Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR ...structures. We reengineered the human A 2A adenosine receptor by replacing its third intracellular loop with apocytochrome b⁵⁶² RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp 2.50 . Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.
Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras ...of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
► A method was developed for the selection of fusion domains for GPCR crystallization ► Apocytochrome b562RIL has advantages over previously utilized T4 lysozyme ► Diffraction quality crystals of two engineered GPCRs were successfully grown ► The method led to the crystal structure of the A2A adenosine receptor at 1.8 Å
Oxidative injury to retinal pigment epithelium (RPE) and retinal photoreceptors has been linked to a number of retinal diseases, including age-related macular degeneration (AMD). Reactive oxygen ...species (ROS)-mediated gene expression has been extensively studied at transcriptional levels. Also, the post-transcriptional control of gene expression at the level of translational regulation has been recently reported. However, the microRNA (miRNA/miR)-mediated post-transcriptional regulation in human RPE cells has not been thoroughly looked at. Increasing evidence points to a potential role of miRNAs in diverse physiological processes.
We demonstrated for the first time in a human retinal pigment epithelial cell line (ARPE-19) that the post-transcriptional control of gene expression via miRNA modulation regulates human catalase, an important and potent component of cell's antioxidant defensive network, which detoxifies hydrogen peroxide (H(2)O(2)) radicals. Exposure to several stress-inducing agents including H(2)O(2) has been reported to alter miRNA expression profile. Here, we demonstrated that a sublethal dose of H(2)O(2) (200 µM) up-regulated the expression of miR-30b, a member of the miR-30 family, which inhibited the expression of endogenous catalase both at the transcript and protein levels. However, antisense (antagomirs) of miR-30b was not only found to suppress the miR-30b mimics-mediated inhibitions, but also to dramatically increase the expression of catalase even under an oxidant environment.
We propose that a microRNA antisense approach could enhance cytoprotective mechanisms against oxidative stress by increasing the antioxidant defense system.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser ...(XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A
adenosine receptor (A
AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites
and
enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A
AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A
AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.
There is a lack of literature on indirect decompression in uniportal endoscopic posterolateral transforaminal lumbar interbody fusion (EPTLIF). Our aim is to evaluate the dimensions of the spinal ...canal and contralateral foramen before and after EPTLIF.
This is a retrospective study of patients who underwent EPTLIF in a tertiary spine centre over a 2-year period. The cross-sectional area of the spinal canal and the contralateral foramen at the level of fusion were measured on magnetic resonance imaging scan at 1-day postoperation and at the final follow-up. Patients were grouped according to the decompression performed as per the clinician's judgement.
One hundred fifty-two levels of fusion were performed in 120 patients. There was a statistically significant clinical improvement in visual analogue scale and Oswestry Disability Index scores postoperation. The measurements of the spinal canal area were 106.0 mm2, 138.8 mm2, and 195.5 mm2; while contralateral foraminal area were 73.2 mm2, 104.4 mm2, and 120.7 mm2 at preoperation, 1-day postoperation, and at the final follow-up, respectively (p < 0.001). For the subgroup analyses, spinal canal area measurements for the bilateral decompression cohort (n = 35) were 57.0 mm2, 123.9 mm2, and 191.8 mm2; for the ipsilateral decompression cohort (n = 42) were 89.3 mm2, 128.9 mm2, 183.3 mm2; and for the cohort without any decompression and only cage inserted (n = 75) were 138.3 mm2, 151.2 mm2, and 204.1 mm2 (p < 0.001). Contralateral foraminal area measurements were 73.3 mm2, 106.4 mm2 and 120.4 mm2 in the bilateral decompression cohort; 69.5 mm2, 99.0 mm2, 116.9 mm2 in the ipsilateral decompression cohort; and 75.1 mm2, 106.5 mm2, 122.9 mm2 in the cohort without any decompression (p < 0.001).
Indirect decompression of both the spinal canal and the contralateral foramen can be achieved via EPTLIF. Decompression on an asymptomatic contralateral side is not necessary.
Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons ...can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments. The structures of proteinase K (PK) and A
adenosine receptor (A
AR) were determined to resolutions of 1.8 and 4.2 Å using 4 and 24 consecutive 100 ps X-ray pulse exposures, respectively. Strong PK data were processed using existing Laue approaches, while weaker A
AR data required an alternative data-processing strategy. This demonstration of the feasibility presents new opportunities for time-resolved experiments with microcrystals to study structural changes in real time at pink-beam synchrotron beamlines worldwide.
Cervical radiculopathy is a common and disabling cervical condition characterized by symptoms including axial neck pain, radicular pain, weakness, and numbness in one or both arms. Common causes ...include herniated discs and foraminal stenosis, often accompanied by varying degrees of degenerative disc disease and uncovertebral joint hypertrophy. In the treatment of cervical radiculopathy, there is an increasing preference for posterior foraminotomy over anterior cervical discectomy and fusion due to the avoidance of fusion-related complications. As endoscopic spine surgery techniques continue to evolve, there is a rising interest in posterior endoscopic cervical foraminotomy and posterior endoscopic cervical discectomy as effective treatments for cervical radiculopathy. Because these procedures can performed through a single subcentimeter incision with minimal soft tissue damage, they can often be carried out as ambulatory procedures. In this narrative review, we examined current literature addressing the indications, surgical techniques, outcomes, and potential complications associated with posterior cervical endoscopic approaches.
Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes ...promises to enable the discovery and production of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the production of such proteins for academic purposes and is now transitioning to commercialization for the production of more optimal protein therapeutics. Focusing on E. coli, we review the history and current state of the field. We also provide a review of the first generation commercialization efforts, the lessons learned, and how those lessons are guiding new efforts. With continued academic and industrial progress, GCE methodologies promise to make possible the routine optimization of proteins for therapeutic use in a way that has only previously been possible with small-molecule therapeutics.
Prospective radiographic comparative study.
To compare and understand the load-bearing properties of each functional spinal unit (FSU) using three commonly assumed, physiological, spinal postures, ...namely, the flexed (slump sitting), erect (standing) and extended (backward bending) postures.
Sagittal spinal alignment is posture-dependent and influences the load-bearing properties of the spine. The routine placement of intervertebral cages "as anterior as possible" to correct deformity may compromise the load-bearing capabilities of the spine, leading to complications.
We recruited young patients with nonspecific low back pain for <3 months, who were otherwise healthy. Each patient had EOS images taken in the flexed, erect and extended positions, in random order, as well as magnetic resonance imaging to assess for disk degeneration. Angular and disk height measurements were performed and compared in all three postures using paired t-tests. Changes in disk height relative to the erect posture were caclulated to determine the alignment-specific load-bearing area of each FSU.
Eighty-three patients (415 lumbar intervertebral disks) were studied. Significant alignment changes were found between all three postures at L1/2, and only between erect and flexion at the other FSUs. Disk height measurements showed that the neutral axis of the spine, marked by zones where disk heights did not change, varied between postures and was level specific. The load-bearing areas were also found to be more anterior in flexion and more posterior in extension, with the erect spine resembling the extended spine to a greater extent.
Load-bearing areas of the lumbar spine are sagittal alignment-specific and level-specific. This may imply that, depending on the surgical realignment strategy, attention should be paid not just to placing an intervertebral cage "as anterior as possible" for generating lordosis, but also on optimizing load-bearing in the lumbar spine.
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