The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

Characterization of two pathological gating-charge substitutions in Cav1.4 L-type calcium channels.

Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.

Intraoperative computed tomography angiography with computed tomography perfusion imaging in vascular neurosurgery: feasibility of a new concept.

OBJECT: In vascular neurosurgery, there is a demand for intraoperative imaging of blood vessels as well as for rapid information about critical impairment of brain perfusion. This study was conducted to analyze the feasibility of intraoperative CT angiography and brain perfusion mapping using an up-to-date multislice CT scanner in a prospective pilot series. METHODS: Ten patients with unruptured aneurysms underwent intraoperative scanning with a 40-slice sliding-gantry CT scanner. Multimodal CT acquisition was obtained in 8 patients consisting of dynamic perfusion CT (PCT) scanning followed by intracranial CT angiography. Two of these patients underwent CT angiography and PCT 2 times in 1 session as a control after repositioning cerebral aneurysm clips. In another 2 patients, CT angiography was performed alone. The quality of all imaging obtained was assessed in a blinded consensus reading performed by an experienced neurosurgeon and an experienced neuroradiologist. A 6-point scoring system ranging from excellent to insufficient was used for quality evaluation of PCT and CT angiography. RESULTS: In 9 of 10 PCT data sets, the quality was rated excellent or good. In the remaining case, the quality was rated insufficient for diagnostic evaluation due to major streak artifacts induced by the titanium pins of the head clamp. In this particular case, the quality of the related CT angiography was rated good and sufficient for intraoperative decision making. The quality of all 12 CT angiography data sets was rated excellent or good. In 1 patient with an anterior communicating artery aneurysm, PCT scanning led to a repositioning of the clip because of an ischemic pattern of the perfusion parameter maps due to clip stenosis of an artery. The subsequent PCT scan obtained in this patient revealed an improved perfusion of the related vascular territory, and follow-up MR imaging showed only minor ischemia of the anterior cerebral artery territory. CONCLUSIONS: Intraoperative CT angiography and PCT scanning were shown to be feasible with short acquisition time, little interference with the surgical workflow, and very good diagnostic imaging quality. Thus, these modalities might be very helpful in vascular neurosurgery. Having demonstrated their feasibility, the impact of these methods on patients' outcomes has now to be analyzed prospectively in a larger series.

Intraoperative computed tomography with integrated navigation system in a multidisciplinary operating suite.

OBJECTIVE: We report our preliminary experience in a prospective series of patients with regard to feasibility, work flow, and image quality using a multislice computed tomographic (CT) scanner combined with a frameless neuronavigation system (NNS). METHODS: A sliding gantry 40-slice CT scanner was installed in a preexisting operating room. The scanner was connected to a frameless infrared-based NNS. Image data was transferred directly from the scanner into the navigation system. This allowed updating of the NNS during surgery by automated image registration based on the position of the gantry. Intraoperative CT angiography was possible. The patient was positioned on a radiolucent operating table that fits within the bore of the gantry. During image acquisition, the gantry moved over the patient. This table allowed all positions and movements like any normal operating table without compromising the positioning of the patient. For cranial surgery, a carbon-made radiolucent head clamp was fixed to the table. RESULTS: Experience with the first 230 patients confirms the feasibility of intraoperative CT scanning (136 patients with intracranial pathology, 94 patients with spinal lesions). After a specific work flow, interruption of surgery for intraoperative scanning can be limited to 10 to 15 minutes in cranial surgery and to 9 minutes in spinal surgery. Intraoperative imaging changed the course of surgery in 16 of the 230 cases either because control CT scans showed suboptimal screw position (17 of 307 screws, with 9 in 7 patients requiring correction) or that tumor resection was insufficient (9 cases). Intraoperative CT angiography has been performed in 7 cases so far with good image quality to determine residual flow in an aneurysm. Image quality was excellent in spinal and cranial base surgery. CONCLUSION: The system can be installed in a preexisting operating environment without the need for special surgical instruments. It increases the safety of the patient and the surgeon without necessitating a change in the existing surgical protocol and work flow. Imaging and updating of the NNS can be performed at any time during surgery with very limited time and modification of the surgical setup. Multidisciplinary use increases utilization of the system and thus improves the cost-efficiency relationship.

Intraoperative computed tomography with integrated navigation system in spinal stabilizations.

STUDY DESIGN.: A prospective interventional case-series study plus a retrospective analysis of historical patients for comparison of data. OBJECTIVE.: To evaluate workflow, feasibility, and clinical outcome of navigated stabilization procedures with data acquisition by intraoperative computed tomography. SUMMARY OF BACKGROUND DATA.: Routine fluoroscopy to assess pedicle screw placement is not consistently reliable. Our hypothesis was that image-guided spinal navigation using an intraoperative CT-scanner can improve the safety and precision of spinal stabilization surgery. METHODS.: CT data of 94 patients (thoracolumbar [n = 66], C1/2 [n = 12], cervicothoracic instability [n = 16]) were acquired after positioning the patient in the final surgical position. A sliding gantry 40-slice CT was used for image acquisition. Data were imported to a frameless infrared-based neuronavigation workstation. Intraoperative CT was obtained to assess the accuracy of instrumentation and, if necessary, the extent of decompression. All patients were clinically evaluated by Odom-criteria after surgery and after 3 months. RESULTS.: Computed accuracy of the navigation system reached <2 mm (0.95 +/- 0.3 mm) in all cases. Additional time necessary for the preoperative image acquisition including data transfer was 14 +/- 5 minutes. The duration of interrupting the surgical process for iCT until resumption of surgery was 9 +/- 2.5 minutes. Control-iCT revealed incorrect screw position >/=2 mm without persistent neurologic or vascular damage in 20/414 screws (4.8%) leading to immediate correction of 10 screws (2.4%). Control-iCT changed the course of surgery in 8 cases (8.5% of all patients). The overall revision rate was 8.5% (4 wound revisions, 2 CSF fistulas, and 2 epidural hematomas). There was no reoperation due to implant malposition. According to Odom-criteria all patients experienced a clinical improvement. A retrospective analysis of 182 patients with navigated thoracolumbar transpedicular stabilizations in the preiCT era revealed an overall revision rate of 10.4% with 4.4% of patients requiring screw revision. CONCLUSION.: Intraoperative CT in combination with neuronavigation provides high accuracy of screw placement and thus safety for patients undergoing spinal stabilization. Reoperations due to implant malpositions could be completely avoided. The system can be installed into a pre-existing operating environment without need for special surgical instruments. The procedure is rapid and easy to perform without restricted access to the patient and-by replacing pre- and postoperative imaging-is not associated with an additional exposure to radiation. Multidisciplinary use increases utilization of the system and thus improves cost-efficiency relation.