Observing the Temperature Dependent Transition of the GP2 Peptide Using Terahertz Spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T _D). In particular, we show that below T _D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T _D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Immunohistochemical Identification of Met-Enkephalin in Digestive System and Its Effect on Digestive Enzyme Activities of the Scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

The study was designed to determine whether methionine-enkephalin (M-ENK) was present in the digestive system of the scallop Chlamys farreri and investigate the effects of M-ENK on the activity of amylase, protease and lipase in the digestive system of C. farreri. The results indicated that M-ENK-like material was present in the epithelium and connective tissue of labial palps, mouth labia, stomach, intestine, rectum, and hepatopancreas of the scallop C. farreri. Moreover, it was also found that many isolated small cells showing M-ENK-like immunoreactivity were scattered in the epithelial layer of intestine, and many isolated big epithelial cells showing M-ENK-like immunoreactivity were scattered in tubules of hepatopancreas of the scallop. The activity levels of amylase and lipase in crystalline style, hepatopancreas and intestine were enhanced at 1 h after injection of exogenous M-ENK into adductor muscle of the scallops, whereas protease activity levels were significantly suppressed. Our report constitutes the first characterization of M-ENK in the digestive system of scallop C. farreri and investigates the effects of M-ENK on the activities of digestive enzymes of mollusk for the first time. The results suggest an involvement of M-ENK in the functional regulation of the digestive system of C. farreri.     

DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.