Causal relationship between inflammatory factors and cerebral small vessel disease: Univariate, multivariate, and summary-data-based mendelian randomization analysis.

OBJECTIVE: To explore the impact of inflammatory factors on the incidence of cerebral small vessel disease (CSVD), we performed a mendelian randomization (MR) study to analyze the causal relationship between multiple inflammatory factors and CSVD imaging markers and utilized summary-data-based mendelian randomization (SMR) analysis to infer whether the impact of instrumental variables (IVs) on disease is mediated by gene expression or DNA methylation. METHODS: Using public databases such as UKB and IEU, and original genome-wide association studies, we obtained IVs related to exposure (inflammatory factors) and outcome (CSVD imaging markers). We performed the inverse variance weighted, weighted median, and MR-Egger methods to assess causal effects between exposure and outcome in univariate MR analysis. To evaluate their heterogeneity, a series of sensitivity analyses were conducted, including the Cochrane Q test, MR-Egger intercept test, MR-Presso, and leave-one-out analysis. We also applied mediation and multivariate MR analysis to explore the interactions between positive exposures on the same outcome. Additionally, we conducted the SMR, which utilizes instruments within or near relevant genes in blood or brain tissues, to elucidate the causal associations with CSVD markers. RESULTS: ABO Univariate MR of multiple cohorts revealed that the risk of small vessel stroke (SVS) increases with elevated levels of TNF-related apoptosis-inducing ligand (TRAIL, OR, 1.23, 95% CI, 1.08-1.39) and interleukin-1 receptor-like 2, (IL-1RL2, OR, 1.29, 95% CI, 1.04-1.61). IL-18 was a potential risk factor for extensive basal ganglia perivascular space burden (BGPVS, OR, 1.02, 95% CI, 1.00-1.05). Moreover, the risk of extensive white matter perivascular space burden (WMPVS) decreased with rising levels of E-selectin (OR, .98, 95% CI, .97-1.00), IL-1RL2 (OR, .97, 95% CI, .95-1.00), IL-3 receptor subunit alpha (IL-3Ra, OR, .98, 95% CI, .97-1.00), and IL-5 receptor subunit alpha (IL-5Ra, OR, .98, 95% CI, .97-1.00). Mediation and multivariate MR analysis indicated that E-selectin and IL-3Ra might interact during the pathogenesis of WMPVS. SMR estimates showed that TRAIL-related IVs rs5030044 and rs2304456 increased the risk of SVS by increasing the expression of gene Kininogen-1 (KNG1) in the cerebral cortex, particularly in the frontal cortex (ßsmr = .10, Psmr = .003, FDR = .04). Instruments (rs507666 and rs2519093) related to E-selectin and IL-3Ra could increase the risk of WMPVS by enhancing DNA methylation of the gene ABO in blood tissue (ßsmr = .01-.02, Psmr = .001, FDR = .01-.03). CONCLUSION: According to MR and SMR analysis, higher levels of TRAIL increased the risk of SVS by upregulating gene expression of KNG1 in brain cortex tissues. In addition, protective effects of E-selectin and IL-3a levels on WMPVS were regulated by increased DNA methylation of gene ABO in blood tissue.

Generative adversarial network-based attenuation correction for 99mTc-TRODAT-1 brain SPECT.

Background: Attenuation correction (AC) is an important correction method to improve the quantification accuracy of dopamine transporter (DAT) single photon emission computed tomography (SPECT). Chang's method was developed for AC (Chang-AC) when CT-based AC was not available, assuming uniform attenuation coefficients inside the body contour. This study aims to evaluate Chang-AC and different deep learning (DL)-based AC approaches on 99mTc-TRODAT-1 brain SPECT using clinical patient data on two different scanners. Methods: Two hundred and sixty patients who underwent 99mTc-TRODAT-1 SPECT/CT scans from two different scanners (scanner A and scanner B) were retrospectively recruited. The ordered-subset expectation-maximization (OS-EM) method reconstructed 120 projections with dual-energy scatter correction, with or without CT-AC. We implemented a 3D conditional generative adversarial network (cGAN) for the indirect deep learning-based attenuation correction (DL-ACµ) and direct deep learning-based attenuation correction (DL-AC) methods, estimating attenuation maps (µ-maps) and attenuation-corrected SPECT images from non-attenuation-corrected (NAC) SPECT, respectively. We further applied cross-scanner training (cross-scanner indirect deep learning-based attenuation correction [cull-ACµ] and cross-scanner direct deep learning-based attenuation correction [call-AC]) and merged the datasets from two scanners for ensemble training (ensemble indirect deep learning-based attenuation correction [eDL-ACµ] and ensemble direct deep learning-based attenuation correction [eDL-AC]). The estimated µ-maps from (c/e)DL-ACµ were then used in reconstruction for AC purposes. Chang's method was also implemented for comparison. Normalized mean square error (NMSE), structural similarity index (SSIM), specific uptake ratio (SUR), and asymmetry index (%ASI) of the striatum were calculated for different AC methods. Results: The NMSE for Chang's method, DL-ACµ, DL-AC, cDL-ACµ, cDL-AC, eDL-ACµ, and eDL-AC is 0.0406 ± 0.0445, 0.0059 ± 0.0035, 0.0099 ± 0.0066, 0.0253 ± 0.0102, 0.0369 ± 0.0124, 0.0098 ± 0.0035, and 0.0162 ± 0.0118 for scanner A and 0.0579 ± 0.0146, 0.0055 ± 0.0034, 0.0063 ± 0.0028, 0.0235 ± 0.0085, 0.0349 ± 0.0086, 0.0115 ± 0.0062, and 0.0117 ± 0.0038 for scanner B, respectively. The SUR and %ASI results for DL-ACµ are closer to CT-AC, Followed by DL-AC, eDL-ACµ, cDL-ACµ, cDL-AC, eDL-AC, Chang's method, and NAC. Conclusion: All DL-based AC methods are superior to Chang-AC. DL-ACµ is superior to DL-AC. Scanner-specific training is superior to cross-scanner and ensemble training. DL-based AC methods are feasible and robust for 99mTc-TRODAT-1 brain SPECT.

Complete genome sequence of a new virus from Allium sativum L in China.

The complete genome of a new virus belonging to the family Betaflexiviridae was identified in garlic and sequenced by next-generation sequencing and reverse transcription PCR. The complete RNA genome (GenBank accession number OP021693) is 8191 nucleotides in length, excluding the 3' poly(A) tail, and contains five open reading frames (ORFs). These open reading frames encode the viral replicase, triple gene block, and coat protein, and the genome organization is typical of members of the subfamily Quinvirinae. The virus has been tentatively named "garlic yellow curl virus" (GYCV). Phylogenetic analysis suggested that it represents an independent evolutionary lineage in the subfamily, clustering with the currently unclassified garlic yellow mosaic associated virus (GYMaV) and peony betaflexivirus 1 (PeV1). Differences between the phylogenies inferred for the replicase and coat protein indicate that the new virus does not belong to any established genus of the family Betaflexiviridae. This is the first report of GYCV in China.

3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage.

Chronic cerebral hypoperfusion is an important pathological factor in many neurodegenerative diseases, such as cerebral small vessel disease (CSVD). One of the most used animal models for chronic cerebral hypoperfusion is the bilateral common carotid artery stenosis (BCAS) mouse. For the therapy of CSVD and other diseases, it will be beneficial to understand the pathological alterations of the BCAS mouse, particularly vascular pathological changes. A mouse model of BCAS was used, and 8 weeks later, cognitive function of the mice was examined by using novel object recognition test and eight-arm radial maze test. 11.7 T magnetic resonance imaging (MRI) and luxol fast blue staining were used to evaluate the injury of the corpus callosum (CC), anterior commissure (AC), internal capsule (IC), and optic tract (Opt) in the cerebral white matter of mice. Three-dimensional vascular images of the whole brain of mice were acquired using fluorescence micro-optical sectioning tomography (fMOST) with a high resolution of 0.32 × 0.32 × 1.00 µm3. Then, the damaged white matter regions were further extracted to analyze the vessel length density, volume fraction, tortuosity, and the number of vessels of different internal diameters. The mouse cerebral caudal rhinal vein was also extracted and analyzed for its branch number and divergent angle in this study. BCAS modeling for 8 weeks resulted in impaired spatial working memory, reduced brain white matter integrity, and myelin degradation in mice, and CC showed the most severe white matter damage. 3D revascularization of the whole mouse brain showed that the number of large vessels was reduced and the number of small vessels was increased in BCAS mice. Further analysis revealed that the vessel length density and volume fraction in the damaged white matter region of BCAS mice were significantly reduced, and the vascular lesions were most noticeable in the CC. At the same time, the number of small vessels in the above white matter regions was significantly reduced, while the number of microvessels was significantly increased in BCAS mice, and the vascular tortuosity was also significantly increased. In addition, the analysis of caudal rhinal vein extraction revealed that the number of branches and the average divergent angle in BCAS mice were significantly reduced. The BCAS modeling for 8 weeks will lead to vascular lesions in whole brain of mice, and the caudal nasal vein was also damaged, while BCAS mice mainly mitigated the damages by increasing microvessels. What is more, the vascular lesions in white matter of mouse brain can cause white matter damage and spatial working memory deficit. These results provide evidence for the vascular pathological alterations caused by chronic hypoperfusion.

DsbA-L mediated renal tubulointerstitial fibrosis in UUO mice.

Recent studies have reported that upregulation of disulfide-bond A oxidoreductase-like protein (DsbA-L) prevented lipid-induced renal injury in diabetic nephropathy (DN). However, the role and regulation of proximal tubular DsbA-L for renal tubulointerstitial fibrosis (TIF) remains unclear. In current study, we found that a proximal tubules-specific DsbA-L knockout mouse (PT-DsbA-L-KO) attenuated UUO-induced TIF, renal cell apoptosis and inflammation. Mechanistically, the DsbA-L interacted with Hsp90 in mitochondria of BUMPT cells which activated the signaling of Smad3 and p53 to produce connective tissue growth factor (CTGF) and then resulted in accumulation of ECM of BUMPT cells and mouse kidney fibroblasts. In addition, the progression of TIF caused by UUO, ischemic/reperfusion (I/R), aristolochic acid, and repeated acute low-dose cisplatin was also alleviated in PT-DsbA-L-KO mice via the activation of Hsp90 /Smad3 and p53/CTGF axis. Finally, the above molecular changes were verified in the kidney biopsies from patients with obstructive nephropathy (Ob). Together, these results suggest that DsbA-L in proximal tubular cells promotes TIF via activation of the Hsp90 /Smad3 and p53/CTGF axis.

Portable Microwave Radar Systems for Short-Range Localization and Life Tracking: A Review.

Short-range localization and life tracking have been hot research topics in the fields of medical care, consumer electronics, driving assistance, and indoor robots/drones navigation. Among various sensors, microwave and mm-wave continuous-wave (CW) radar sensors are gaining more popularity in their intrinsic advantages such as simple architecture, easy system integration, high accuracy, relatively low cost, and penetration capability. This paper reviews the recent advances in CW radar systems for short-range localization and life tracking applications, including system improvement, signal processing, as well as the emerging applications integrated with machine learning.

Endovascular treatment for left innominate vein aneurysm: Case report and literature review.

Innominate vein aneurysms originating from the mediastinum are very rare. Previous treatments for this condition often required thoracotomy. We report a case of a 43-year-old male who presented a mediastinal mass by chest radiography. Contrast-enhanced CT and venography confirmed the diagnosis of left innominate vein aneurysm. The patient underwent endovascular treatment with stent placement and coil embolization of the left innominate vein. The patient remains well 18 months after surgery. The objective of this report is to discuss the diagnosis and endovascular treatment results of innominate vein aneurysm and to review the relevant literature to enhance and expand the pool of knowledge for this abnormality.

Mycobacterium tuberculosis H37Rv infection regulates alternative splicing in Macrophages.

OBJECTIVE: The objective of this study was to evaluate the expression of genes encoding SR proteinsand alternative splicing of IL4 and TLR4 in Mycobacterium tuberculosis (M. tb) H37Rv-infected macrophages. MATERIALS AND METHODS: THP-1 cells were induced to differentiate into macrophages with 200 nM PMA, and H37Rv strains were used for macrophage infection. After RNA extraction, qRT-PCR was performed to evaluate the expression of many SR proteins as well as the alternative splicing of IL4 and TLR4. RESULTS: IL4 and TLR4 play significant roles in host immunity to tuberculosis. The level of IL-4 splice variants in THP-1 cells increased after M. tb H37Rv infection. Three splice variants of TLR4 were detected in M. tb-infected THP-1 cells, when compared with uninfected controls; the expression level of these splicing variants in M. tb-infected THP-1 cell was down-regulated. Since SR proteins are RNA-binding proteins that regulate RNA splicing, the expression of SR proteins was examined, and SRSF2 and SRSF3 were significantly down-regulated. In addition, splicing factors SRp75 and SF3a were also significantly down-regulated post M. tb infection. CONCLUSION: Our findings indicate that alternative splicing may be involved in host gene regulation post M. tb infection of macrophage cells.

Time-Varying Vocal Folds Vibration Detection Using a 24 GHz Portable Auditory Radar.

Time-varying vocal folds vibration information is of crucial importance in speech processing, and the traditional devices to acquire speech signals are easily smeared by the high background noise and voice interference. In this paper, we present a non-acoustic way to capture the human vocal folds vibration using a 24-GHz portable auditory radar. Since the vocal folds vibration only reaches several millimeters, the high operating frequency and the 4 × 4 array antennas are applied to achieve the high sensitivity. The Variational Mode Decomposition (VMD) based algorithm is proposed to decompose the radar-detected auditory signal into a sequence of intrinsic modes firstly, and then, extract the time-varying vocal folds vibration frequency from the corresponding mode. Feasibility demonstration, evaluation, and comparison are conducted with tonal and non-tonal languages, and the low relative errors show a high consistency between the radar-detected auditory time-varying vocal folds vibration and acoustic fundamental frequency, except that the auditory radar significantly improves the frequency-resolving power.

Transcriptional Profiling of Mycobacterium tuberculosis Replicating in the Human Type II Alveolar Epithelial Cell Line, A549.

Alveolar epithelial cells outnumber alveolar macrophages by ~500 fold and increasing evidence suggests Mycobacterium tuberculosis may replicate dramatically in these cells during the initial weeks of infecting the lung [1,2]. Here, we report in experimental detail the transcriptional profiling of Mycobacterium tuberculosis replicating at 72 hr post-infection in the human type II alveolar epithelial cell line, A549, as compared to Mycobacterium tuberculosis growing logarithmically in laboratory broth culture [2]. All resulting transcriptional profiling data was deposited to the Gene Expression Omnibus (GEO) database under the accession number GSE58466.

Transcriptional profile of Mycobacterium tuberculosis replicating in type II alveolar epithelial cells.

Mycobacterium tuberculosis (M. tb) infection is initiated by the few bacilli inhaled into the alveolus. Studies in lungs of aerosol-infected mice provided evidence for extensive replication of M. tb in non-migrating, non-antigen-presenting cells in the alveoli during the first 2-3 weeks post-infection. Alveoli are lined by type II and type I alveolar epithelial cells (AEC) which outnumber alveolar macrophages by several hundred-fold. M. tb DNA and viable M. tb have been demonstrated in AEC and other non-macrophage cells of the kidney, liver, and spleen in autopsied tissues from latently-infected subjects from TB-endemic regions indicating systemic bacterial dissemination during primary infection. M. tb have also been demonstrated to replicate rapidly in A549 cells (type II AEC line) and acquire increased invasiveness for endothelial cells. Together, these results suggest that AEC could provide an important niche for bacterial expansion and development of a phenotype that promotes dissemination during primary infection. In the current studies, we have compared the transcriptional profile of M. tb replicating intracellularly in A549 cells to that of M. tb replicating in laboratory broth, by microarray analysis. Genes significantly upregulated during intracellular residence were consistent with an active, replicative, metabolic, and aerobic state, as were genes for tryptophan synthesis and for increased virulence (ESAT-6, and ESAT-6-like genes, esxH, esxJ, esxK, esxP, and esxW). In contrast, significant downregulation of the DevR (DosR) regulon and several hypoxia-induced genes was observed. Stress response genes were either not differentially expressed or were downregulated with the exception of the heat shock response and those induced by low pH. The intra-type II AEC M. tb transcriptome strongly suggests that AEC could provide a safe haven in which M. tb can expand dramatically and disseminate from the lung prior to the elicitation of adaptive immune responses.

Transcriptional profiling of Mycobacterium tuberculosis replicating ex vivo in blood from HIV- and HIV+ subjects.

Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60-70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.

[Expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain].

OBJECTIVE: To investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain. METHODS: Brain samples were collected from mice with different developmental stages: 9, 12, 14 d before birth (E9, E12, E14) and 1 d, 3 weeks and 3 months after birth. The expression of NSSR1 in mouse brain at different developmental stages was detected by Western blot and the distribution of NSSR1 was analyzed by immunohistochemical staining. The expression and distribution of NSSR1 in mouse brain were compared among embryos, neonatal and adult animals. RESULTS: During embryogenesis, the expression of NSSR1 proteins increases significantly from 0.186(E9) to 0.445(E14) and reached a high level after birth. Immunohistochemical analysis showed that in E12 embryos, NSSR1 was specifically distributed in the marginal and mantle layers. The expression of NSSR1 in hippocampus was very low in neonatal animals but stronger in adults. In cerebellar cortex, NSSR1 was widely expressed in purkinje and granule cells of adult animals, but mainly expressed in Purkinje cells in neonates. CONCLUSION: The expression of NSSR1 is regulated by the development of mouse brain and presents dynamic changes.

Dephosphorylated NSSR1 is induced by androgen in mouse epididymis and phosphorylated NSSR1 is increased during sperm maturation.

NSSR1 (Neural salient serine/arginine rich protein 1, alternatively SRp38) is a newly identified RNA splicing factor and predominantly expressed in neural tissues. Here, by Western blot analysis and immunofluorescent staining, we showed that the expression of dephosphorylated NSSR1 increased significantly during development of the caput epididymis. In adult mice, phosphorylated NSSR1 was mainly expressed in the apical side of epithelial cells, and dephosphorylated NSSR1 in caput epididymis was upregulated in a testosterone dependent manner. In addition, subcellular immunoreactive distribution of NSSR1 varied in different regions of the epididymis. With respect to the sperm, phosphorylated NSSR1 was detected in the mid-piece of the tail as well as the acrosome. Furthermore, NSSR1 was released from the sperm head during the capacitation and acrosome reaction. These findings for the first time provide the evidence for the potential roles of NSSR1 in sperm maturation and fertilization.

[Establishment of NCAM L1 minigene model and its splicing patterns in different cell lines].

OBJECTIVE: To establish a minigene model of neural cell adhesion molecule L1 (NCAM L1) gene and to study its splicing patterns in different cell lines. METHODS: Using human genetic cDNA as template, the NCAM L1 minigene fragment was amplified and inserted into eukaryotic expression vector. The minigene was transfected into 4 cell lines and the splicing patterns of NCAM L1 minigene in these cell lines were studied. RESULTS: The splicing patterns of NCAM L1 minigene were different in individual cell lines. In PFSK and Hela cell lines, two splicied isoforms were generated but in COS-1 and R28 cell lines, only one isoform existed. CONCLUSION: NCAM L1 minigene model can be used in alternative splicing analysis.

NSSR1 is regulated by testosterone in the mouse uterus and extensively expressed in endometrial carcinoma.

Neural salient serine/arginine-rich protein 1 (NSSR1) has been found to play important roles in inhibiting alternative splicing during heat shock and mitosis and is predominantly expressed in neural tissues such as cerebral neurons, cerebellar Purkinje cells and bipolar cells of the retina. Recently, NSSR1 has also been shown to be highly expressed in the testes, suggesting its potential roles in reproductive system. In this report, the expression of NSSR1 in the columnar epithelium of the endometrium and gland epithelium during the development of the mouse uterus, the regulation of NSSR1 level by testosterone in the adult mouse uterus, and expression level of NSSR1 in both human endometrial carcinomas and ovarian cancers were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry. We demonstrated that the expression of NSSR1 was developmentally regulated in the columnar epithelium of the endometrium and gland epithelium in the mouse uterus. Additionally, the NSSR1 level in the mouse uterus was maintained and regulated by testosterone. Interestingly, an enhanced level of NSSR1 was observed in both human endometrial carcinomas and ovarian cancers. Our results suggest that expression and distribution of NSSR1 is developmentally and hormonally regulated and up-regulated in endometrial carcinomas as well as ovarian cancers, indicating its potential involvement in uterine development and tumorgenesis.

The expression pattern of heterogeneous nuclear ribonucleoprotein R in rat retina.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) play important roles in DNA repairing, cell signaling, telomere biogenesis, and in regulating gene expression at both transcriptional and translational levels. In the present study, we demonstrated that the expression of hnRNP-R1 and hnRNP-R2 is developmentally regulated in rat retina. The neural specific isoform hnRNP-R2 is expressed in 7-day postnatal rat retina, but not in the adult retina. The positive immunohistochemistry signal of hnRNP-R1 is extensively distributed in the outer plexiform layer, inner nuclear layer and ganglion cell layer of rat retina. Double staining experiments showed that the positive signal of hnRNP-R1 is distributed in ON-type bipolar cells and localized in the cytoplasm, dendrites and axon terminals. In addition, the hnRNP-R1 distribution is regulated in rat retina during circadian. The present investigation suggests that hnRNP-R may play roles in retinal development and light-elicited cellular activities.

The expression and distribution of neural salient serine/arginine-rich protein 1 in rat retina.

Neural salient serine/arginine-rich protein 1 (NSSR1) expression has been found in mouse cerebral neurons, cerebellar Purkinje cells, pyramidal neurons and granule cells of dentate gyrus and regulates the pre-mRNA splicing of genes important for neural functions. In this study, we demonstrated that NSSR1 is expressed in rat retina and extensively distributed in the outer and inner plexiform layers. Double staining experiments showed that NSSR1 distributed mainly in ON-type bipolar cells and localized in the dendrites, somata and axon terminals. The result suggests that NSSR1 may play important roles in retinal function, possibly via regulating the neural-specific alternative splicing of genes.

A binding free energy hot spot in the ankyrin repeat protein GABPbeta mediated protein-protein interaction.

The frequently observed ankyrin repeat motif represents a structural scaffold evolved for mediating protein-protein interactions. As such, these repeats modulate a diverse range of cellular functions. We thermodynamically characterized the heterodimeric GA-binding protein (GABP) alphabeta complex and focused specifically on the interaction mediated by the ankyrin repeat domain of the GABPbeta. Our isothermal titration calorimetric analysis of the interaction between the GABP subunits determined an association constant (K(A)) of 6.0 x 10(8) M(-1) and that the association is favorably driven by a significant change in enthalpy (DeltaH) and a minor change in entropy (-TDeltaS). A total of 16 GABPbeta interface residues were chosen for alanine scanning mutagenesis. The calorimetrically measured differences in the free energy of binding were compared to computationally calculated values resulting in a correlation coefficient r = 0.71. We identified three spatially contiguous hydrophobic and aromatic residues that form a binding free energy hot spot (DeltaDeltaG > 2.0 kcal/mol). One residue provides structural support to the hot spot residues. Three non-hot spot residues are intermediate contributors (DeltaDeltaG approximately 1.0 kcal/mol) and create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. The remaining interface residues are located peripherally and have weak contributions. Finally, our mutational analysis revealed a significant entropy-enthalpy compensation for this interaction.

The ankyrin repeat as molecular architecture for protein recognition.

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.