Integrated photoacoustic and diffuse optical tomography system for imaging of human finger joints in vivo

In this study, we developed a dual‐modality tomographic system that integrated photoacoustic imaging (PAI) and diffuse optical tomography (DOT) into a single platform for imaging human finger joints with fine structures and associated optical properties. In PAI, spherical focused transducers were utilized to collect acoustic signals, and the concept of virtual detector was applied in a conventional back‐projection algorithm to improve the image quality. A finite‐element based reconstruction algorithm was employed to quantitatively recover optical property distribution in the objects for DOT. The phantom results indicate that PAI has a maximum lateral resolution of 70 µm in resolving structures of targets. DOT was able to recover both optical absorption and reduced scattering coefficients of targets accurately. To validate the potential of this system in clinical diagnosis of joint diseases, the distal interphalangeal (DIP) joints of 4 healthy female volunteers were imaged. We successfully obtained high‐resolution images of the phalanx and the surrounding soft tissue via PAI, and recovered both optical absorption and reduced scattering coefficients of phalanx using DOT. The in vivo results suggest that this dual‐modality system has the potential for the early diagnosis of joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA).

Integrated PAI/DOT imaging interface (top) and typical reconstruction of structures and associated optical properties of a female finger joint via PAI and DOT (bottom).     

Detecting hemodynamic changes in the foot vessels of diabetic patients by photoacoustic tomography

Limb perfusion monitoring is critical for diabetes mellitus (DM) patients as they are vulnerable to vascular complications due to prolonged hyperglycemia. However, current clinical approaches are ineffective in vascular imaging and in assessing vascular function in lower limbs. In this work, a concave ultrasound transducer array‐based photoacoustic tomography (PAT) system was used to image the foot dorsal section of a subject, and a total of seven DM patients and seven healthy volunteers were enrolled in this study. Hemodynamic changes in foot vessels during vascular occlusion as well as oxygen saturation (SO₂) in rest were analyzed for both groups. The results obtained showed that DM patients have a unique peripheral hemodynamic response to occlusion and a lower level SO₂, compared to that for healthy subjects. This suggests that PAT has the potential to detect vascular dysfunction in DM patients and to measure the effect of treatment.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.     

In vivo study of endometriosis in mice by photoacoustic microscopy

Endometriosis (EM) impacts the healthcare and the quality of life for women of reproductive age. However, there is no reliable noninvasive diagnosis method for either animal study or clinical use. In this work, a novel imaging method, photoacoustic microscopy (PAM) was employed to study the EM on the mouse model. Our results demonstrated the PAM noninvasively provided the high contrast and 3D imaging of subcutaneously implanted EM tissue in the nude mouse in vivo. The statistical study also indicated PAM had high sensitivity and specificity in the diagnosis of EM in this animal study. In addition, we also discussed the potential clinical application for PAM in the diagnosis of EM. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Solution structure of the PHD finger from the human KIAA1045 protein

Cross‐brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin‐conjugating enzyme (E2)‐binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131–190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C₄HC₃‐type PHD fold belonging to the cross‐brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.     

Quantitative investigation of vascular response to mesenteric venous thrombosis using large‐field‐of‐view photoacoustic microscopy

Mesenteric venous thrombosis (MVT) is one of major causes leading to severe mesenteric ischemia. Vascular network plays an important role during the occurrence and development of MVT. However, there lacks an appropriate imaging method, which features advanced volumetric resolving capability, superior sensitivity to hemoglobin, and ultra‐large field‐of‐view (FOV), to investigate vascular response of MVT. In this study, we developed and applied a large‐FOV optical resolution photoacoustic microscopy to quantify the vascular response during the entire course of two different MVT models in which we ligated the superior mesenteric vein and inferior mesenteric vein, respectively. Furthermore, we developed a quantitative algorithm to derive total vascular length, relative concentration of total hemoglobin and vascular density over the FOV to reveal different vascular responses in different MVT models.     

Solution structure of the zinc finger domain of human RNF144A ubiquitin ligase

RNF144A is involved in protein ubiquitination and functions as an ubiquitin‐protein ligase (E3) via its RING finger domain (RNF144A RING). RNF144A is associated with degradation of heat‐shock protein family A member 2 (HSPA2), which leads to the suppression of breast cancer cell proliferation. In this study, the solution structure of RNF144A RING was determined using nuclear magnetic resonance. Moreover, using a metallochromic indicator, we spectrophotometrically determined the stoichiometry of zinc ions and elucidated that RNF144A RING binds two zinc atoms. This structural analysis provided the position and range of the active site of RNF144A RING at the atomic level, which contributes to the creation of artificial RING fingers having the specific ubiquitin‐conjugating enzyme (E2)‐binding capability.     

The unique N‐terminal zinc finger of synaptotagmin‐like protein 4 reveals FYVE structure

Synaptotagmin‐like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N‐terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N‐terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross‐brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C₄C₄‐type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin‐conjugating enzyme (E2)‐binding capability, cross‐brace structures with eight zinc‐ligating residues are needed as the scaffold. The cross‐brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs.     

Relationship between1H and13C NMR chemical shifts and the secondary and tertiary structure of a zinc finger peptide

Summary Essentially complete assignments have been obtained for the¹H and protonated¹³C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the¹H and¹³C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone C^aH and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their¹H chemical shifts and secondary structure.     

Improving taxonomy-based protein fold recognition by using global and local features

Fold recognition from amino acid sequences plays an important role in identifying protein structures and functions. The taxonomy-based method, which classifies a query protein into one of the known folds, has been shown very promising for protein fold recognition. However, extracting a set of highly discriminative features from amino acid sequences remains a challenging problem. To address this problem, we developed a new taxonomy-based protein fold recognition method called TAXFOLD. It extensively exploits the sequence evolution information from PSI-BLAST profiles and the secondary structure information from PSIPRED profiles. A comprehensive set of 137 features is constructed, which allows for the depiction of both global and local characteristics of PSI-BLAST and PSIPRED profiles. We tested TAXFOLD on four datasets and compared it with several major existing taxonomic methods for fold recognition. Its recognition accuracies range from 79.6 to 90% for 27, 95, and 194 folds, achieving an average 6.9% improvement over the best available taxonomic method. Further test on the Lindahl benchmark dataset shows that TAXFOLD is comparable with the best conventional template-based threading method at the SCOP fold level. These experimental results demonstrate that the proposed set of features is highly beneficial to protein fold recognition.     

Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.

The nucleic acid interactive properties of a synthetic peptide with sequence of the N-terminal CCHC zinc finger (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence-dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3' side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding to a portion of the HIV-1 psi-packaging signal, d(ACGCC), forms a relatively tight complex with Zn(HIV1-F1) (Kd = 5 x 10(-6) M). Two-dimensional nuclear Overhauser effect (NOESY) data indicate that the bound nucleic acid exists predominantly in a single-stranded, A-helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three-dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site-directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.     

芦荟维管束的结构与芦荟素积累的相关性

应用半薄切片、组织化学、荧光显微镜观察和薄层层析 (TLC)相结合的方法研究了中华芦荟 (Aloeve-ra L.var.chinensis)、木立芦荟 (Aloe arborescens)叶和茎内维管束的结构及其与芦荟素积累的关系。结果表明 ,木立芦荟叶内维管束和中华芦荟叶内外轮的维管束中有大型韧皮薄壁细胞 ,而木立芦荟茎和中华芦荟叶中内轮维管束无大型韧皮薄壁细胞。组织化学结果表明 ,用醋酸铅处理过的上述材料 ,大型韧皮薄壁细胞内出现沉淀物 ;在荧光显微镜下经蓝光激发 ,大型韧皮薄壁细胞发出桔黄色荧光 ,都显示出芦荟素反应。薄层层析(TLC)结果证明 ,木立芦荟和中华芦荟叶含有大型韧皮薄壁细胞的维管束都含芦荟素 ,而木立芦荟茎及中华芦荟叶中内轮维管束都不含芦荟素。为此 ,维管束中的大型韧皮薄壁细胞与芦荟素的积累密切相关 ,维管束中是否有大型韧皮薄壁细胞可作为判断是否含有芦荟素的解剖学指标。     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Solution structure of the zinc finger HIT domain in protein FON

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.     

The recognition of multi-class protein folds by adding average chemical shifts of secondary structure elements

The recognition of protein folds is an important step in the prediction of protein structure and function. Recently, an increasing number of researchers have sought to improve the methods for protein fold recognition. Following the construction of a dataset consisting of 27 protein fold classes by Ding and Dubchak in 2001, prediction algorithms, parameters and the construction of new datasets have improved for the prediction of protein folds. In this study, we reorganized a dataset consisting of 76-fold classes constructed by Liu et al. and used the values of the increment of diversity, average chemical shifts of secondary structure elements and secondary structure motifs as feature parameters in the recognition of multi-class protein folds. With the combined feature vector as the input parameter for the Random Forests algorithm and ensemble classification strategy, we propose a novel method to identify the 76 protein fold classes. The overall accuracy of the test dataset using an independent test was 66.69%; when the training and test sets were combined, with 5-fold cross-validation, the overall accuracy was 73.43%. This method was further used to predict the test dataset and the corresponding structural classification of the first 27-protein fold class dataset, resulting in overall accuracies of 79.66% and 93.40%, respectively. Moreover, when the training set and test sets were combined, the accuracy using 5-fold cross-validation was 81.21%. Additionally, this approach resulted in improved prediction results using the 27-protein fold class dataset constructed by Ding and Dubchak.     

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.     

Fold recognition by concurrent use of solvent accessibility and residue depth

Recognizing the structural similarity without significant sequence identity (called fold recognition) is the key for bridging the gap between the number of known protein sequences and the number of structures solved. Previously, we developed a fold-recognition method called SP(3) which combines sequence-derived sequence profiles, secondary-structure profiles and residue-depth dependent, structure-derived sequence profiles. The use of residue-depth-dependent profiles makes SP(3) one of the best automatic predictors in CASP 6. Because residue depth (RD) and solvent accessible surface area (solvent accessibility) are complementary in describing the exposure of a residue to solvent, we test whether or not incorporation of solvent-accessibility profiles into SP(3) could further increase the accuracy of fold recognition. The resulting method, called SP(4), was tested in SALIGN benchmark for alignment accuracy and Lindahl, LiveBench 8 and CASP7 blind prediction for fold recognition sensitivity and model-structure accuracy. For remote homologs, SP(4) is found to consistently improve over SP(3) in the accuracy of sequence alignment and predicted structural models as well as in the sensitivity of fold recognition. Our result suggests that RD and solvent accessibility can be used concurrently for improving the accuracy and sensitivity of fold recognition. The SP(4) server and its local usage package are available on http://sparks.informatics.iupui.edu/SP4.