Genetic and Functional Analyses of the Novel KLF11 Pro193Thr Variant in a Three-Generation Family with MODY7.

KLF11 regulates insulin gene expression through binding to the insulin promoter and has been reported as a causative gene for maturity-onset diabetes of the young 7 (MODY7). Here, we report a novel KLF11 variant associated with a three-generation family with early childhood-onset diabetes and explore its clinical and functional characteristics. The three-generational pedigree contains five patients affected by diabetes. The pathogenic variant identified by whole-exome sequencing was further confirmed by Sanger sequencing and pedigree verification. Luciferase reporter assays and glucose-stimulated insulin secretion were used to examine whether the KLF11 variant binds to the insulin promoter and regulate insulin secretion in vitro. The proband, his son, and his uncle exhibited hyperglycemia at ages 32, 13 and 71 years, respectively. All three patients showed characteristics of metabolic syndrome (obesity, dyslipidemia, and diabetes), but the insulin secretion of islet ß-cells was impaired. A novel heterozygous missense variant, c.577 C>A (p.Pro193Thr) of the KLF11 gene was detected in all three patients. This variant co-segregates with the diabetes phenotype, consistent with an autosomal dominant disorder. The identified KLF11 p.Pro193Thr variant drastically decreased the transcriptional activity of KLF11, as demonstrated by luciferase reporter assay. Functional analyses revealed that the KLF11 Pro193Thr variant inhibited glucose-stimulated insulin secretion. We identified a novel KLF11 Pro193Thr variant in a three generation family with MODY7. These findings shed light on the molecular mechanisms underlying the pathogenesis of MODY7 and expand the genotype and clinical spectrum of MODY7.

Identification of a novel mutation in patients with type A insulin resistance syndrome.

INTRODUCTION: Type A insulin resistance syndrome is a rare type of congenital insulin resistance often caused by heterozygous mutations in the insulin receptor gene (INSR). The aim of this study is to explore the clinical and genetic characteristics of three patients with type A insulin resistance syndrome from two Chinese families. METHODS: The peripheral blood samples were collected from each family members. Whole-exome sequencing were performed on three patients. RESULTS: Patient #1 was diagnosed with hyperinsulinemia at the age of 11 years and presented with hirsutism, acanthosis nigricans, and polycystic ovaries by 13 years. A heterozygous c.3470A > G mutation in the INSR gene was identified in patient #1. Patient #2 was a 13-year-old girl who presented with insulin resistance, polycystic ovary, and hyperandrogenemia. A novel c.3601C > G INSR mutation was identified in patient #2. Co-segregated analysis showed that the c.3601C > G mutation was also found in her father, who had hyperinsulinemia and diabetes mellitus, which was consistent with autosomal dominant inheritance. SIFT and PolyPhen-2 predicted that the c.3470A > G and c.3601C > G mutations in INSR had damaging effects. CONCLUSION: Our study expands the genotypic and phenotypic spectrum of type A insulin resistance syndrome. Awareness of the clinical features coupled with INSR gene screening is key to early detection and active intervention.

[Analysis of a child with Marfan syndrome due to a novel variant of FBN1 gene].

OBJECTIVE: To carry out genetic testing for a child with Marfan syndrome (MFS) and explore its genotype-phenotype correlation. METHODS: Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA and subjected to whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing. Functional impact of the variant was predicted by using bioinformatic software. RESULTS: The child, a 13-year-old male, has featured Marfanoid habitus, with arm span exceeding his height, tapering fingers and toes, pectus excavatum and scoliosis, but absence of typical cardiovascular system diseases such as aortic dilation, thoracic-abdominal aortic aneurysm, mitral valve prolapse, and lens dislocation. The child has harbored a novel splice site variant c.7383_7413del (p. N2461Kfs*211) of the FBN1 gene, which was not found in his parents and younger brother. The variant was unreported previously. CONCLUSION: The novel variant of p. N2461Kfs*211 of the FBN1 gene probably underlay the MFS in this child. Above finding has enriched the genotypic and phenotypic spectrum of MFS.

A case of early onset diabetes with myotonic dystrophy type 1. / ä»¥æ—©å‘ç³–å°¿ç— é¦–è¯Šçš„å¼ºç›´æ€§è‚Œè¥å »ä¸è‰¯1型1例.

Myotonic dystrophy type 1 (DM1, OMIM 160900) is a rare autosomal dominant hereditary disease. A case of DM1 patient with early onset diabetes and decreased muscle strength was treated in the Department of Endocrinology, Third Xiangya Hospital, Central South University. The peripheral blood of the patient was collected to extract DNA for gene detection. It was found that the triple nucleotide CTG repeat in the 3'-untranslated region (3'-UTR) of the dystrophia myotonica protein kinase (DMPK) gene was more than 100 times, and the diagnosis of DM1 was clear. For diabetes patients with multiple system abnormalities such as muscle symptoms, attention should be paid to the screening of DM1, a rare disease.

Methylprednisolone pulse therapy promotes the differentiation of regulatory T cells by inducing the apoptosis of CD4+ T cells in patients with systemic lupus erythematosus.

OBJECTIVES: To investigate the differentiation of regulatory T cells (Tregs) induced by methylprednisolone (MP) pulse therapy in patients with Systemic Lupus Erythematosus (SLE). METHODS: We enrolled 30 patients with SLE and analyzed peripheral blood mononuclear cells (PBMCs) before and after MP pulse therapy. Peripheral Tregs, apoptosis of PBMCs subsets, and TGFß production by monocytes was quantified by flow cytometry. Proliferation and IFN-γ production of CD4+ T cells were measured. Furthermore, TGFß1 production by human monocyte-derived macrophages (HMDM) stimulated with MP-treated CD4+ T cells were quantified by ELISA. RESULTS: Peripheral Tregs was significantly increased after MP pulse therapy (6.76 ± 1.46% vs. 3.82 ± 1.02%, p < 0.01), with an expansion of Nrp1- induced Tregs (4.54 ± 0.46% vs. 1.75 ± 0.38%, p < 0.01). Proliferation and IFN-γ production of CD4+ T cells were significantly decreased after MP pulse therapy. MP pulse therapy induced CD4+ T cell apoptosis (early apoptosis, 26.34 ± 3.54% vs. 14.81 ± 2.89%, p < 0.01) and TGFß expression on monocytes (6.02% vs. 2.45%, p < 0.01). Furthermore, MP induced CD4+ T cell apoptosis in vitro, which stimulated HMDM to produce TGFß. Moreover, elevated TGFß level in supernatant from HMDM stimulated with MP-treated CD4+ T cells promoted Tregs differentiation. CONCLUSIONS: MP pulse therapy induces CD4+ T cell apoptosis, which promotes monocytes to produce TGFß and further facilitates Tregs differentiation. Newly-differentiated Tregs suppress proliferation and IFN-γ production of CD4+ T cells and contribute to immunoregulatory milieu after MP pulse therapy.

Downregulation of Programmed Death-1 Pathway Promoting CD8 + T Cell Cytotoxicity in Primary Biliary Cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is an autoimmune disease. CD8 + T cell (CTLs) cytotoxicity played a crucial rule in of PBC with unclear detailed pathogenesis. AIMS: The role of the programmed death-1 (PD-1) pathway in CD8 + T cell cytotoxicity in patients with PBC was determined. METHODS: We recruited 69 patients with PBC and 57 healthy controls (HCs). PD-1 pathway in peripheral CD8 + T cells and related cytokines were detected, and gene expression levels were detected. Immunofluorescence staining of PD-1/PD-L1 was performed on liver tissue. PD-1 ± CTLs were cocultured with human intrahepatic biliary epithelial cells (HiBECs) to measure CTL cytotoxicity, proliferation and cytokine levels and HiBEC apoptosis. The upstream signaling pathway of PD-1 was detected. RESULTS: PBC patients exhibited Tbet gene upregulation and PD-1 downregulation in CTLs, with PD-1 expression reduced in CTLs and PD-L1 reduced in the liver portal region relative to HCs. Higher plasma IL-10, interferon-γ and transforming growth factor-ß concentrations were observed in the PBC group than the HC group. In CTL and HiBEC coculture experiment, compared with PD-1- CTLs, PD-1 + CTLs exhibited weaker cytotoxicity, less proliferation and lower cytokine production. When the system was blocked by anti-PD-1 antibodies, these effects were antagonized. CONCLUSIONS: PD-1 expression in CD8 + T cells decreased, and PD-1 pathway-related cytokines changed in patients with PBC. PD-1/PD-L1 pathway silencing increased CD8 + T cell proliferation, related cytokine production and CTL cytotoxic effects on HiBECs in coculture experiment. The PD-1/PD-L1 pathway might represent an important pathway in the immunological mechanism underlying PBC.

Clinical and genetic characteristics for 4 patients with Type Ib pseudohypoparathyroidism. / 4例Ibåž‹å‡æ€§ç”²çŠ¶æ—è ºåŠŸèƒ½å‡é€€ç—‡çš„ä¸´åºŠå’Œé—ä¼ å­¦ç‰¹å¾.

Pseudohypoparayhyroidism (PHP) is a rare autosomal dominant or recessive genetic disorder characterized by low calcium, high phosphorus, and target organ resistance to parathyroid. The clinical characteristics and genetic features in 4 patients with Type Ib PHP in the Third Xiangya Hospital, Central South University, have been reviewed. All 4 patients had low calcium, high phosphorus, and parathyroid resistance. Among them, 2 patients had slightly elevated thyroid stimulating hormone and mild features of Albright's hereditary osteodystrophy, and one patient had hypokalemia. No guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (GNAS) and gene variant associated with hypokalemia were identified using the whole exome sequencing. The results of the methylation-specific multiple ligation-dependent probe amplification showed that there were abnormal methylation of the upstream differentially methylated regions of GNAS in the 4 patients. There were phenotype overlap among the various subtypes of PHP. Detection of GNAS gene methylation in patients with clinical suspicion of Type Ib PHP is helpful for the diagnosis and treatment of PHP.

Aberrant distribution of CD3+CD56+ NKT-like cells in patients with primary Sjögren's syndrome.

OBJECTIVES: To elucidate the potential role of CD3+CD56+ NKT-like cells in the pathogenesis of primary Sjögren's syndrome (pSS). METHODS: We enrolled pSS patients and healthy controls and examined the peripheral population, the surface chemokine receptors and the proinflammatory cytokine production of NKT-like cells by flow cytometry. The infiltration of NKT-like cells in the labial salivary gland (LSG) was examined by immunofluorescence. Serum and tissue levels of CX3CL1 were detected by Cytometric Bead Array and immunohistochemistry, respectively. The chemotaxis of NKT-like cells was examined by transwell migration assay. RESULTS: Peripheral NKT-like cells from pSS patients were significantly lower than those from HC (3.09±2.35% vs. 5.37±4.06%, p=0.0002), which was negatively correlated with European League Against Rheumatism Sjögren's Syndrome Disease Activity index. NKT-like cells infiltrated into the LSG of pSS patients. Serum and LSG epithelial CX3CL1 levels were higher in pSS patients than those in HC, which promoted the chemotaxis of the NKT-like cells. NKT-like cells from pSS patients expressed a higher level of CD69, and secreted high level of TNF-α and IFN-γ, which was promoted by CX3CL1 in vitro. CONCLUSIONS: NKT-like cells decreased in peripheral and infiltrated into the LSG of the pSS patients, which could be driven by CX3CL1-CX3CR1 axis. NKT-like cells might be implicated in the pathogenesis of pSS.

Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood. METHODS: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS. RESULTS: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1. CONCLUSION: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS.

Influence of Different Aromatic Hydrophobic Residues on the Antimicrobial Activity and Membrane Selectivity of BRBR-NH2 Tetrapeptide.

The ultrashort linear antimicrobial tetrapeptide BRBR-NH2 with an unnatural residue biphenylalanine (B) has potent and rapid antimethicillin-resistant Staphylococcus aureus (MRSA) activity but lacks hemolytic activity. The anti-MRSA activity of BRBR-NH2 is 8-fold more potent than that of WRWR-NH2 and 16-fold more potent than that of FRFR-NH2. However, how to influence their antimicrobial activities and mechanisms through the substitution of different aromatic hydrophobic residues is still unclear. In this work, to study the effects of varying hydrophobic interactions and membrane selectivities of BRBR-NH2, we performed multiple long-time (1000 ns) molecular dynamics (MD) simulations to investigate the interactions of a red blood cell (RBC) membrane and a Gram-positive bacterial cell membrane with three different tetrapeptides (BRBR-NH2, WRWR-NH2, and FRFR-NH2) under different ratios of peptides and lipids and also explored the changes in the membrane and structural characteristics of peptides. The binding energy results show that BRBR-NH2 interacts weakly with the RBC membrane, while not all BRBR-NH2 can be adsorbed to the RBC membrane surface. The MD simulation results produced significant local membrane thinning of multiBRBR-NH2 peptides in the Gram-positive bacterial cell membrane. An in-depth analysis of structural features and peptide-membrane interactions suggests that the aggregation of BRBR-NH2 on the membrane surface plays a crucial role in the destruction of the cell membrane. Taken together with the observed local membrane thinning, the in-depth analysis demonstrated that the interactions between the lipid bilayer and the BRBR-NH2 aggregation surface result in a local disturbance of the membrane structure. It can be concluded that the high anti-MRSA activity of BRBR-NH2 is attributed to the aggregation of BRBR-NH2 on the membrane surface. On the other hand, WRWR-NH2 and FRFR-NH2 peptides tend to bind with the membrane surface in a monomeric form and cover the membrane surface in a carpet-like manner. Therefore, these results provide an advanced microscopic understanding of how hydrophobic interactions or hydrophobic residues affect the antimicrobial activity and mechanism of antimicrobial peptides (AMPs).

Different effects of cholesterol on membrane permeation of arginine and tryptophan revealed by bias-exchange metadynamics simulations.

Experiments have shown that cholesterol influences the membrane permeability of small molecules, amino acids, and cell-penetrating peptides. However, their exact translocation mechanisms under the influence of cholesterol remain poorly understood. Given the practical importance of cell-penetrating peptides and the existence of varied cholesterol contents in different cell types, it is necessary to examine the permeation of amino acids in cholesterol-containing membranes at atomic level of details. Here, bias-exchange metadynamics simulations were employed to investigate the molecular mechanism of the membrane permeation of two amino acids Arg and Trp important for cell-penetrating peptides in the presence of different concentrations of cholesterol. We found that the free energy barrier of Arg+ (the protonated form) permeation increased linearly as the cholesterol concentration increased, whereas the barrier of Trp permeation had a rapid increase from 0 mol. % to 20 mol. % cholesterol-containing membranes and nearly unchanged from 20 mol. % to 40 mol. % cholesterol-containing membranes. Arg0 becomes slightly more stable than Arg+ at the center of the dipalmitoylphosphatidylcholine (DPPC) membrane with 40 mol. % cholesterol concentrations. As a result, Arg+ has a similar permeability as Trp at 0 mol. % and 20 mol. % cholesterol, but a significantly lower permeability than Trp at 40 mol. % cholesterol. This difference is caused by the gradual reduction of water defects for Arg+ as the cholesterol concentration increases but lack of water defects for Trp in cholesterol-containing membranes. Strong but different orientation dependence between Arg+ and Trp permeations is observed. These results provide an improved microscopic understanding of amino-acid permeation through cholesterol-containing DPPC membrane systems.

Fast-Folding Pathways of the Thrombin-Binding Aptamer G-Quadruplex Revealed by a Markov State Model.

G-quadruplex structures participate in many important cellular processes. For a better understanding of their functions, knowledge of the mechanism by which they fold into the functional native structures is necessary. In this work, we studied the folding process of the thrombin-binding aptamer G-quadruplex. Enabled by a computational paradigm that couples an advanced sampling method and a Markov state model, four folding intermediates were identified, including an antiparallel G-hairpin, two G-triplex structures, and a double-hairpin conformation. Likewise, a misfolded structure with a nonnative distribution of syn/anti guanines was also observed. Based on these states, a transition path analysis revealed three fast-folding pathways, along which the thrombin-binding aptamer would fold to the native state directly, with no evidence of potential nonnative competing conformations. The results also showed that the TGT-loop plays an important role in the folding process. The findings of this research may provide general insight about the folding of other G-quadruplex structures.

Complete genome sequence of a new bipartite begomovirus infecting Boehmeria leiophylla in China.

A bipartite begomovirus was identified from a Boehmeria leiophylla plant sample exhibiting yellow mosaic symptoms collected in Nabanhe National Nature Reserve, Xishuangbanna, Yunnan, China. Five complete DNA-A and four DNA-B genome sequences were obtained by rolling-circle amplification (RCA), cloned, and sequenced. All DNA-A sequences were determined to be 2759 nucleotides in size, sharing 99.9%-100% nucleotide sequence identity with each other. The DNA-B sequences were comprised of 2673 nucleotides, sharing 98.6-100% nucleotide sequence identity with each other. Genomic organization of the begomovirus was typical of Old World bipartite begomoviruses. Sequence analysis revealed 81.84% nucleotide sequence identity to tomato leaf curl Hsinchu virus (ToLCHsV) from China for the DNA A component and 67.23% identity to the squash leaf curl China virus (SLCCNV) from India for the DNA B component. The sequence comparisons suggest that this bipartite begomovirus represents a novel species for which we propose the name "Ramie yellow mosaic virus".

Two distinct begomoviruses associated with an alphasatellite coinfecting Emilia sonchifolia in Thailand.

Emilia sonchifolia is a traditionally used medicinal plant that is widespread in tropical and subtropical regions of the world. Yellow vein symptoms were observed in E. sonchifolia plants in fields in the county of Koh Samui, Surat Thani Province, Thailand, in August 2015. Two distinct begomoviruses, designated TH4872-6 and TH4872-9, and an associated alphasatellite were obtained from an E. sonchifolia leaf sample (TH4872). Sequence analysis showed that the full-length sequence of TH4872-6 was most closely related to that of ageratum yellow vein China virus (AYVCNV), with 85.7% identity, suggesting that it is a novel begomovirus, while the TH4872-9 sequence closely resembled cotton leaf curl Multan virus (CLCuMuV) with 99.1% identity. The alphasatellite sequence showed the highest nucleotide sequence identity (92.8%) to an isolate of tobacco curly shoot alphasatellite (TbCSA) originating from China. Recombination analysis revealed that the isolate TH4872-6 is a potential recombinant begomovirus, derived from ageratum yellow vein virus (AYVV) and tobacco leaf curl Thailand virus (TbLCTHV). This study represents the first report of begomoviruses identified in E. sonchifolia in Thailand.

Molecular Dynamics Simulations of Human Antimicrobial Peptide LL-37 in Model POPC and POPG Lipid Bilayers.

Cathelicidins are a large family of cationic antimicrobial peptides (AMPs) found in mammals with broad spectrum antimicrobial activity. LL-37 is the sole amphipathic α-helical AMP from human Cathelicidins family. In addition to its bactericidal capability, LL-37 has antiviral, anti-tumor, and immunoregulatory activity. Despite many experimental studies, its molecular mechanism of action is not yet fully understood. Here, we performed three independent molecular dynamics simulations (600 ns or more) of a LL-37 peptide in the presence of 256 lipid bilayers with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) mimicking bacterial and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mimicking mammalian membranes. We found that LL-37 can be quickly absorbed onto the POPG bilayer without loss of its helical conformation in the core region and with the helix lying in parallel to the bilayer. The POPG bilayer was deformed. In contrast, LL-37 is slower in reaching the POPC surface and loss much of its helical conformation during the interaction with the bilayer. LL-37 only partially entered the POPC bilayer without significant deformation of the membrane. The observed difference for different bilayers is largely due to the fact that LL-37 is positively charged, POPG is negatively charged, and POPC is neutral. Our simulation results demonstrated the initial stage of disruption of the bacterial membrane by LL-37 in atomic details. Comparison to experimental results on LL-37 and simulation studies in other systems was made.

Atomistic Analysis of ToxN and ToxI Complex Unbinding Mechanism.

ToxIN is a triangular structure formed by three protein toxins (ToxNs) and three specific noncoding RNA antitoxins (ToxIs). To respond to stimuli, ToxI is preferentially degraded, releasing the ToxN. Thus, the dynamic character is essential in the normal function interactions between ToxN and ToxI. Here, equilibrated molecular dynamics (MD) simulations were performed to study the stability of ToxN and ToxI. The results indicate that ToxI adjusts the conformation of 3' and 5' termini to bind to ToxN. Steered molecular dynamics (SMD) simulations combined with the recently developed thermodynamic integration in 3nD (TI3nD) method were carried out to investigate ToxN unbinding from the ToxIN complex. The potentials of mean force (PMFs) and atomistic pictures suggest the unbinding mechanism as follows: (1) dissociation of the 5' terminus from ToxN, (2) missing the interactions involved in the 3' terminus of ToxI without three nucleotides (G31, A32, and A33), (3) starting to unfold for ToxI, (4) leaving the binding package of ToxN for three nucleotides of ToxI, (5) unfolding of ToxI. This work provides information on the structure-function relationship at the atomistic level, which is helpful for designing new potent antibacterial drugs in the future.

Bias-Exchange Metadynamics Simulation of Membrane Permeation of 20 Amino Acids.

Thermodynamics of the permeation of amino acids from water to lipid bilayers is an important first step for understanding the mechanism of cell-permeating peptides and the thermodynamics of membrane protein structure and stability. In this work, we employed bias-exchange metadynamics simulations to simulate the membrane permeation of all 20 amino acids from water to the center of a dipalmitoylphosphatidylcholine (DPPC) membrane (consists of 256 lipids) by using both directional and torsion angles for conformational sampling. The overall accuracy for the free energy profiles obtained is supported by significant correlation coefficients (correlation coefficient at 0.5-0.6) between our results and previous experimental or computational studies. The free energy profiles indicated that (1) polar amino acids have larger free energy barriers than nonpolar amino acids; (2) negatively charged amino acids are the most difficult to enter into the membrane; and (3) conformational transitions for many amino acids during membrane crossing is the key for reduced free energy barriers. These results represent the first set of simulated free energy profiles of membrane crossing for all 20 amino acids.

Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor.

Drug resistance of mutations in HIV-1 protease (PR) is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A) and inhibitor (GRL-0519) complexes, we have performed five molecular dynamics (MD) simulations and calculated the binding free energies using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT) complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors.

Developments in Methods for Measuring the Intestinal Absorption of Nanoparticle-Bound Drugs.

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative that the intestinal absorption of oral NPs be investigated. This review examines the various literature on the oral absorption of polymeric NPs, and provides an overview of the intestinal absorption models that have been developed for the study of oral nanoparticles. Three major categories of models including a total of eight measurement methods are described in detail (in vitro: dialysis bag, rat gut sac, Ussing chamber, cell culture model; in situ: intestinal perfusion, intestinal loops, intestinal vascular cannulation; in vivo: the blood/urine drug concentration method), and the advantages and disadvantages of each method are contrasted and elucidated. In general, in vitro and in situ methods are relatively convenient but lack accuracy, while the in vivo method is troublesome but can provide a true reflection of drug absorption in vivo. This review summarizes the development of intestinal absorption experiments in recent years and provides a reference for the systematic study of the intestinal absorption of nanoparticle-bound drugs.

Effect of pH on the Aggregation of α-syn12 Dimer in Explicit Water by Replica-Exchange Molecular Dynamics Simulation.

The dimeric structure of the N-terminal 12 residues drives the interaction of α-synuclein protein with membranes. Moreover, experimental studies indicated that the aggregation of α-synuclein is faster at low pH than neutral pH. Nevertheless, the effects of different pH on the structural characteristics of the α-syn12 dimer remain poorly understood. We performed 500 ns temperature replica exchange molecular dynamics (T-REMD) simulations of two α-syn12 peptides in explicit solvent. The free energy surfaces contain ten highly populated regions at physiological pH, while there are only three highly populated regions contained at acidic pH. The anti-parallel ß-sheet conformations were found as the lowest free energy state. Additionally, these states are nearly flat with a very small barrier which indicates that these states can easily transit between themselves. The dimer undergoes a disorder to order transition from physiological pH to acidic pH and the α-syn12 dimer at acidic pH involves a faster dimerization process. Further, the Lys6-Asp2 contact may prevent the dimerization.