CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Unconventional Facial Entry Points Confirmed Using a 3D CT Reconstruction-Guided Stereotactic Approach to Radiofrequency Thermocoagulation for the Treatment of Trigeminal Neuralgia: A Series of Case Reports.

OBJECTIVES: Patients with trigeminal neuralgia who are refractory to medical therapy may choose to undergo Gasserian ganglion percutaneous radiofrequency thermocoagulation. However, in cases where the foramen ovale is difficult to access due to various anatomical anomalies, the typical estimation of the facial entry point is suboptimal. METHODS: Three-dimensional computed tomography reconstruction imaging performed before surgery revealed anatomical variations in each of the four adult patient cases that made it more difficult to successfully access the foramen ovale (FO) for percutaneous radiofrequency thermocoagulation. Using measurements collected from preoperative imaging that showed each specific anatomical variation in the FO, researchers marked alternate facial entry points that would allow successful probe placement into the FO and recorded the arc angle data in the stereotactic instrument. RESULTS: Patients were evaluated during follow-up visits ranging from seven to 26 months after surgery and asked to rate postoperative pain using a visual analog scale. These scores decreased from 10 to 3 in all four patients by the third day after the procedure. There were no permanent complications or morbidities from the surgery. One patient experienced mild facial numbness; however, this side effect subsided within three months after surgery. During the follow-up period, no patient reported pain recurrence. CONCLUSIONS: The expectation for clinicians approaching trigeminal nerve block using a peri-oral approach should be to expect a great degree of potential variability in terms of both distances from the corner of the mouth and needle angle taken to successfully navigate the anatomy and access the foramen ovale.

Stereotactic Approach Combined with 3D CT Reconstruction for Difficult-to-Access Foramen Ovale on Radiofrequency Thermocoagulation of the Gasserian Ganglion for Trigeminal Neuralgia.

OBJECTIVES: The authors describe a technique that includes a stereotactic approach in the preoperative plan in cases where the foramen ovale is difficult to access for radiofrequency thermocoagulation of the Gasserian ganglion. METHODS: The study included 395 patients for whom three-dimensional computed tomographic reconstruction of the skull base, maxilla, and mandible was conducted before surgery. Accessibility of the foramen ovale was defined using numerical data from the three-dimensional computed tomographic reconstruction images. In those patients for whom accessibility of the foramen ovale was considered difficult, the authors used a stereotactic frame to design an individual operative plan. Adjustments of a single point of data,-that is, a change in X axis, Y axis, or an arc angle-were guided by radiographic fluoroscopy images. After verifying successful cannulation and electroneurophysiology, thermocoagulation targets-especially multiple targets recorded as data on the Z axis of the stereotactic approach-were identified and treated. RESULTS: There were 24 patients who met the predetermined criteria for having a difficult-to-access foramen ovales-that is, they had at least two contributing factors and/or involvement of division V1 . Twenty-one of the 24 patients required a single satisfactory puncture; three patients required two to three punctures to successfully access the foramen ovale. There were no permanent complications from the procedure. CONCLUSIONS: The authors conclude that this stereotactic approach combined with three-dimensional computed tomographic reconstruction model can improve the accuracy, safety, and efficiency of percutaneous radiofrequency thermocoagulation in patients with trigeminal neuralgia for whom the foramen ovale is difficult to access.

Targeting the activity of T cells by membrane surface redox regulation for cancer theranostics.

T cells play a determining role in the immunomodulation and prognostic evaluation of cancer treatments relying on immune activation. While specific biomarkers determine the population and distribution of T cells in tumours, the in situ activity of T cells is less studied. Here we designed T-cell-targeting fusogenic liposomes to regulate and quantify the activity of T cells by exploiting their surface redox status as a chemical target. The T-cell-targeting fusogenic liposomes equipped with 2,2,6,6-tetramethylpiperidine (TEMP) groups neutralize reactive oxygen species protecting T cells from oxidation-induced loss of activity. Meanwhile, the production of paramagnetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) radicals allows magnetic resonance imaging quantification of the T cell activity. In multiple mouse models, the T-cell-targeting fusogenic liposomes led to efficient tumour inhibition and to early prediction of radiotherapy outcomes. This study uses a chemical targeting strategy to measure the in situ activity of T cells for cancer theranostics and may provide further understanding on engineering T cells for cancer treatment.

Artificial transcription factors which mediate double-strand DNA cleavage.

A new family of artificial transcription factor (ATF)-based conjugates have been designed and synthesized as potent chemical nucleases. Polyamides as the important and efficient ATFs were used to modify and activate several anchor compounds. The results demonstrate that the resulting conjugates remarkably promote the rate accelerations and non-random double-strand DNA cleavage activity. Interestingly, the cleavage activity of both the hydrolytic and oxidative agents was promoted efficiently through the modification of the ATFs.

Trigeminal Cardiac Reflex Caused by Onyx Embolization of Intracranial Dural Arteriovenous Fistula.

Trigeminocardiac reflex (TCR) is a reflexive response of bradycardia, hypotension and gastric hypermotility which is observed upon mechanical stimulation in the distribution of the trigeminal nerve. Previous articles have described TCR during intracranial operations, ophthalmic surgery, microcompression of the trigeminal ganglion and radiofrequency lesioning of the trigeminal ganglion. TCR may occur during transarterial embolization of dural arteriovenous fistula (DAVF) with Onyx, leading to a significant decrease in heart rate under a standard anesthetic protocol. TCR may also occur due to chemical stimulus of dimethyl sulfoxide (DMSO) in transvenous Onyx embolization of dural cavernous sinus fistula. Slow rate of injection may give DMSO enough time to dissipate in the blood stream which is important for the prevention of toxicity. This report confirms that the reflex was blunted by the anticholinergic effects of atropine and there was no harm to patients if stopped immediately.

Hollow multilayer microcapsules for pH-/thermally responsive drug delivery using aliphatic poly(urethane-amine) as smart component.

Hollow multilayer microcapsules made of aliphatic poly(urethane-amine) (PUA) and sodium poly(styrene sulfonate) (PSS), templated on PSS-doped CaCO3 particles, are prepared for pH-/thermally responsive drug delivery. The electrostatic interaction and hydrogen bonding under weak-acid conditions between aliphatic PUA and PSS contribute to the formation of multilayer microcapsules. Scanning electron microscopy (SEM) results demonstrate an obvious variation of the hollow multilayer microcapsules in response to changes in temperature and pH value. Drug-release behaviors using DOX as a model drug demonstrate that the drug release increases on decreasing the pH value because of the interaction weakness between aliphatic PUA and PSS in acidic conditions. Moreover, the drug release is higher at 55 °C than that at 37 °C for the sake of the shrinkage of aliphatic PUA above its lower critical solution temperature (LCST).

PUA/PSS multilayer coated CaCO3 microparticles as smart drug delivery vehicles.

Hybrid CaCO3 microparticles coated by sodium poly(styrene sulfonate) (PSS) and aliphatic poly(urethane-amine) (PUA) were developed as thermal-/pH-responsive drug delivery vehicles via LbL self-assembly technique. The DOX release from the CaCO3 microparticles was higher than 60% within 36 h, whereas the value of PUA/PSS-coated microparticles was only 20%. The results demonstrated that the PUA/PSS multilayer coating could reduce the drug release rate and significantly assuage the initial burst release of DOX. In addition, the drug release of the hybrid microparticles was found to be thermal-/pH-dual responsive. More interestingly, more than 90% of DOX was released in 36 h at pH2.1 and 55 °C owing to the combined action of the dissolution of the CaCO3 core and the shrinkage of aliphatic PUA.

In vitro permeability of poorly aqueous soluble compounds using different solubilizers in the PAMPA assay with liquid chromatography/mass spectrometry detection.

PURPOSE: This study compares the use of UV-VIS detection with liquid chromatography/mass spectrometry (LC/MS) detection for the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability determination of compounds in the drug discovery stage. LC/MS detection offers a selective and sensitive method for the determination of the PAMPA permeability for compounds that do not contain a UV chromophore or possess a low UV extinction coefficient. To enhance the reliability of our permeability measurements for compounds with low aqueous solubility, we demonstrated the use of LC/MS detection as a means for facilitating the study of solubilizing agents to enhance aqueous solubility that normally would interfere with UV-VIS detection. In doing so, the PAMPA assay can be expanded to study the in vitro permeability of poorly water soluble compounds and evaluate the effects of solubilizers' on the membrane permeability of different compounds. This might be useful in selecting solubilizers for poorly water soluble compounds to be used for further in vivo studies. METHODS: A diverse set of 20 drugs using UV-VIS detection were compared with data using LC/MS detection. A PAMPA screening method was designed which used solubilizers (Brij 35, Cremophor EL, ethanol, and Tween 80) for compounds with low aqueous solubility. The stability of the artificial membrane was determined using various solubilizer concentrations (0.1-5% w/v) to ensure the phospholipid membrane was not disrupted. Two compounds, amiodarone and miconazole, with low aqueous solubility yielding an undetected response in the PAMPA assay using UV-VIS detection were subjected to the different solubilizing agents and their PAMPA permeability was measured using LC/MS detection. RESULTS: Most of the compounds showed similar PAMPA permeability using the two detection systems. However, for compounds lacking a UV chromophore or with a low UV extinction coefficient, LC/MS was the detection method of choice for determination of PAMPA permeability values. LC/MS also gave reliable quantification data for compounds containing impurities, as well as compounds that were not stable during the assay. Although many solubilizers were found to interfere with UV-VIS detection, the LC/MS approach was applicable to determine the permeability values of compounds with normally low aqueous solubility. CONCLUSIONS: LC/MS detection offered greater sensitivity and selectivity as compared with UV-VIS detection for the PAMPA assay. With this added versatility in detection, PAMPA can be used in both discovery and pre-formulation applications, which has not been described before.