TGN46 indicates a delayed formation of post-Golgi vesicles containing LyzC in Retention-using-selective-hooks (RUSH)-based experiments and missorting by reduced co-localization with sphingomyelin (SM)-rich vesicles. Results that are similar to the phenotypes described previously for dysfunctional Cab45 and depletion of other components of the Cab45-dependent sorting mechanism (Deng et al., 2016; Hecht et al., 2020). In addition to this secretion-delayed phenotype of Cab45 clients upon deletion of TGN46, a novel hypersecretion of lysosomal hydrolases was observed with depletion of Cab45 in secretome analysis with subsequent mass spectrometric analysis. Secretion assays confirmed the influence of Cab45 depletion on increased secretion of prosaposin (PSAP), progranulin (PGRN) and with lesser degree Cathepsin D (CatD) implying a new role of Cab45 in sorting of lysosomal proteins. Secretion assays using cells with loss of lysosomal protein receptor sortilin and cation-independent mannose-6-phosphate receptor (M6PR) and Cab45 siRNA treatment indicated an influence of Cab45 on receptor-cargo interactions necessary for correct sorting. This hypothesis was substantiated by RUSH-based live-cell imaging observing the TGN export of PSAP in Cab45 knockout, -Ca2+-binding mutant and -wt rescue compared to control cells. Additionally, lysosomal positioning and co-localization of PSAP with lysosomes was altered with Cab45 deletion. The study gives evidence for the herein new proposed role of Cab45 in receptor-dependent lysosomal sorting in addition to the already reported receptor-independent sorting of soluble secretory proteins, which is extended with TGN46 as a new component

Postural Support Device for Children in Low-Income Families

ME450 Capstone Design and Manufacturing Experience: Winter 2021Alison and Monica, two girls in Nicaragua with cerebral palsy, are in need of a low-cost, adjustable, and accessible seating device to provide them proper postural support by promoting alignment in their head, neck, spine, and legs, in order to prevent further side effects that result from improper posture over long periods of time due to cerebral palsy. This project aims to create a solution for this need and create a prototype that will be further developed by Bluelab to send down to the girls in the future.Bluelab Nicaragua: FNE International, Salud Para Todos Los Niñoshttp://deepblue.lib.umich.edu/bitstream/2027.42/167637/1/Team_20-Postural_Support_Device_for_Children_in_Low-Income_Families.pd

COPPER-MODIFIED MCM-22 AS CATALYSTS FOR HYDROCARBON SELECTIVE CATALYTIC REDUCTION OF NOX

Joint Research on Environmental Science and Technology for the Eart

Fe-MCM-22 zeolites: synthesis and study about the states of iron

The Fe-MCM-22 zeolite was successfully synthesized with hexametylenimine template. Several physicochemical techniques (XRD, SEM, BET, AAS, IR and ESR) have been used to characterize this zeolite. Iron exists under three states: isolated ions in tetrahedral lattice positions, in octahedral coordination as isolated ions at cationic positions and as aggregated oxide species or hydroxide phases.Keywords: Fe-MCM-22 zeolite, synthesis, characterization, framework iron

Randomized controlled trial of artesunate or artemether in Vietnamese adults with severe falciparum malaria

<p>Abstract</p> <p>Background</p> <p>Both artemether and artesunate have been shown to be superior to quinine for the treatment of severe falciparum malaria in Southeast Asian adults, although the magnitude of the superiority has been greater for artesunate than artemether. These two artemisinin derivatives had not been compared in a randomized trial.</p> <p>Methods</p> <p>A randomized double blind trial in 370 adults with severe falciparum malaria; 186 received intramuscular artesunate (2.4 mg/kg immediately followed by 1.2 mg/kg at 12 hours then 24 hours then daily) and 184 received intramuscular artemether (3.6 mg per kilogram immediately followed by 1.8 mg per kilogram daily) was conducted in Viet Nam. Both drugs were given for a minimum of 72 hours.</p> <p>Results</p> <p>There were 13 deaths in the artesunate group (7 percent) and 24 in the artemether group (13 percent); P = 0.052; relative risk of death in the patients given artesunate, 0.54; (95 percent confidence interval 0.28-1.02). Parasitaemia declined more rapidly in the artesunate group. Both drugs were very well tolerated.</p> <p>Conclusions</p> <p>Intramuscular artesunate may be superior to intramuscular artemether for the treatment of severe malaria in adults.</p

Performance of Sampling/Resampling-based Particle Filters Applied to Non-Linear Problems

In this work, we propose a wireless body area sensor network (WBASN) to monitor patient position. Localization and tracking are enhanced by improving the effect of the received signal strength (RSS) variation. First, we propose a modified particle filter (PF) that adjusts resampling parameters for the Kullback-Leibler distance (KLD)-resampling algorithm to ameliorate the effect of RSS variation by generating a sample set near the high-likelihood region. The key issue of this method is to use a resampling parameter lower bound for reducing both the root mean square error (RMSE) and the mean number of particles used. To determine this lower bound, an optimal algorithm is proposed based on the maximum RMSE between the proposed algorithm and the KLD-resampling algorithm or based on the maximum mean number of particles used of these algorithms. Finally, PFs based on KLD-sampling and KLD-resampling are proposed to minimize the efficient number of particles and to reduce the estimation error compared to traditional algorithms

ATM induces MacroD2 nuclear export upon DNA damage

ADP-ribosylation is a dynamic post-translation modification that regulates the early phase of various DNA repair pathways by recruiting repair factors to chromatin. ADP-ribosylation levels are defined by the activities of specific transferases and hydrolases. However, except for the transferase PARP1/ARDT1 little is known about regulation of these enzymes. We found that MacroD2, a mono-ADP-ribosylhydrolase, is exported from the nucleus upon DNA damage, and that this nuclear export is induced by ATM activity. We show that the export is dependent on the phosphorylation of two SQ/TQ motifs, suggesting a novel direct interaction between ATM and ADP-ribosylation. Lastly, we show that MacroD2 nuclear export temporally restricts its recruitment to DNA lesions, which may decrease the net ADP-ribosylhydrolase activity at the site of DNA damage. Together, our results identify a novel feedback regulation between two crucial DNA damage-induced signaling pathways: ADP-ribosylation and ATM activation

Combination Antifungal Therapy for Cryptococcal Meningitis

Background Combination antifungal therapy (amphotericin B deoxycholate and flucytosine) is the recommended treatment for cryptococcal meningitis but has not been shown to reduce mortality, as compared with amphotericin B alone. We performed a randomized, controlled trial to determine whether combining flucytosine or high-dose fluconazole with high-dose amphotericin B improved survival at 14 and 70 days. Methods We conducted a randomized, three-group, open-label trial of induction therapy for cryptococcal meningitis in patients with human immunodeficiency virus infection. All patients received amphotericin B at a dose of 1 mg per kilogram of body weight per day; patients in group 1 were treated for 4 weeks, and those in groups 2 and 3 for 2 weeks. Patients in group 2 concurrently received flucytosine at a dose of 100 mg per kilogram per day for 2 weeks, and those in group 3 concurrently received fluconazole at a dose of 400 mg twice daily for 2 weeks. Results A total of 299 patients were enrolled. Fewer deaths occurred by days 14 and 70 among patients receiving amphotericin B and flucytosine than among those receiving amphotericin B alone (15 vs. 25 deaths by day 14; hazard ratio, 0.57; 95% confidence interval [CI], 0.30 to 1.08; unadjusted P=0.08; and 30 vs. 44 deaths by day 70; hazard ratio, 0.61; 95% CI, 0.39 to 0.97; unadjusted P=0.04). Combination therapy with fluconazole had no significant effect on survival, as compared with monotherapy (hazard ratio for death by 14 days, 0.78; 95% CI, 0.44 to 1.41; P=0.42; hazard ratio for death by 70 days, 0.71; 95% CI, 0.45 to 1.11; P=0.13). Amphotericin B plus flucytosine was associated with significantly increased rates of yeast clearance from cerebrospinal fluid (−0.42 log10 colony-forming units [CFU] per milliliter per day vs. −0.31 and −0.32 log10 CFU per milliliter per day in groups 1 and 3, respectively; P<0.001 for both comparisons). Rates of adverse events were similar in all groups, although neutropenia was more frequent in patients receiving a combination therapy. Conclusions Amphotericin B plus flucytosine, as compared with amphotericin B alone, is associated with improved survival among patients with cryptococcal meningitis. A survival benefit of amphotericin B plus fluconazole was not found

A preliminary study to establish the transfected CHO cell lines which highly express Trastuzumab - A biosimilar product of Herceptin

Human epidermal growth factor receptor 2 (HER2) has been identified as a molecular target for breast cancer therapy, such as Trastuzumab (Herceptin®). This has been shown to improve patient survival substantially. The current study is aiming to locally produce an anti-HER2 monoclonal antibody (named Trastuzumab) which has an equivalent biological properties in comparison with the original version, Herceptin®). In silico design and construction of recombinant vectors, as well as the establishment of transfected cell lines with high expression of Trastuzumab were performed. Based on the protein sequences obtained from the Drugbank, the DNA sequences encoding for the light chain (Tras-Lc) and heavy chain (Tras-Hc) of Trastuzumab were optimized and integrated into pNanogen-Hygro and pNanogen-Puro vectors, respectively. The Neon Transfection System was used to co-transfect the pNanogen-Tras-Lc-Hygro and pNanogen-Tras-Hc-Puro constructs into CHO cells. Different co-transfected single-cell-colonies selected on media supplemented with hygromycin and puromycin were used for ELISA and SDS-PAGE assays to identify the CHO cell lines which highly express Trastuzumab. Based on the present results, 30μg of both constructs were suitable for DNA co-transfection. After 07 days of culture, the highest amount of Trastuzumab (561 µg/ml) was obtained from the H06LD68 cell line