scholarly journals Characterization of the Modulatory Effect of Hydroxychloroquine on ACE2 Activity: New Insights in relation to COVID-19

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Juan E. Tichauer ◽  
Dagoberto Soto ◽  
Max Andresen
Keyword(s):  
Structural Changes ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
In Vitro System ◽  
Trial Outcomes ◽  
A Cell ◽  
The One ◽  
First Time

Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown the ability to inhibit in vitro viral replications of coronaviridae viruses such as SARS-CoV and SARS-CoV-2. However, clinical trial outcomes have been disparate, suggesting that CQ and HCQ antiviral mechanisms are not fully understood. Based on three-dimensional structural similarities between HCQ and the known ACE2 specific inhibitor MLN-4760, we compared their modulation on ACE2 activity. Here we describe, for the first time, in a cell-free in vitro system that HCQ directly and dose-dependently inhibits the activity of recombinant human ACE2, with a potency similar to the MLN-4760. Further analysis suggests that HCQ binds to a noncompetitive site other than the one occupied by MLN-4760. We also determined that the viral spike glycoprotein segment that comprises the RBD segment has no effect on ACE2 activity but unexpectedly was able to partially reverse the inhibition induced by HCQ but not that by MLN-4760. In summary, here we demonstrate the direct inhibitory action of HCQ over the activity of the enzyme ACE2. Then, by determining the activity of ACE2, we reveal that the interaction with the spike protein of SARS-CoV-2 leads to structural changes that at least partially displace the interaction of the said enzyme with HCQ. These results may help to explain why the effectiveness of HCQ in clinical trials has been so variable. Additionally, this knowledge could be used for to develop techniques for the detection of SARS-CoV-2.

Thermal characterization of ABS-Graphene blended three dimensional printed functional prototypes for electro chemical energy storage

2018 ◽  
Vol 1 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Kamaljit Singh Boparai ◽  
Rupinder Singh
Keyword(s):  
Energy Storage ◽  
Structural Changes ◽  
Fused Deposition Modeling ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Thermal Characterization ◽  
Chemical Energy ◽  
Flow Index ◽  
Fused Deposition

This study highlights the thermal characterization of ABS-Graphene blended three dimensional (3D) printed functional prototypes by fused deposition modeling (FDM) process. These functional prototypes have some applications as electro-chemical energy storage devices (EESD). Initially, the suitability of ABS-Graphene composite material for FDM applications has been examined by melt flow index (MFI) test. After establishing MFI, the feedstock filament for FDM has been prepared by an extrusion process. The fabricated filament has been used for printing 3D functional prototypes for printing of in-house EESD. The differential scanning calorimeter (DSC) analysis was conducted to understand the effect on glass transition temperature with the inclusion of Graphene (Gr) particles. It has been observed that the reinforced Gr particles act as a thermal reservoir (sink) and enhances its thermal/electrical conductivity. Also, FT-IR spectra realized the structural changes with the inclusion of Gr in ABS matrix. The results are supported by scanning electron microscopy (SEM) based micrographs for understanding the morphological changes.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates From Commercially Stored Apple Fruit

2017 ◽  
Vol 107 (3) ◽  
pp. 362-368 ◽  
Author(s):  
Wayne M. Jurick ◽  
Otilia Macarisin ◽  
Verneta L. Gaskins ◽  
Eunhee Park ◽  
Jiujiang Yu ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Gray Mold ◽  
Apple Fruit ◽  
Sublethal Dose ◽  
Long Term Storage ◽  
Pose Control ◽  
First Time

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 μg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals Characterization of Each St and Y Genome Chromosome of Roegneria grandis Based on Newly Developed FISH Markers

2021 ◽  
pp. 213-222
Author(s):  
Dandan Wu ◽  
Xiaoxia Zhu ◽  
Lu Tan ◽  
Haiqin Zhang ◽  
Lina Sha ◽  
...  
Keyword(s):  
Structural Changes ◽  
Distribution Patterns ◽  
Proximal Region ◽  
Genome Chromosome ◽  
High Affinity ◽  
Complete Set ◽  
Genome Analyses ◽  
First Time ◽  
Homoeologous Chromosomes

The genera of the tribe Triticeae (family Poaceae), constituting many economically important plants with abundant genetic resources, carry genomes such as St, H, P, and Y. The genome symbol of <i>Roegneria</i> C. Koch (Triticeae) is StY. The St and Y genomes are crucial in Triticeae, and tetraploid StY species participate extensively in polyploid speciation. Characterization of St and Y nonhomologous chromosomes in StY-genome species could help understand variation in the chromosome structure and differentiation of StY-containing species. However, the high genetic affinity between St and Y genome and the deficiency of a complete set of StY nonhomologous probes limit the identification of St and Y genomes and variation of chromosome structures among <i>Roegneria</i> species. We aimed to identify St- and Y-enhanced repeat clusters and to study whether homoeologous chromosomes between St and Y genomes could be accurately identified due to high affinity. We employed comparative genome analyses to identify St- and Y-enhanced repeat clusters and generated a FISH-based karyotype of <i>R. grandis</i> (Keng), one of the taxonomically controversial StY species, for the first time. We explored 4 novel repeat clusters (StY_34, StY_107, StY_90, and StY_93), which could specifically identify individual St and Y nonhomologous chromosomes. The clusters StY_107 and StY_90 could identify St and Y addition/substitution chromosomes against common wheat genetic backgrounds. The chromosomes V_St, VII_St, I_Y, V_Y, and VII_Y displayed similar probe distribution patterns in the proximal region, indicating that the high affinity between St and Y genome might result from chromosome rearrangements or transposable element insertion among V_St/Y, VII_St/Y, and I_Y chromosomes during allopolyploidization. Our results can be used to employ FISH further to uncover the precise karyotype based on colinearity of Triticeae species by using the wheat karyotype as reference, to analyze diverse populations of the same species to understand the intraspecific structural changes, and to generate the karyotype of different StY-containing species to understand the interspecific chromosome variation.

Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽  
1980 ◽  
Vol 28 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Luanne M. Deal ◽  
J. T. Reeves ◽  
B. A. Larkins ◽  
F. D. Hess
Keyword(s):  
Protein Synthesis ◽  
Sand Culture ◽  
Leucine Incorporation ◽  
Insoluble Protein ◽  
In Vitro System ◽  
A Cell ◽  
Protein Incorporation ◽  
Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

Scaffold topography alters intracellular calcium dynamics in cultured cardiomyocyte networks

2004 ◽  
Vol 287 (3) ◽  
pp. H1276-H1285 ◽  
Author(s):  
Lihong Yin ◽  
Harold Bien ◽  
Emilia Entcheva
Keyword(s):  
Ventricular Myocytes ◽  
Three Dimensional ◽  
Triggered Release ◽  
In Vitro System ◽  
Functional Changes ◽  
Cardiac Electromechanics ◽  
Intracellular Ca ◽  
Intracellular Calcium Dynamics ◽  
Cell Networks

Structural and functional changes ensue in cardiac cell networks when cells are guided by three-dimensional scaffold topography. We report enhanced synchronous pacemaking activity in association with slow diastolic rise in intracellular Ca2+ concentration ([Ca2+]i) in cell networks grown on microgrooved scaffolds. Topography-driven changes in cardiac electromechanics were characterized by the frequency dependence of [Ca2+]i in syncytial structures formed of ventricular myocytes cultured on microgrooved elastic scaffolds (G). Cells were electrically paced at 0.5–5 Hz, and [Ca2+]i was determined using microscale ratiometric (fura 2) fluorescence. Compared with flat (F) controls, the G networks exhibited elevated diastolic [Ca2+]i at higher frequencies, increased systolic [Ca2+]i across the entire frequency range, and steeper restitution of Ca2+ transient half-width ( n = 15 and 7 for G and F, respectively, P < 0.02). Significant differences in the frequency response of force-related parameters were also found, e.g., overall larger total area under the Ca2+ transients and faster adaptation of relaxation time to pacing rate ( P < 0.02). Altered [Ca2+]i dynamics were paralleled by higher occurrence of spontaneous Ca2+ release and increased sarcoplasmic reticulum load ( P < 0.02), indirectly assessed by caffeine-triggered release. Electromechanical instabilities, i.e., Ca2+ and voltage alternans, were more often observed in G samples. Taken together, these findings 1) represent some of the first functional electromechanical data for this in vitro system and 2) demonstrate direct influence of the microstructure on cardiac function and susceptibility to arrhythmias via Ca2+-dependent mechanisms. Overall, our results substantiate the idea of guiding cellular phenotype by cellular microenvironment, e.g., scaffold design in the context of tissue engineering.

scholarly journals Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

2005 ◽  
Vol 391 (2) ◽  
pp. 185-190 ◽  
Author(s):  
Renu Wadhwa ◽  
Syuichi Takano ◽  
Kamaljit Kaur ◽  
Satoshi Aida ◽  
Tomoko Yaguchi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Molecular Interactions ◽  
Heat Shock Protein 60 ◽  
Hsp60 Expression ◽  
Mitochondrial Hsp70 ◽  
Shock Proteins ◽  
First Time

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

Three-Dimensional Microfabrication System for Biodegradable Microdevices With High-Resolution and Bio-Applicability

2005 ◽  
Author(s):  
Akira Yamada ◽  
Fuminori Niikura ◽  
Koji Ikuta
Keyword(s):  
Tensile Strength ◽  
Biodegradable Polymers ◽  
Conventional Method ◽  
Three Dimensional ◽  
Single Layer ◽  
Batch Process ◽  
Potential Applications ◽  
A Cell ◽  
The One ◽  
Almost All

Biodegradable polymers are employed in medicine and its further application is expected with eagerness. But the lack of an appropriate processing method retards the progress. To overcome this problem, we have developped a novel three-dimensional microfabrication system. The system design allows us the processing of the free three-dimensional micro-level forms by stacking up melted polymers from the nozzle. Different from the conventional method, we adopted a batch process to supply materials in order to eliminate the prior process that required toxic solvents. In addition, it is possible to handle almost all biodegradable thermoplastic resins by adopting this system. A single layer from the piled-up layers of extruded lines was observed to evaluate the resolution. The lateral and depth resolutions attained are 40 μm and 45 μm, respectively. Biodegradable polymers enable three-dimensional microstructures such as micro-pipes, micro-bends, and micro-coil springs to be manufactured in less than 15 min. The biocompatibility of the newly fabricated structure was evaluated using a cell line (PC12). For this purpose, a small vessel, with a transparent base, was fabricated using PLA and cells were cultivated in it. The results were then compared with the results obtained using the standard method. The mechanical strength of our microstructures was evaluated using a tensile strength test. The tensile strength of the microstructure was lower than the one obtained from the conventional method, but has enough strength for fabrication of medical devices. Our system renders it possible to produce toxic-free, as well as transparent and leakage-free devices. Our system is expected to have potential applications in optimum design and fabrication of implantable devices, especially in tissue engineering.

scholarly journals THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

2018 ◽  
pp. 26-31
Author(s):  
I. V. Yakovleva ◽  
E. A. Kurbatova ◽  
E. A. Akhmatova ◽  
E. V. Sukhova ◽  
D. V. Yashunsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Solid Phase ◽  
Synthetic Analogue ◽  
Immunochemical Characterization ◽  
Carbohydrate Epitopes ◽  
First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

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