Controlling Solvent Polarity to Regulate Protein Self-Assembly Morphology and Its Universal Insight for Fibrillation Mechanism.

The mechanism of ethanol-induced fibrillation of ß-lactoglobulin (ß-lg) in the acidic aqueous solution upon heating was investigated using various techniques, mainly thioflavin T fluorescence, atomic force microscopy, nonreducing electrophoresis, mass spectrometry, Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. The results showed that fibrillation occurred with a heating time increase, but high ethanol content slowed down the process. At a low ethanol volume fraction, peptides existed after heating for 2 h, with long and straight fibrils formed after 4-6 h, while at a high ethanol volume fraction, the proteins aggregated with very few peptides appeared at the early stage of heating, and short and curved fibrils formed after heating for 8 h. Ethanol weakened the hydrophobic interactions between proteins in the aqueous solution; therefore the latter could not completely balance the electrostatic repulsion, and thus suppressing the fibrillation process. It is believed that the fibrillation of ß-lg in the acidic solution upon heating is mainly dominated by the polypeptide model; however, ethanol inhibited the hydrolysis of proteins, and the self-assembly mechanism changed to the monomer model.

Segmental analysis of liver cirrhosis with different etiologies: a study based on iodine mixed imaging in port-venous phase.

BACKGROUND: Computed tomography (CT) in port-venous phase can display the intra-hepatic vessels, and may provide the possibility for segment function evaluation for cirrhosis. PURPOSE: To assess the value of iodine mixed imaging of dual-source dual-energy CT in port-venous phase in segmental evaluation of liver cirrhosis with different etiologies. MATERIAL AND METHODS: Patients diagnosed with liver cirrhosis were enrolled. Patients without cirrhosis were included as a control group. Each patient underwent iodine-contrast enhanced multi-phase dual-energy CT scanning. Parameters were analyzed by SPSS, version 22.0, and Medcalc. RESULTS: In total, 256 patients were investigated, including 114 Child-Pugh A, 51 Child-Pugh B, 41 Child-Pugh C and 50 control patients. Total iodine content (ICt)/body surface area (BSA) in the cirrhosis group was significantly lower than the control group (P < 0.05) and the standardized-iodine parameter (SI) of each segment decreased with cirrhosis progression. In Child-Pugh A and B, SI increased more significantly in the caudal and lateral segment in A (alcholism) than in the V (virus-related) and N (non-alcoholic steatohepatitis) groups (P < 0.001). ICt/BSA showed the best diagnosis power of cirrhosis with an area under the curve of 0.765, sensitivity of 76.0% and specificity of 71.8%. CONCLUSION: Blood flow compensated in the left lateral and caudal lobe in the early stage of liver cirrhosis. The compensation in alcoholism in the middle and early stages is significantly higher than that of V and N cirrhosis. Iodine mixed imaging in portal phase may provide the possibility of an incremental value in segmented blood flow perfusion and functional evaluation of liver cirrhosis on a morphological basis.

Targeted curation of the gut microbial gene content modulating human cardiovascular disease.

IMPORTANCE: One of the most-cited examples of the gut microbiome modulating human disease is the microbial metabolism of quaternary amines from protein-rich foods. By-products of this microbial processing promote atherosclerotic heart disease, a leading cause of human mortality globally. Our research addresses current knowledge gaps in our understanding of this microbial metabolism by holistically inventorying the microorganisms and expressed genes catalyzing critical atherosclerosis-promoting and -ameliorating reactions in the human gut. This led to the creation of an open-access resource, the Methylated Amine Gene Inventory of Catabolism database, the first systematic inventory of gut methylated amine metabolism. More importantly, using this resource we deliver here, we show for the first time that these gut microbial genes can predict human disease, paving the way for microbiota-inspired diagnostics and interventions.

Insights into pyrrolysine function from structures of a trimethylamine methyltransferase and its corrinoid protein complex.

The 22nd genetically encoded amino acid, pyrrolysine, plays a unique role in the key step in the growth of methanogens on mono-, di-, and tri-methylamines by activating the methyl group of these substrates for transfer to a corrinoid cofactor. Previous crystal structures of the Methanosarcina barkeri monomethylamine methyltransferase elucidated the structure of pyrrolysine and provide insight into its role in monomethylamine activation. Herein, we report the second structure of a pyrrolysine-containing protein, the M. barkeri trimethylamine methyltransferase MttB, and its structure bound to sulfite, a substrate analog of trimethylamine. We also report the structure of MttB in complex with its cognate corrinoid protein MttC, which specifically receives the methyl group from the pyrrolysine-activated trimethylamine substrate during methanogenesis. Together these structures provide key insights into the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC.

Investigation of the Effect of Rice Bran Content on the Antioxidant Capacity and Related Molecular Conformations of Plant-Based Simulated Meat Based on Raman Spectroscopy.

At present, plant-based simulated meat is attracting more and more attention as a meat substitute. This study discusses the possibility of partial substitution of rice bran (RB) for soybean protein isolate (SPI) in preparing plant-based simulated meat. RB was added to SPI at 0%, 5%, 10%, 15%, and 20% to prepare RB-SPI plant-based simulated meat by the high moisture extrusion technique. RB-SPI plant-based simulated meat revealed greater polyphenol content and preferable antioxidant capacity (DPPH radical scavenging capacity, ABTS scavenging ability, and FRAP antioxidant capacity) compared to SPI plant-based simulated meat. The aromatic amino acids (tryptophan and tyrosine) of RB-SPI plant-based simulated meats tend to be masked first, and then the hydrophobic groups are exposed as RB content increases and the polarity of the surrounding environment increases due to the change in the disulfide conformation of RB-SPI plant-based simulated meats from a stable gauche-gauche-gauche conformation to a trans-gauche-trans conformation.

Effects of rice bran content on plant-based simulated meat: From the aspects of apparent properties and structural characteristics.

Rice Bran (RB) was added to soybean protein isolate (SPI) at 0%, 5%, 10%, 15% and 20% as addition to produce simulated meat by high moisture extrusion, and the apparent properties and structural characteristics of RB-SPI simulated meat were studied. The addition of 10% RB weakened the interaction among hydrogen bond (HB), hydrophobic bond (HI) and disulfide bond (DB), further increasing the hardness of simulated meat. Meanwhile, it decreased the content of intermolecular hydrogen bonding and enhanced the interaction between HI and HB, resulted in an increased tension. Adding 5% RB weakened the interaction between HB, HI and DB, decreased the content of random coils in the secondary structure, but strengthened the DB and ultimately increased the thermal stability of simulated meat.

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase.

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

Kinetic and substrate complex characterization of RamA, a corrinoid protein reductive activase from Methanosarcina barkeri.

In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.

Diffusion tensor imaging of breast lesions: evaluation of apparent diffusion coefficient and fractional anisotropy and tissue cellularity.

OBJECTIVE: To investigate the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) measured by diffusion tensor imaging (DTI), tissue cellularity and their relationship in breast malignant/benign lesions. METHODS: 88 patients with 88 breast lesions who underwent DTI and dynamic contrast-enhanced MR scanning between November 2013 and December 2014 were retrospectively analyzed. The diagnosis was confirmed pathologically. ADC and FA values as well as histopathological cellularity of different pathological types of lesions were analyzed and compared statistically. The Pearson's correlation between cellularity and ADC and FA was calculated. RESULTS: There were 59 cases of breast cancer and 29 cases of benign lesions included in the study. ADC values of breast cancers were statistically lower than that of benign lesions (p < 0.001). FA and cellularity were higher in cancers than in benign lesions with statistical significance (p < 0.05 and p < 0.001, respectively). The mean FA values in the patients with invasive ductal carcinoma (IDC) were higher than that in the patients with ductal carcinoma in situ (DCIS) without statistical difference (p > 0.05). The ADC and the cellularity in the IDC of grade III were statistically lower (p < 0.05) and higher (p < 0.05) than that in the DCIS and IDC of grade I-II, respectively. ADC was negatively correlated to cellularity (r = -0.8319, p < 0.001) and FA was positively correlated to cellularity (r = 0.4231, p < 0.001). CONCLUSION: ADC and FA values were statistically different between benign and malignant breast lesions and were significantly correlated to tissue cellularity. ADC and FA may help to discriminate malignant from benign breast lesions and to predict cellularity. ADC is helpful in the prediction of the grade of breast cancer. ADVANCES IN KNOWLEDGE: ADC and FA values were statistically different between benign and malignant breast lesions and were significantly correlated to tissue cellularity.

Assessing Detection, Discrimination, and Risk of Breast Cancer According to Anisotropy Parameters of Diffusion Tensor Imaging.

BACKGROUND The aim of this study was to investigate whether the anisotropy parameters are helpful in the detection and discrimination of breast cancers, and to determine its value in predicting the risk of cancers. MATERIAL AND METHODS There were 56 patients with 56 lesions (34 malignant, 22 benign) included in the study. DTI was performed in every patient and apparent diffusion coefficient (ADC), fractional anisotropy (FA), and eigenvalues E1, E2, and E3 were measured in every lesion and the normal breast tissue. RESULTS ADC, FA, and eigenvalues of E1, E2, E3, and E1-E3 in breast cancers were all significantly lower than in normal tissue (P<0.001 for all) with mean reduction of (32 ± 17)%, (24 ± 13)%, (33 ± 19)%, (32 ± 17)%, (31 ± 18)%, and (37 ± 20)% for ADC, FA, E1, E2, E3, and E1-E3, respectively. These parameters were also statistically lower in cancers than in benign lesions (P<0.01 for all), except FA (P>0.05). ADC, E1, E2, and E3 were very similar in discriminating breast cancers and benign lesions, with area under the curve (AUC) 0.885-0.898, sensitivity 73.5-85.3%, and specificity 90.9-100%. CONCLUSIONS ADC, E1, E2, E3, and E1-E3 are much lower in breast cancers than in normal tissue and benign lesions. The reduction of ADC, E1, E2, E3, and E1-E3 of a mass in the breast is highly associated with the risk of breast cancer, but the FA has no utility in breast cancer risk prediction.

PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine.

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA(Pyl). Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA(Pyl) in EMSA with apparent K(d) values of 0.12 and 0.13 µM, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA(Pyl) binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA(Pyl) binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA(Pyl) binding. Further indicating the specificity of PylSn interaction with tRNA(Pyl), substitutions of conserved residues in tRNA(Pyl) in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA(Pyl).

Functional context, biosynthesis, and genetic encoding of pyrrolysine.

In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In Escherichia coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two molecules of lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.

Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase.

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1A resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.

Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide.

AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer. METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis. RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax. CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.