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2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Lin Lin ◽  
Jie Chen ◽  
Yingying Hu ◽  
Huapan Fang ◽  
Kui Wang ◽  
...  

Polyethyleneimine (PEI) has been extensively used as a common gene carrier due to its high gene transfection efficiency. PEI1.8k shows significantly lower cytotoxicity than its high molecular weight counterparts. However, it also has the problem of low gene transfection efficiency. To address the dilemma, a highly effective crosslinker (DTME) was synthesized to react with PEI1.8k to obtain CS-PEI1.8k. The reaction showed several advantages, such as a fast process in room temperature within nine hours with the product which can directly complex with DNA after removing the solvent. The ability of CS-PEI1.8k to agglomerate with DNA was proven by particle size, zeta potential, and gel retardation assays. The cytotoxic in vitro transfection ability and cell internalization capacity of CS-PEI1.8k were tested to verify the transfection capacity of CS-PEI1.8k. Moreover, we also studied the mechanism of the relatively high level of gene transfection by this binary complex compared with PEI25k.


PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260786
Author(s):  
Bhargavi Bhat ◽  
Shuhao Liu ◽  
Yu-Ting Lin ◽  
Martin L. Sentmanat ◽  
Joseph Kwon ◽  
...  

Hydraulic fracturing of unconventional reservoirs has seen a boom in the last century, as a means to fulfill the growing energy demand in the world. The fracturing fluid used in the process plays a substantial role in determining the results. Hence, several research and development efforts have been geared towards developing more sustainable, efficient, and improved fracturing fluids. Herein, we present a dynamic binary complex (DBC) solution, with potential to be useful in the hydraulic fracturing domain. It has a supramolecular structure formed by the self-assembly of low molecular weight viscosifiers (LMWVs) oleic acid and diethylenetriamine into an elongated entangled network under alkaline conditions. With less than 2 wt% constituents dispersed in aqueous solution, a viscous gel that exhibits high viscosities even under shear was formed. Key features include responsiveness to pH and salinity, and a zero-shear viscosity that could be tuned by a factor of ~280 by changing the pH. Furthermore, its viscous properties were more pronounced in the presence of salt. Sand settling tests revealed its potential to hold up sand particles for extended periods of time. In conclusion, this DBC solution system has potential to be utilized as a smart salt-responsive, pH-switchable hydraulic fracturing fluid.


2021 ◽  
Vol 8 ◽  
Author(s):  
Jongseo Park ◽  
Hyung-Seop Youn ◽  
Jun Yop An ◽  
Youngjin Lee ◽  
Soo Hyun Eom ◽  
...  

DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.


2021 ◽  
Author(s):  
Jiemin Shen ◽  
Gang Wu ◽  
Ah-Lim Tsai ◽  
Ming Zhou

Mammalian cytochrome b5 (cyt b5) and cytochrome b5 reductase (b5R) are electron carrier proteins required for many membrane-embedded oxidoreductases. Both cyt b5 and b5R have a cytosolic domain anchored to the membrane by a single transmembrane helix (TM). It is not clear if b5R, cyt b5 and their partner oxidoreductases assemble as binary or ternary complexes. Here we show that b5R and cyt b5 form a stable binary complex, and that b5R, cyt b5 and a membrane-embedded oxidoreductase, stearoyl-CoA desaturase 1 (SCD1) form a stable ternary complex. The formation of the complexes significantly enhances electron transfer rates, and that the single TM of cyt b5 and b5R mediated assembly of the complexes. These results reveal a novel functional role of TMs in cyt b5 and b5R and suggest that an electron transport chain composed of a stable ternary complex may be a general feature in oxidoreductases that require the participation of cyt b5 and b5R.


2021 ◽  
Author(s):  
Thibaud Dieudonne ◽  
Sara Abad Herrera ◽  
Michelle Juknaviciute Laursen ◽  
Maylis Lejeune ◽  
Charlott Stock ◽  
...  

P-type ATPases from the P4 subfamily (P4-ATPases) are primary active transporters that maintain lipid asymmetry in eukaryotic cell membranes by flipping lipids from the exoplasmic to the cytosolic leaflet. Mutations in several human P4-ATPase genes are associated with severe diseases. For instance, mutations in the ATP8B1 gene result in progressive familial intrahepatic cholestasis, a rare inherited disorder that usually progresses toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 angstrom resolution. The lipid flippase complex is autoinhibited by the N- and C-termini of ATP8B1, which in concert form extensive interactions with the catalytic sites and flexible domain interfaces of ATP8B1. Consistently, ATP hydrolysis by the ATP8B1-CDC50A complex requires truncation of its C-terminus as well as the presence of phosphoinositides, with a marked preference for phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminus further suggests molecular communication between N- and C-termini in the autoinhibition process and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A conserved (G/A)(Y/F)AFS motif in the C-termini of several P4-ATPase subfamilies suggests that this mechanism is employed widely across P4-ATPase lipid flippases, including both plasma membrane and endomembrane P4-ATPases.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi76-vi76
Author(s):  
Lakshmi Bollu ◽  
Prashant Bommi ◽  
Derek Wainwright ◽  
Erik Ladomersky ◽  
Lijie Zhai ◽  
...  

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes tryptophan and is expressed in >90% of patient-resected glioblastoma (GBM). IDO-mediated tryptophan metabolism has been the proposed mechanism for suppressing the immune response in GBM. Recently, we discovered that IDO also possesses non-enzymic functions that contribute to suppress the anti-GBM immune response. This finding motivated us to develop IDO-Proteolysis Targeting Chimeras (IDO-PROTACs) to degrade IDO protein rather than simply inhibiting IDO enzyme activity. METHODS Western blot analysis was used to determine IDO-PROTAC efficiency of IDO protein degradation among human and mouse GBM cell lines and PDX. Our lead IDO-PROTAC was tested for toxicity, blood brain barrier penetration, and pharmacokinetics (PK) in wild-type C57BL/6 mice. RESULTS IDO-PROTACs degrade IDO protein in both tumor and non-tumor cells with a DC50 value of ~0.5µM in human GBM tumor cells. Biolayer interferometry (BLI) studies show that IDO-PROTAC forms a binary complex with IDO protein with similar affinity comparable to parental compound – BMS986205. IDO-PROTACs induced IDO ubiquitination and the pretreatment with ubiquitin proteasome inhibitors, MG132 or MLN4924, inhibited IDO protein degradation. In vivo toxicity studies showed that treatment with IDO-PROTAC at 25 mg/kg for 3-weeks did not develop any apparent toxicity in C57BL/6 mice. PK analysis revealed that IDO-PROTAC bioavailabilty reached a peak serum and brain concentration within 30 minutes after intraperitoneal administration. CONCLUSIONS This study developed a lead IDO-PROTAC compound that efficiently degrades IDO protein in human GBM cells with a moderate bioavailability and blood-brain barrier (BBB) penetration. Future work will focus on the enhancement of BBB penetration, increased bioavailability, and route of administration to improve IDO-PROTAC potency for combination with other forms of immunotherapy for GBM patient treatment.


Author(s):  
Kaushik Bhattacharya ◽  
Didier Picard

AbstractThe Hsp70 and Hsp90 molecular chaperone systems are critical regulators of protein homeostasis (proteostasis) in eukaryotes under normal and stressed conditions. The Hsp70 and Hsp90 systems physically and functionally interact to ensure cellular proteostasis. Co-chaperones interact with Hsp70 and Hsp90 to regulate and to promote their molecular chaperone functions. Mammalian Hop, also called Stip1, and its budding yeast ortholog Sti1 are eukaryote-specific co-chaperones, which have been thought to be essential for substrate (“client”) transfer from Hsp70 to Hsp90. Substrate transfer is facilitated by the ability of Hop to interact simultaneously with Hsp70 and Hsp90 as part of a ternary complex. Intriguingly, in prokaryotes, which lack a Hop ortholog, the Hsp70 and Hsp90 orthologs interact directly. Recent evidence shows that eukaryotic Hsp70 and Hsp90 can also form a prokaryote-like binary chaperone complex in the absence of Hop, and that this binary complex displays enhanced protein folding and anti-aggregation activities. The canonical Hsp70-Hop-Hsp90 ternary chaperone complex is essential for optimal maturation and stability of a small subset of clients, including the glucocorticoid receptor, the tyrosine kinase v-Src, and the 26S/30S proteasome. Whereas many cancers have increased levels of Hop, the levels of Hop decrease in the aging human brain. Since Hop is not essential in all eukaryotic cells and organisms, tuning Hop levels or activity might be beneficial for the treatment of cancer and neurodegeneration.


2021 ◽  
pp. 104359
Author(s):  
Yanfei Li ◽  
Tong Geng ◽  
Ang Li ◽  
Huimin Yu

2021 ◽  
Author(s):  
Seyed Iraj Sadraei ◽  
Greg Yousif ◽  
S. Maryamdokht Taimoory ◽  
Maryam Kosar ◽  
Samaneh Mehri ◽  
...  

Invariant natural killer T cells (iNKT) are responsible for the production of pro-inflammatory cytokines which induce a systemic immune response. They are distinctive in possessing an invariant T-cell receptor that recognizes glycolipid antigens presented by the class I major histocompatibility complex-related protein CD1d, conserved across multiple mammalian species in a class of proteins well-renowned for their high degree of polymorphism. This receptor’s first identified antigen is the potent α-galactose ceramide, KRN7000, a glycosphingolipid isolated from bacteria that were found on a Japanese marine sponge. The corresponding terrestrial antigen remained unidentified until quite recently, when diacylglycerol-containing glycolipids, reported to activate iNKT cells, were isolated from Streptococcus pneumoniae. We report the total synthesis and immunological re-evaluation of these two glycolipids. The compounds are unable to meaningfully activate iNKT cells. Computational modelling shows that these ligands, while being capable of interacting with the CD1d receptor, create a different surface for the binary complex that makes formation of the ternary complex with the iNKT T-cell receptor difficult. Together these results suggest that the reported activity might have been due to an impurity in the original isolated sample and highlights the importance of taking care when reporting biological activity from isolated natural products.


Author(s):  
S. N. Chebotarev ◽  
R. R. Maksyutov

The article presents the results of experimental research on the development of a formulation and technology for the industrial production of kumis drink enriched with an iodine-glycoside complex, which is a source of molecular iodine with antioxidant activity. Study of the iodine level in patients with tuberculosis showed the following results: iodine deficiency was diagnosed in 86,7% of the total number of examined patients - the median ioduria in this group of patients ranged from 20 ?g/l to 99 ?g/l. The article describes a technology of production of kumis drink enriched with iodine-glycoside complex, including following operations: acceptance and preparation of raw materials; normalization and preparation of the mixture; pasteurization, homogenization, cooling and insertion of "iodine-glycoside"; preparation of the fermentation culture; fermentation and souring, bottling, maturation of the drink; packaging, labeling and cooling. It also includes the results of evaluating the organoleptic indicators of kumis drink - consistency, taste, color, smell and appearance, as well as the physical and chemical parameters: weight fraction of fat, protein, dry fat-free substances; the volume of settled whey, antioxidant balance in respiratory pathology by activating the KEAP1 /Nrf2 /ARE redox-sensitive system inducing the gene expression that control the endogenous antioxidants synthesis. Binary complex (kumis drink + molecular iodine) is considered here as a non-pharmacological pathogenetic agent. The technological process of the production of a functional product with anti-tuberculosis activity ensured the preservation of the physical and chemical parameters of the kumis drink, reducing the titratable acidity to 44,8% in the iodine-enriched drink against 57,6% in the control sample. The author elaborates on mechanisms of the participation of the “iodine-glycoside complex” and low-molecular organic substances, in particular, the amino acids of the kumis drink, in the regulation of pro-, with pleiotropic properties for the treatment and prevention of tuberculosis.


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