Engineering three-dimensional cardiac microtissues for potential drug screening applications.

Heart disease is one of the major global health issues. Despite rapid advances in cardiac tissue engineering, limited successful strategies have been achieved to cure cardiovascular diseases. This situation is mainly due to poor understanding of the mechanism of diverse heart diseases and unavailability of effective in vitro heart tissue models for cardiovascular drug screening. With the development of microengineering technologies, three-dimensional (3D) cardiac microtissue (CMT) models, mimicking 3D architectural microenvironment of native heart tissues, have been developed. The engineered 3D CMT models hold greater potential to be used for assessing effective drugs candidates than traditional two-dimensional cardiomyocyte culture models. This review discusses the development of 3D CMT models and highlights their potential applications for high-throughput screening of cardiovascular drug candidates.

Genome-wide copy number variation association study suggested VPS13B gene for osteoporosis in Caucasians.

UNLABELLED: Osteoporotic fracture (OF) is a serious outcome of osteoporosis. Important risk factors for OF include reduced bone mineral density and unstable bone structure. This genome-wide copy number variation association study suggested VPS13B gene for osteoporosis in Caucasians. INTRODUCTION: Bone mineral density (BMD) and femoral neck cross-sectional geometric parameters (FNCSGPs) are under strong genetic control. DNA copy number variation (CNV) is an important source of genetic diversity for human diseases. This study aims to identify CNVs associated with BMD and FNCSGPs. METHODS: Genome-wide CNV association analyses were conducted in 1,000 unrelated Caucasian subjects for BMD at the spine, hip, femoral neck, and for three FNCSGPs -cortical thickness (CT), cross-section area (CSA), and buckling ratio (BR). BMD was measured by dual energy X-ray absorptiometry (DEXA). CT, CSA, and BR were estimated using DEXA measurements. Affymetrix 500K arrays and copy number analysis tool was used to identify CNVs. RESULTS: A CNV in VPS13B gene was significantly associated with spine, hip and FN BMDs, and CT, CSA, and BR (p < 0.05). Compared to subjects with two copies of the CNV, carriers of one copy had an average of 14.6%, 12.4%, and 13.6% higher spine, hip, and FN BMD, 20.0% thicker CT, 10.6% larger CSA, and 12.4% lower BR. Thus, a decrease of the CNV consistently produced stronger bone, thereby reducing osteoporotic fracture risk. CONCLUSIONS: VPS13B gene, via affecting BMD and FNCSGPs, is a novel osteoporosis risk gene.

Herpes simplex virus hepatitis: expanding the spectrum of disease.

We describe 2 transplant patients with herpes simplex virus (HSV) hepatitis who were minimally symptomatic throughout their illness. The spectrum of disease caused by HSV hepatitis is more variable than previously reported in this population. HSV hepatitis should be considered in immunocompromised hosts with elevated transaminases without evidence of fulminant hepatic necrosis.

Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 ClpB N-terminal domain.

Escherichia coli Hsp100 ClpB plays critical roles in multi-chaperone systems in cell physiology. After the ATPase activity is stimulated by protein or peptide binding, ClpB disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to more efficiently refold the non-native polypeptides. The mechanisms by which the ClpB acts as a molecular chaperone to disaggregate non-native polypeptides are unknown. The N-terminal domain of ClpB has been proposed to interact with non-native polypeptides. To investigate whether the N-terminal domain participates in polypeptide recognition and binding or modulates the activity of ClpB, the ClpB N-terminal domain has been cloned, purified and crystallized. The ClpB N-terminal domain crystals diffract to 1.95 A using a synchrotron X-ray source and belong to the space group P1, with unit-cell parameters a = 50.2, b = 52.6, c = 56.8 A, alpha = 90.5, beta = 111.8, gamma = 107.1 degrees. Structure determination by the multiple anomalous dispersion (MAD) method is under way.

The crystal structure of the influenza matrix protein M1 at neutral pH: M1-M1 protein interfaces can rotate in the oligomeric structures of M1.

The influenza matrix protein (M1) forms a protein layer under the viral membrane and is essential for viral stability and integrity. M1 mediates the encapsidation of the viral RNPs into the viral membrane by its membrane and RNP-binding activities. In order to understand the roles of M1-M1 protein interactions in forming the M1 layer, X-ray crystallographic studies of a M1 fragment (1-162) were carried out at neutral pH and compared with an acidic pH structure. At neutral pH the asymmetric unit was a stacked dimer of M1. A long molecular ribbon of neutral stacked dimers was formed by translation as dictated by the P1 space group. The elongated ribbon had a positively charged stripe on one side of the ribbon. A similar M1-M1 stacking interface was also found in the acidic asymmetric unit. However, within the acidic stacked dimer the molecules were not straight, but rotated in relation to each other by slightly changing the M1-M1 stacking interface. The acidic structure possessed an additional M1-M1 twofold interface. Protein docking confirmed that the M1-M1 stacking and M1-M1 twofold interfaces could be used to form a double ribbon of M1 molecules. By iterative repetition of the rotated relationship among the M1 molecules, a helix of M1 was generated. These studies suggest that M1 has the ability to form straight or bent elongated ribbons and helices. These oligomers are consistent with previous electron microscopic studies of M1, which demonstrated that isolated M1 formed elongated and flexible ribbons when isolated from what appeared to be a helical shell of M1 in the influenza virus.

Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 ClpB nucleotide-binding domain 1 (NBD1).

Escherichia coli Hsp100 ClpB plays critical roles in multi-chaperone systems in cell physiology. After being activated by protein or peptide binding, ClpB disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to refold the non-native polypeptides. ClpB contains two nucleotide-binding domains with Walker A and B motifs within their primary sequences. Therefore, ClpB can be classified as a member of the large ATPase family known as ATPases associated with various cellular activities (AAAs). The mechanisms by which the ClpB acts as a molecular chaperone to disaggregate denatured polypeptides are unknown. To investigate how the nucleotide-binding domain participates in ClpB chaperone activity, we have cloned and crystallized ClpB nucleotide-binding domain 1 (NBD1). The ClpB NBD1 crystals diffract to 1.80 A using a synchrotron X-ray source and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 38.41, b = 65.48, c = 79.13 A. Structure determination by the MAD method is under way.

Cloning, expression, purification and preliminary X-ray crystallographic studies of yeast Hsp40 Sis1 complexed with Hsp70 Ssa1 C-terminal lid domain.

Heat-shock protein 70 (Hsp70) plays essential roles in a number of cellular processes such as protein folding, assembly and translocation. Heat-shock protein 40 (Hsp40) transiently interacts with Hsp70 and facilitates Hsp70 functions in these processes within cells. Hsp40 recognizes and binds non-native polypeptide and delivers it to Hsp70. Hsp40 can then stimulate the ATPase activity of Hsp70 to refold the polypeptide. To investigate the molecular mechanism by which Hsp40 interacts with Hsp70 to transport the non-native polypeptide, Saccharomyces cerevisiae Hsp40 Sis1 C-terminal peptide-binding fragment complexed with Hsp70 Ssa1 C-terminal lid domain has been produced and crystallized. The complex crystals diffract to 3.3 A and belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 112.17, c = 171.31 A. Structure determination by the MAD method is under way.

Myeloid-related protein (MRP)-8 from cervico-vaginal secretions activates HIV replication.

OBJECTIVES: To identify a substance found in female genital tract secretions that enhances HIV expression in infected cells. DESIGN: Cervico-vaginal lavages (CVL), collected in sterile normal saline, were fractionated and tested for HIV-inducing activity using HIV-infected monocytes. METHODS: To purify the component(s) of CVL that enhance HIV production, Mono-Q ion exchange chromatography followed by Superose-12 molecular sieve analysis, and SDS--PAGE were performed. The purified protein was identified by amino acid sequence analysis. RESULTS: SDS--PAGE of bioactive fractions showed a 14 kDa polypeptide band. Amino acid sequence analysis of selected peptides from the 14 kDa band showed 100% homology with the myeloid-related protein (MRP)-8, an inflammatory protein found in mucosal secretions. Western blot analysis revealed that bioactive CVL contained more immunoreactive MRP-8 than samples without bioactivity. The HIV-inducing activity of MRP-8 was further confirmed by showing that human recombinant MRP-8 increased HIV expression by up to 40-fold. CONCLUSIONS: MRP-8 in cervico-vaginal secretions stimulates HIV production. Strategies aimed at blocking MRP-8 activity in the genital tract could reduce risk of sexual as well as maternal--infant transmission of HIV.

HIV-1 reverse transcriptase mutations found in a drug-experienced patient confer reduced susceptibility to multiple nucleoside reverse transcriptase inhibitors.

HIV-1 reverse transcriptase (RT) genotypes were obtained from 13 patients treated with stavudine. No previously-reported mutations indicative of stavudine resistance were found in these patients and no novel mutations occurred in more than two patients. One patient, treated with stavudine for 1 month and treated previously with zidovudine, zalcitabine and lamivudine, carried a mutation at codon 75 of the RT (V75M). A chimeric virus, including the patient's RT sequence from codon 25 to codon 220, which carried the resistance mutations M41 L, D67N, T69D, K70R, L210W and T215Y in addition to V75M, displayed reduced susceptibility to multiple nucleoside RT inhibitors (NRTIs). Removal of V75M from this RT background resulted in a return of susceptibility to didanosine and lamivudine. Our data are in agreement with previous studies demonstrating the rarity of stavudine resistance mutations in stavudine-treated patients. However, we describe a new set of mutations, found in the RT of a heavily-treated patient, that can confer reduced susceptibility to multiple NRTIs. These results underscore the importance of increased vigilance for possible multiple-drug resistance in patients who have been heavily treated with NRTIs.

The crystal structure of the peptide-binding fragment from the yeast Hsp40 protein Sis1.

BACKGROUND: Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding. How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue. RESULTS: To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae. The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis. Sis1 monomers are elongated and consist of two domains with similar folds. Sis1 dimerizes through a short C-terminal stretch. The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids. CONCLUSIONS: Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity. Thus, dimer formation is critical for Sis1 chaperone function. We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.

Force production of the genioglossus as a function of muscle length in normal humans.

Resting muscle length affects both maximum force production and force maintenance. The strength and force maintenance characteristics of the genioglossus as a function of resting muscle length have not been described. We hypothesized that genioglossus optimum length (L(o)) could be defined in vivo and that the ability of the genioglossus to sustain a given workload would decrease as resting length deviated from L(o). To test this, 11 normal men repeated maximal isometric genioglossus protrusions at different muscle lengths to determine L(o). L(o) was also obtained by using submaximal efforts while simultaneously recording electromyographic activity of the genioglossus, with L(o) defined as the length at which the force-to-genioglossus electromyographic activity ratio was maximum. Both methods provided similar results. Force maintenance was measured at four muscle lengths on separate days. Target efforts representing 60% of each subject's maximum at L(o) and lasting 5 s were performed at 12-s intervals. Time limit of endurance of the genioglossus was defined as the time from trial onset at which 90% of the target could not be maintained for three consecutive efforts. Time limit of endurance was greatest at L(o) and fell to 47.5% at L(o) + 1 cm, 53.8% at L(o) - 1 cm, and 47.4% at L(o) - 1.5 cm. We conclude that L(o) of the genioglossus can be determined in vivo and that force maintenance of the genioglossus is decreased when operating length deviates from L(o).

Body composition in HIV-infected women.

Although loss of lean body mass is a common complication of human immunodeficiency virus (HIV) infection that can occur across the disease trajectory, few studies have characterized the body composition of HIV-infected women. We used bioelectrical impedance analysis to characterize the body composition of HIV-infected (n = 56) and uninfected (n = 12) women who were matched on percentage of ideal body weight. The HIV-infected women did not differ from the uninfected women by height-adjusted fat mass or body cell mass. Intergroup comparisons among the HIV-infected women showed that underweight women had significantly less fat mass than did normal-weight women but did not significantly differ with respect to body cell mass. Among all HIV-infected women, CD4(+) lymphocyte count was positively correlated with fat mass (r = 0.32, P = 0.01) but not with body cell mass. No significant correlations were found between any body-composition parameter and plasma viral load. Our findings suggest that, unlike men, HIV-infected underweight women show a preferential loss of fat mass and a relative preservation of body cell mass. This altered pattern of weight loss may relate to higher premorbid fat stores in women and/or hormonal differences.

PI transfer protein: the specific recognition of phospholipids and its functions.

Phosphatidylinositol transfer proteins (PITPs) can bind specifically and transfer a single phosphatidylinositol (PI) molecule between phospholipid membranes in an ATP-independent manner in vitro. PITPs exist in all the eukaryotic systems from yeast to human. PITP plays an essential role in intracellular vesicle flow and inositol lipid signaling. The crystal structure of yeast PITP Sec14p reveals a large hydrophobic pocket to accommodate the acyl chains of phospholipid molecules. At the opening of the pocket, a hydrogen bond network may render Sec14p the binding specificity to PI molecules. The structure suggests that the PI-binding ability may play an important role in the in vivo function of PITPs.

Association of indicators of bacterial vaginosis with a female genital tract factor that induces expression of HIV-1.

OBJECTIVE: The aim of this study was to determine the relationship of bacterial vaginosis and bacterial vaginosis-associated microorganisms with an HIV-inducing factor (HIF) found in cervicovaginal lavage. DESIGN: A total of 26 cervicovaginal lavage specimens collected from 17 women were used in this study to determine if HIF was significantly associated with features consistent with bacterial vaginosis. METHODS: Patients were evaluated for various clinical features including age, HIV status and stage, CD4 cell counts, clinical diagnosis of gynecological infections, vaginal pH, Gram stains of vaginal fluid, phase of menstruation, and presence of cervical dysplasia. Cervicovaginal lavage specimens were analyzed for the presence of HIF by U1 bioassay. The presence of Gardnerella vaginalis, and general Mycoplasmataceae, and specifically Mycoplasma hominis, Ureaplasma urealyticum, M. fermentans, M. genitalium in cervicovaginal lavage were determined by semiquantitative PCR. RESULTS: Eleven cervicovaginal lavage samples from seven women were HIF-positive and 15 cervicovaginal lavage samples from 11 women were HIF-negative (patient No. 8 had two HIF-negative cervicovaginal lavage and one HIF-positive cervicovaginal lavage). The following parameters were significantly associated with HIF: abnormal vaginal fluid pH (>4.5) (P = 0.006), Gram stains indicative of bacterial vaginosis (P = 0.007), normal menstrual cycle (P = 0.0007) and PCR detection and relative quantity of M. hominis (P = 0.0003, P = 0.002). CONCLUSIONS: This study indicates that HIF is closely associated with features of bacterial vaginosis.