Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

Analysis of a broad range of perfluoroalkyl acids in accipiter feathers: method optimization and their occurrence in Nam Co Basin, Tibetan Plateau.

Perfluoroalkyl acids (PFAAs) are ubiquitous in the environment. They are prone to accumulate in organisms and have raised public attention in recent decades. Feather samples have been successfully applied as nondestructive indicators for several contaminants. However, a sophisticated analytical method for determining PFAAs in feathers is still lacking. In the present study, a series of conditions, such as the use of the solid-phase extraction cartridge type and extraction/digestion methods, were optimized for the analysis of 13 PFAAs in feathers. According to the spiked recoveries, a weak-anion exchange cartridge was chosen and the methanol was selected as the extraction solvent. In the present study, an optimized pretreatment procedure combined with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for the determination of PFAAs in feathers. The recoveries and method detection limits of the PFAAs ranged from 71 to 120% and 0.16 to 0.54 ng/g, respectively. Finally, 13 PFAAs in four accipiter feather samples from Nam Co Basin, Tibetan Plateau, were analyzed, indicating that PFOS was the predominant PFAA in accipiter feathers, with an average of 4.67 ng/g, followed by the short-chain PFAAs, PFBS and PFBA, with averages of 1.91 and 1.39 ng/g, respectively. These results partly indicated the current situation of PFAA pollution in the Nam Co Basin, especially the existence of short-chain PFAAs in this region.

Two Paralogous Genes Encoding Auxin Efflux Carrier Differentially Expressed in Bitter Gourd (Momordica charantia).

The phytohormone auxin regulates various developmental programs in plants, including cell growth, cell division and cell differentiation. The auxin efflux carriers are essential for the auxin transport. To show an involvement of auxin transporters in the coordination of fruit development in bitter gourd, a juicy fruit, we isolated novel cDNAs (referred as McPIN) encoding putative auxin efflux carriers, including McPIN1, McPIN2 (allele of McPIN1) and McPIN3, from developing fruits of bitter gourd. Both McPIN1 and McPIN3 genes possess six exons and five introns. Hydropathy analysis revealed that both polypeptides have two hydrophobic regions with five transmembrane segments and a predominantly hydrophilic core. Phylogenetic analyses revealed that McPIN1 shared the highest homology to the group of Arabidopsis, cucumber and tomato PIN1, while McPIN3 belonged to another group, including Arabidopsis and tomato PIN3 as well as PIN4. This suggests different roles for McPIN1 and McPIN3 in auxin transport involved in the fruit development of bitter gourd. Maximum mRNA levels for both genes were detected in staminate and pistillate flowers. McPIN1 is expressed in a particular period of early fruit development but McPIN3 continues to be expressed until the last stage of fruit ripening. Moreover, these two genes are auxin-inducible and qualified as early auxin-response genes. Their expression patterns suggest that these two auxin transporter genes play a pivotal role in fruit setting and development.

Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the ß-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the ß-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

Structure of NKp65 bound to its keratinocyte ligand reveals basis for genetically linked recognition in natural killer gene complex.

The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65-KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65-KACL interaction compared with other cell-cell interaction pairs (KD = 6.7 × 10(-10) M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor-ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1-Clr, NKRP1A-LLT1, and NKp80-AICL.

Structural basis for self-recognition by autoimmune T-cell receptors.

T-cell receptors (TCRs) recognize peptides presented by major histocompatibility complex molecules (pMHC) to discriminate between foreign and self-antigens. Whereas T-cell recognition of foreign peptides is essential for protection against microbial pathogens, recognition of self-peptides by T cells that have escaped negative selection in the thymus can lead to autoimmune disease. Structural studies of autoimmune TCR-pMHC complexes have provided insights into the mechanisms underlying self-recognition and escape from thymic deletion. Two broad categories of self-reactive TCRs can be clearly distinguished: (i) TCRs with altered binding topologies to self-pMHC and (ii) TCRs that bind self-pMHC in the canonical diagonal orientation, but where there are structural defects or suboptimal anchors in the self-ligand. For both categories, however, the overall stability of the autoimmune TCR-pMHC complex is markedly reduced compared to anti-microbial complexes, allowing the autoreactive T cells to evade negative selection, yet retain the ability to be activated by self-antigens in target organs. Additionally, the structures provide insights into TCR cross-reactivity, which can contribute to autoimmunity by increasing the likelihood of self-pMHC recognition. Efforts are now underway to understand the impact of structural alterations in autoimmune TCR-pMHC complexes on higher order assemblies involved in TCR signaling, as well as on immunological synapse formation.

Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection.

The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3ß conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

Structure-based design of altered MHC class II-restricted peptide ligands with heterogeneous immunogenicity.

Insights gained from characterizing MHC-peptide-TCR interactions have held the promise that directed structural modifications can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineering might thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study, we determined the crystal structure of HLA-DR4 in complex with the nonmutated dominant gp100 epitope gp10044-59, associated with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of gp100 in HLA-DR4(+) melanoma patients. Increased MHC binding of several APLs was observed, validating this approach biochemically. Nevertheless, heterogeneous preferences of CD4(+) T cells from several HLA-DR4(+) melanoma patients for different gp100 APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild-type gp100 peptide. This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma. Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial, effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled before structural and in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders.

Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4-HLA-DR1 complex.

Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K(D) = ∼200 µM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K(D) = 8.8 µM, compared with >400 µM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and ß2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.

Ferroptosis: emerging roles in lung cancer and potential implications in biological compounds.

Lung cancer has high metastasis and drug resistance. The prognosis of lung cancer patients is poor and the patients' survival chances are easily neglected. Ferroptosis is a programmed cell death proposed in 2012, which differs from apoptosis, necrosis and autophagy. Ferroptosis is a novel type of regulated cell death which is driven by iron-dependent lipid peroxidation and subsequent plasma membrane ruptures. It has broad prospects in the field of tumor disease treatment. At present, multiple studies have shown that biological compounds can induce ferroptosis in lung cancer cells, which exhibits significant anti-cancer effects, and they have the advantages in high safety, minimal side effects, and less possibility to drug resistance. In this review, we summarize the biological compounds used for the treatment of lung cancer by focusing on ferroptosis and its mechanism. In addition, we systematically review the current research status of combining nanotechnology with biological compounds for tumor treatment, shed new light for targeting ferroptosis pathways and applying biological compounds-based therapies.

Structure of the F-spondin domain of mindin, an integrin ligand and pattern recognition molecule.

Mindin (spondin-2) is an extracellular matrix protein of unknown structure that is required for efficient T-cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N-terminal F-spondin (FS) domain and C-terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8-A resolution. The structure revealed an eight-stranded antiparallel beta-sandwich motif resembling that of membrane-targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C-mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.

Effects of work-family conflict and anxiety in the relationship between work-related stress and job burnout in Chinese female nurses: A chained mediation modeling analysis.

BACKGROUND: Job burnout among nurses has been a challenging problem in recent years globally and in China. Work-related stress, work-life interference and mental health have been shown to be associated with nurse job burnout. However, the underlying mechanisms remain not fully understood. This study aims to examine the complex relationships linking work-related stress to nurse burnout among Chinese nurses. METHODS: Study data were collected from female nurses (n = 2172) in cities of Wuhan, Shiyan and Jingzhou, Hubei Province of China. Job burnout was used as outcome variable, work-related stress was the predictor, work-life interference and anxiety symptoms were mediators. Mediation and chained mediation modeling analysis were used for data analysis. RESULTS: The association between work-related stress and job burnout was significantly mediated by work-family conflict (indirect effect[95%CI] = 0.05[0.05,0.06]) and anxiety symptoms (indirect effect = 0.42[0.36,0.49]), respectively. Further, a chained mediation mechanism was observed with work-family conflict and anxiety symptoms consecutively mediated the relationship between work-related stress and job burnout (indirect effect = 0.02[0.01,0.02]). LIMITATIONS: The data were collected in one province in central China, so it needs caution when generalizing the study findings to other regions within or outside of China. CONCLUSION: Work-related stress exerts effects on job burnout through work-family conflict and anxiety symptoms among female nurses in China. Work-related stress-based burnout prevention must consider both work-family conflict and mental health problems.

Risk factors and prognosis of acute lactation mastitis developing into a breast abscess: A retrospective longitudinal study in China.

BACKGROUND: Breast abscess is developed on the basis of acute mastitis, which will cause damage to the physical and mental health of lactating women and is an important factor affecting the rate of breastfeeding. This study examined the risk factors for mastitis to develop into breast abscess, and analyzed the distribution of pathogenic bacteria, bacterial resistance, and treatment outcome. METHODS: The medical records of 316 cases of mastitis and 219 cases of breast abscess were retrospectively collected. We analyzed the bacterial distribution of mastitis and breast abscess, and compared the differences of bacterial drug resistance. Univariate analysis and binary logistic regression were used to analyze the following aspects: age, primiparity or not, history of breast surgery, body temperature, puerperium or not, onset time, located in the nipple/areolar complexe area or not, history of massage by non-professionals, staphylococcus aureus/methicillin-resistant staphylococcus aureus (MRSA) infection or not, diabetes and white blood cell count. RESULTS: Of the 535 patients, 203 (37.9%) were positive for staphylococcus aureus. There were 133 (65.5%) cases of methicillin-sensitive staphylococcus aureus (MSSA) and 70 (34.5%) cases of MRSA. Concerning bacterial drug resistance, a statistical analysis showed that MSSA had high resistance rate to penicillin (96.2%), ampicillin (91%), clindamycin (42.9%) and erythromycin (45.9%). MRSA had a high resistance rate to penicillin (100%), ampicillin (98.6%), oxacillin (95.7%), erythromycin (81.4%), clindamycin (80%), and amoxicillin (31.7%). Risk factors for progression of mastitis to breast abscess include a body temperature<38.5°C, a postpartum time ≥ 42 days, an onset time ≥ 2 days, lesions in the nipple/areolar complex area, a history of massage by non-medical staff and bacterial cultures for milk or pus that test positive for staphylococcus aureus or MRSA (P < 0.001). CONCLUSIONS: The most common pathogenic bacteria of mastitis and breast abscess is staphylococcus aureus. There are many risk factors for mastitis to develop into breast abscess. We should take effective measures for its risk factors and select sensitive antibiotics according to the results of bacterial culture to reduce the formation of breast abscess.

[Double-door laminoplasty combined with C2 dome decompression for the treatment of cervical spinal stenosis].

OBJECTIVE: To investigate the clinical effect of double-door laminoplasty combined with C2 dome decompression in treatment of cervical spinal stenosis. METHODS: The clinical data of 28 patients with cervical spinal stenosis who underwent double-door laminoplasty combined with C2 dome decompression from June 2016 to June 2018 were retrospectively analyzed, including 17 males and 11 females, aged 39 to 74 years with an average of (61.0±6.7) years. The clinical effects were evaluated by JOA score, axial symptoms, cervical spine activity, cervical spinal cord compression degree and so on. RESULTS: All patients were followed up for 6 to12 months with an average of 10.2 months. The JOA score in the final follow-up was significantly improved (P<0.05). The range of cervical activity before and after surgery was respectively (41.8±15.3) °, (36.3±18.2) °, and there was no significant difference (P>0.05). After operation, sagittal diameter at the narrowest level of C2-C3 spinal canal was (16.20±1.82) mm, which was significantly higher than (8.38±1.16) mm before operation (P<0.05). There were 4 cases with axial symptoms in 24 patients with the incidence rate of 14.29% (4/24). CONCLUSION: Double-door laminoplasty combined with C2 dome decompression can directly expand the volume of C2-C3 spinal canal, relieve the compression of spinal cord and nerve root, reduce the damage to the posterior cervical ligament complex as much as possible, maintain the stability of cervical spine sequence, reduce the occurrence of axial symptoms, and the operation is relatively simple, without the need of metal internal fixation.

A novel loop domain in superantigens extends their T cell receptor recognition site.

Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the alpha3-beta8 loop, a unique approximately 15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hVbeta5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the alpha3-beta8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR Vbeta domain specificity of SEK and other group V SAGs.

An integrated method for simultaneously determining 10 classes of per- and polyfluoroalkyl substances in one drop of human serum.

Per- and polyfluoroalkyl substances (PFASs) represent a group of synthetic chemicals, and they have quite different physicochemical properties, which result in difficulties of their simultaneous determination in a single injection. A sensitive, reliable, and fully automated method was developed for simultaneously detecting 10 classes of PFASs (total of 43) in human serum using online Turboflow SPE-UHPLC-MS/MS. This method provided high linearity of matrix-matched calibration standards (R > 0.99), excellent method limits of detection (MLODs) (0.013-0.089 ng mL-1), satisfactory matrix spiked recoveries (84.3-109%) and relative standard deviations (RSDs) (intra-day RSDs: 1.3-12.6%, inter-day RSDs: 1.7-13.8%, inter-week RSDs: 1.8-13.5%, inter-month RSDs: 3.1-12.4%), short analysis time (19 min per sample) and small sample amount requirement (25 µL), which were suitable for large-scale epidemiologic studies. Moreover, the method provided the feasibility of real-time monitoring for the degradation kinetics of PFASs precursors both in vitro and in vivo. The quality of the present method was further verified by repetitive analysis of a standard reference material (SRM 1957), with the deviations of the targeted PFAS concentrations ranging from 1.9% to 14.2% (n = 5) between the detected and reference values. The present study also determined values for several PFASs in SRM 1957 other than those on the certificate, for the first time, such as N-EtFOSA, 6:2 Cl-PFESA, and PFBA. Finally, the established method was applied to detect PFASs in serum samples of 15 ordinary people and 15 occupational workers, and 6:2 FTSA was found as the dominant precursor.

So many ways of getting in the way: diversity in the molecular architecture of superantigen-dependent T-cell signaling complexes.

Superantigens (SAGs) elicit massive T-cell proliferation through simultaneous interaction with MHC and TCR molecules. SAGs have been implicated in toxic shock syndrome and food poisoning, and they may also play a pathogenic role in autoimmune diseases. The best-characterized group of SAGs are the pyrogenic bacterial SAGs, which utilize a high degree of genetic variation on a common structural scaffold to achieve a wide range of MHC-binding and T-cell-stimulating effects while assisting pathogen evasion of the adaptive immune response. Several new structures of SAG-MHC and SAG-TCR complexes have significantly increased understanding of the molecular bases for high-affinity peptide/MHC binding by SAGs and for TCR Vbeta domain specificity of SAGs. Using the currently available SAG-MHC and SAG-TCR complex structures, models of various trimolecular MHC-SAG-TCR complexes may be constructed that reveal wide diversity in the architecture of SAG-dependent T-cell signaling complexes, which nevertheless may result in similar signaling outcomes.

Understanding antibody-antigen associations by molecular dynamics simulations: detection of important intra- and inter-molecular salt bridges.

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure-function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize 'coincident epitopes'. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63- HEL reveals the presence of functionally important saltbridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing saltbridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed saltbridges are present in one or more of the other three complexes and/or involve \"hot-spot\" epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The "hot spot" epitope residue Lys97Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this intermolecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed saltbridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97Y in the formation of inter-molecular triad further validates the functional importance of Lys97Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights "hot-spot" molecular interactions in HyHEL63-HEL complex.

Magnitude of the hydrophobic effect at central versus peripheral sites in protein-protein interfaces.

Hydrophobic interactions are essential for stabilizing protein-protein complexes, whose interfaces generally consist of a central cluster of hot spot residues surrounded by less important peripheral residues. According to the O-ring hypothesis, a condition for high affinity binding is solvent exclusion from interacting residues. This hypothesis predicts that the hydrophobicity at the center is significantly greater than at the periphery, which we estimated at 21 cal mol(-1) A(-2). To measure the hydrophobicity at the center, structures of an antigen-antibody complex where a buried phenylalanine was replaced by smaller hydrophobic residues were determined. By correlating structural changes with binding free energies, we estimate the hydrophobicity at this central site to be 46 cal mol(-1) A(-2), twice that at the periphery. This context dependence of the hydrophobic effect explains the clustering of hot spots at interface centers and has implications for hot spot prediction and the design of small molecule inhibitors.

A rapid and fully automatic method for the accurate determination of a wide carbon-chain range of per- and polyfluoroalkyl substances (C4-C18) in human serum.

A rapid and fully automatic method for determining 21 per- and polyfluoroalkyl substances (with carbon chains ranging from C4 to C18, including 13 PFCAs, 5 PFSAs, 2 Cl-PFESAs, and PFOSA) in human serum samples was developed. The HPLC parameters, Turboflow column, mobile phase, sample injection volume, loading flow rate, and sample cleanup and elution time were optimized. 25µL serum sample was directly injected into the developed on-line Turboflow SPE HPLC-MS/MS system for analysis after dilution. Matrix effects were corrected due to the matrix removal efficiency of the Turboflow column and sufficient types of internal isotope standards that were used. The established method showed a good linearity (r2>0.99), rapid processing time (20min per sample), satisfactory recoveries (matrix spiked recoveries range from 84.6% to 114%) and precision (intra-day and inter-day RSDs ranged from 1.5% to 9.2% and from 1.1% to 7.0%, respectively). The limits of detection (LODs) of the 21 analyzed PFASs were between 0.008 and 0.19ngmL-1. The LODs of short- and long-chain PFASs, such as PFBA, PFPeA, PFHxDA, and PFODA, were 0.008, 0.022, 0.15 and 0.19ngmL-1, respectively; the spiked recoveries of these PFASs were 101, 105, 87.1, and 85.8%, respectively. Both the LODs and recoveries were better than previous studies. Further, serum PFASs concentrations detected by the presented on-line SPE method were consistent with the traditional off-line SPE method (r: 0.98-0.99), which verified the accuracy and applicability of the present method. The method shows good practical prospects in the analysis of trace per- and polyfluoroalkyl substances in human serum.