Forces hold everything together and determine its structure and dynamics. In
particular, tiny forces of 1-100 piconewtons govern the structures and dynamics
of biomacromolecules. These forces enable folding, assembly, conformational
fluctuations, or directional movements of biomacromolecules over sub-nanometer
to micron distances. Optical tweezers have become a revolutionary tool to probe
the forces, structures, and dynamics associated with biomacromolecules at a
single-molecule level with unprecedented resolution. In this review, we
introduce the basic principles of optical tweezers and their latest applications
in studies of protein folding and molecular motors. We describe the folding
dynamics of two strong coiled coil proteins, the GCN4-derived protein pIL and
the SNARE complex. Both complexes show multiple folding intermediates and
pathways. ATP-dependent chromatin remodeling complexes translocate DNA to
remodel chromatin structures. The detailed DNA translocation properties of such
molecular motors have recently been characterized by optical tweezers, which are
reviewed here. Finally, several future developments and applications of optical
tweezers are discussed. These past and future applications demonstrate the
unique advantages of high-resolution optical tweezers in quantitatively
characterizing complex multi-scale dynamics of biomacromolecules.
optical tweezers; single-molecule manipulation; protein folding; molecular motors; DNA translocation; SNARE proteins
Subarachnoid hemorrhage is a common and dangerous disease with an unfavorable prognosis. Patients with poor-grade subarachnoid hemorrhage (Hunt & Hess Grades 4–5) are unconscious on admission. Because of the high mortality and disability rate associated with poor-grade subarachnoid hemorrhage, it is often treated conservatively. Timing of surgery for poor-grade aneurysmal subarachnoid hemorrhage is still controversial, therefore this study aims to identify the optimal time to operate on patients admitted in poor clinical condition.
Ninety-nine patients meeting the inclusion criteria were randomly assigned into three treatment groups. The early surgery group received operation within 3 days after onset of subarachnoid hemorrhage (day of SAH = day 1); the intermediate surgery group received operation from days 4 to 7, and surgery was performed on the late surgery group after day 7. Follow-up was performed 1, 3, and 6 months after aneurysm clipping. Primary indicators of outcome included the Extended Glasgow Outcome Scale and the Modified Rankin Scale, while secondary indicators of outcome were assessed using the Barthel Index and mortality.
This is the first prospective, single-center, observer-blinded, randomized controlled trial to elucidate optimal timing for surgery in poor-grade subarachnoid hemorrhage patients. The results of this study will be used to direct decisions of surgical intervention in poor-grade subarachnoid hemorrhage, thus improving clinical outcomes for patients.
Chinese Clinical Trial Registry: ChiCTR-TRC-12002917
Timing of surgery; Poor-grade; Subarachnoid hemorrhage; ICP; Prognosis
SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here we used optical tweezers to observe in real time a long-sought SNARE assembly intermediate in which only the membrane-distal N-terminal half of the bundle is assembled. Our finding supports the zippering hypothesis, but suggests that zippering proceeds through three sequential binary switches, not continuously, in the N- and C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force which mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion.
Renal hematuria is caused by glomerular disease. Under pathological conditions, the distribution of interleukin-6 (IL-6) in kidney tissue is abnormal and urinary IL-6 levels are increased. Abnormal IL-6 secretion promotes the hyperplasia of mesangial cells and matrix and, thus, affects the permeability of the glomerular filtration membrane. Therefore, the detection of urinary IL-6 levels in patients with renal hematuria is beneficial for disease evaluation. A total of 82 patients with primary renal hematuria were divided into group 1 (UPr/24 h < 150 mg; pure hematuria group), group 2 (150 mg ≤ UPr/24 h ≤ 1,000 mg) and group 3 (UPr/24 h > 1,000 mg). A total of 30 normal individuals were selected as the controls. The urinary IL-6 levels were detected by the enzyme-linked immunosorbent assay (ELISA) method and a renal biopsy was conducted. The urinary IL-6 levels and renal pathological damage scores in groups 1 and 2 were significantly reduced compared with those in group 3, (P<0.001 and 0.01, respectively), with no significant difference between groups 1 and 2 (P>0.05). The correlation coefficient (r) of urinary IL-6 with 24 h urinary protein (UPr/24 h) in groups 1, 2 and 3 was 0.017, 0.045 and 0.747, respectively, and that of urinary IL-6 with renal pathological damage score was 0.627, 0.199 and 0.119, respectively. The UPr/24 h was significantly correlated with IL-6 level (r=0.7320, P<0.000). In group 1, the urinary IL-6 levels were correlated with the degree of renal pathological damage. A positive correlation was observed between urinary IL-6 levels and UPr/24 h.
interleukin-6; renal hematuria; 24 h urinary protein
CD and UV resonance Raman measurements surprisingly find that the charge screening of even 2 M concentrations of NaCl and KCl do not alter the unfolded PPII and 2.51-helix conformations of poly-L-glutamate. These salts appear to be excluded from the region between the side chain charges and the peptide backbone. Furthermore, no direct ion pairing occurs between these salts and the side chain carboxylates.
poly-L-glutamate; PPII; 2.51-helix; salt exclusion; UV resonance Raman
We used 204 nm excitation UV Resonance Raman (UVRR) spectroscopy to examine the effects of NaClO4 on the conformation of poly–L–lysine (PLL). The presence of NaClO4 induces the formation of α–helix, π–helix/bulge and turn conformations. The dependence of the AmIII3 frequency on the peptide Ψ Ramachandran angle allows us to experimentally determine the conformational population distributions and the energy landscape of PLL along the Ramachandran Ψ angle. We also used UVRR to measure the NaClO4 concentration dependence of PLL amide hydrogen exchange kinetics. Exchange rates were determined by fitting the D2O exchanging PLL UVRR AmII′ band time evolution. Hydrogen exchange is slowed at high NaClO4 concentrations. The PLL AmII′ band exchange kinetics at 0.0, 0.2 and 0.35 M NaClO4 can be fit by single exponentials, but the AmII′ band kinetics of PLL at 0.8 M NaClO4 requires a double exponential fit. The exchange rates for the extended conformations were monitored by measuring the Cα–H band kinetics. These kinetics are identical to those of the AmII′ band until 0.8 M NaClO4 whereupon the extended conformation exchange becomes clearly faster than that of the α–helix–like conformations. Our results indicate that ClO4− binds to the PLL backbone to protect it from OH− exchange catalysis. In addition, ClO4− binding also slows the conformational exchange between the extended and α–helix–like conformations, probably by increasing the activation barriers for conformational interchanges.
Conformational distribution; Energy landscape; Conformational dynamics; Salt binding
The previous meta-analysis on the use of endothelin-receptor antagonists (ETRAs) to treat aneurysmal subarachnoid hemorrhage (SAH) has become outdated due to recently published phase 3 clinical trials. An up-to-date meta-analysis is needed to provide the best available evidence for the efficacy of ETRAs for aneurysmal SAH.
We performed a systematic review and meta-analysis of published randomized controlled trials that investigate efficacy of ETRAs in patients with aneurysmal SAH. Mortality, unfavorable outcome, delayed ischemic neurological deficit (DIND), delayed cerebral infarction (DCI), angiographic vasospasm and adverse events were analyzed. Meta-analysis was performed in terms of the risk ratio (RR) and 95% confidence interval (CI).
Five eligible studies were reviewed and analyzed, involving 2,595 patients. The pooled RRs of mortality and unfavorable outcome after SAH were 1.03 (95% CI = 0.77 to 1.36) and 1.07 (95% CI = 0.93 to 1.22), respectively. The pooled RRs were 0.87 (95% CI = 0.74 to 1.03) for DCI, 0.77 (95% CI = 0.66 to 0.90) for DIND, and 0.66 (95% CI = 0.57 to 0.77) for angiographic vasospasm. There were significant increases in lung complications (RR = 1.80, 95% CI = 1.55 to 2.09), hypotension (RR = 2.42, 95% CI = 1.78 to 3.29) and anemia (RR = 1.47, 95% CI = 1.19 to 1.83) in patients administered ETRAs.
There is no evidence that ETRAs could benefit clinical outcome in patients with SAH. Owing to the increased adverse events, further clinical trials of ETRAs in SAH patients should be more carefully formulated and designed. The present results also suggest that DCI may be a better outcome measure than vasospasm and DIND in SAH clinical trials and observational studies.
Recently, a multicenter randomized controlled trial (RCT) by Cooper and colleagues indicated that decompressive craniectomy (DC) may be associated with a worse functional outcome in patients with diffuse traumatic brain injury (TBI), although DC can immediately and constantly reduce intracranial pressure (ICP). As this trial is well planned and of high quality, the unexpected result is meaningful. However, the evidence of the study is insufficient and the effect of DC in severe TBI is still uncertain. Additional multicenter RCTs are necessary to provide class I evidence on the role of DC in the treatment of refractory raised ICP after severe TBI.
We used 204 nm excitation UV Resonance Raman (UVRR) spectroscopy to examine the role of side chain electrostatic interactions in determining the conformation of poly-L-lysine (PLL). We examined the pH and ionic strength dependence of the UVRR. The pH dependence of PLL UVRR spectra between pH 7.1 and 11.7 cannot be described by a two-state model, but requires at least one additional state. The AmIII3 region fitting with pH 7.1 and 11.7 basis spectra reveals a small pH induced decrease in the relative fraction of the 2.51-helix conformation compared to the PPII conformation. We performed a 2D general correlation analysis on the PLL pH dependence UVRR spectra. The asynchronous spectrum shows enhanced spectral resolution. The 2D asynchronous spectrum reveals multiple components in the Cα-H b band and the AmII band whose origins are unclear. The cross peaks in the 2D asynchronous spectrum between the AmIII band and the other bands reveals that increasing pH induces three new structures: π-helix, α-helix and some turn structure. We find that 2.5 M NaCl does not change the equilibrium between the PPII and 2.51-helix conformations by screening sidechain electrostatic repulsion. The result indicates that NaCl does not penetrate the region between the sidechain and the peptide backbone. We also compared PLL conformations induced by high pH to that induced by 0.8 M ClO4−. Both conditions induce α-helix-like conformations. 0.8 M ClO4− induces 6% more α-helix-like conformations than at pH 12.4. Higher pH gives rise to longer α-helices and less turn structures.
The X protein (HBx) of hepatitis B virus (HBV) is involved in the development of hepatocellular carcinoma (HCC), and methionine adenosyltransferase 2A (MAT2A) promotes the growth of liver cancer cells through altering S-adenosylmethionine homeostasis. Thus, we speculated that a link between HBx and MAT2A may contribute to HCC development. In this study, the effects of HBx on MAT2A expression and cell apoptosis were investigated, and the molecular mechanism by which HBx and MAT2A regulate tumorigenesis was evaluated. Results from immunohistochemistry analyses of 37 pairs of HBV-associated liver cancer tissues/corresponding peritumor tissues showed that HBx and MAT2A are highly expressed in most liver tumor tissues. Our in vitro results revealed that HBx activates MAT2A expression in a dose-dependent manner in hepatoma cells, and such regulation requires the cis-regulatory elements NF-κB and CREB on the MAT2A gene promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) further demonstrated that HBx facilitates the binding of NF-κB and CREB to MAT2A gene promoter. In addition, overexpression of HBx or MAT2A inhibits cell apoptosis, whereas knockdown of MAT2A expression stimulates apoptosis in hepatoma cells. Furthermore, we demonstrated that HBx reduces MAT1A expression and AdoMet production but enhances MAT2β expression. Thus, we proposed that HBx activates MAT2A expression through NF-κB and CREB signaling pathways to reduce AdoMet production, inhibit hepatoma cell apoptosis, and perhaps enhance HCC development. These findings should provide new insights into our understanding how the molecular mechanisms underline the effects of HBV infection on the production of MAT2A and the development of HCC.
Apoptosis; Cancer Tumor Promoter; Chromatin Immunoprecipitation (ChiP); CREB; DNA-Protein Interaction; DNA Viruses; Gene Regulation; Hepatitis Virus; Oncogene; S-Adenosylmethionine (AdoMet)
To obtain and analyze early retinal changes at the molecular level 24 h after a radiation injury to the ipsilateral intraorbital nerve using gamma knife surgery (GKS), and to examine the morphological changes in bilateral optic nerves.
Unilateral intraorbital optic nerves of three rhesus macaques were treated by GKS with irradiated doses of 15 Gy, while contralateral optic nerves and retinas served as the control. Gene expression profiles of the control and affected retinas were analyzed with Affymetrix Rhesus Macaque Genome arrays. To verify the results, a quantitative real-time polymerase chain reaction (qRT–PCR) was performed to test the expression patterns of five function-known genes. Morphological changes in the bilateral optic nerves were examined using a transmission electron microscope (TEM) and light microscopy. The glial cell reaction in bilateral optic nerves was studied using immunohistochemistry.
Of the probe sets, 1,597 (representing 1,081 genes) met the criteria for differential expression, of which 82 genes were significantly up-or down-regulated in treated retinas. There was prominent upregulation of genes associated with glial cell activation in the treated retina. Genes related to an early inflammatory reaction and to cell death were also significantly regulated in response to a radiation injury to the intraorbital optic nerve. In contrast, the messenger ribonucleic acid (mRNA) expression levels of retinal ganglion cell (RGC)-specific genes were low. Morphologically, cytoplasmic processes of astrocytes in treated nerves were shorter than those of the control and were not straight, while also being accompanied by decreased GFAP immunostaining. More oligodendrocytes and inflammatory cells were apparent in treated nerves than in the control. In addition, swollen mitochondria and slight chromation condensation could be seen in the glial cells of treated nerves.
We conclude that the current irradiated dose of 15 Gy was sufficient to lead to a radiation injury of the optic nerve and retina. Several transcripts deregulated in retinas after a radiation injury play a key role in radiation-induced neurogenic visual loss, especially for genes associated with RGC, glial cell, and cell death. Glial cells in optic nerves might be the primary target of a radiation injury in the optic nerve.
Modifications of DNA and histones in various combinations are correlated with many cellular processes. In this study, we investigated the possible relationship between histone H4 tetraacetylation, DNA methylation and histone H3 dimethylation at lysine 9 during mitosis in maize root meristems.
Treatment with trichostatin A, which inhibits histone deacetylases, resulted in increased histone H4 acetylation accompanied by the decondensation of interphase chromatin and a decrease in both global H3K9 dimethylation and DNA methylation during mitosis in maize root tip cells. These observations suggest that histone acetylation may affect DNA and histone methylation during mitosis. Treatment with 5-azacytidine, a cytosine analog that reduces DNA methylation, caused chromatin decondensation and mediated an increase in H4 acetylation, in addition to reduced DNA methylation and H3K9 dimethylation during interphase and mitosis. These results suggest that decreased DNA methylation causes a reduction in H3K9 dimethylation and an increase in H4 acetylation.
The interchangeable effects of 5-azacytidine and trichostatin A on H4 acetylation, DNA methylation and H3K9 dimethylation indicate a mutually reinforcing action between histone acetylation, DNA methylation and histone methylation with respect to chromatin modification. Treatment with trichostatin A and 5-azacytidine treatment caused a decrease in the mitotic index, suggesting that H4 deacetylation and DNA and H3K9 methylation may contain the necessary information for triggering mitosis in maize root tips.
Galectin-1, a member of a family of evolutionarily conserved glycan-binding proteins, binds specifically to poly-N-acetyllactosamine-enriched glycoconjugates. Through interactions with these glycoconjugates, this protein modulates inflammatory responses and contributes to tumor progression and immune cell homeostasis. The carbohydrate recognition domain includes the single protein tryptophan (Trp68). UV Resonance Raman spectroscopy and molecular dynamic simulation were used to examine the change in the environment of the Trp on ligand binding. The UV Raman spectra and the calculated water radial distribution functions show that, while no large structural changes in the protein follows lactose binding, substantial solvent reorganization occurs. These new insights into the microscopic role of water molecules on Gal-1 binding to its specific carbohydrate ligands provides a better understanding of the physicochemical properties of Gal-1-saccharide interactions, which will be useful for the design of synthetic inhibitors for therapeutic purposes.
In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.
Methods and Results
During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.
The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.
Particulate air pollution is a serious problem all over the world, and the development of a method to evaluate the health effects of ambient particles is necessary. In this study, cells cultured in vitro were exposed to particles sampled at the side of a main road, and their protein expression levels were examined.
Ambient particles were collected at the side of a main road using a high-volume air sampler. Some of the collected particles (crude particles) were treated with an organic solvent to remove chemical components, and the resulting residues were used as residual particles. Cells from the mouse alveolar epithelial cell line LA-4 were inoculated into tissue-culture dishes at 1.4×104/cm2, exposed to each type of particle or artificial carbon particles (Printex 90) that were dispersed using an ultrasonic homogenizer by mixing in the medium twice at 24 and 48 hours, and incubated for up to 72 hours after the start of inoculation. After exposure, the number of cells and intracellular dehydrogenase activity were measured. Proteins extracted from the cells were subjected to two-dimensional gel electrophoresis with isoelectric focusing at pHs 4–7 using a 10% acrylamide gel, and their expression levels were analyzed after fluorescent staining.
The intracellular dehydrogenase activity of the cells significantly decreased as a result of exposure to the residual (0.70-fold) and crude (0.84-fold) particles compared with that of the control, but it showed no change as a result of exposure to Printex 90. The protein expression levels in the cells exposed to the particles increased or decreased similarly, but different expression levels were also observed. There were differences in the effects observed between the cells exposed to the artificial carbon particles and those exposed to particles collected from ambient air.
This study indicates that protein expression levels in cells change in response to exposure to particles collected from ambient air. To evaluate the effects of particles on health, it is considered necessary to use particles collected from ambient air.
particulate matter; air pollution; health effect; biomarker; two-dimensional gel electrophoresis
Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.