Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.

Crimean-Congo Hemorrhagic Fever Beyond Ribavirin: A Systematic Review.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne virus endemic to a vast geographical area spanning from Africa to the shores of the Mediterranean Sea and north to the Balkans. The infection carries a high case fatality rate, which prompts the development of new treatment and prophylactic measures. This review explores the different treatment and prophylactic measures found in recent literature. For this purpose, we used Medical Subject Headings (MeSH) as well as PubMed advanced search. The inclusion criteria included full-text studies conducted on humans and in the English language. We found that plasma exchange was associated with a decrease in mortality rates. Similarly, the use of immunoglobulins proved effective in decreasing the severity and mortality risk. Ribavirin use was determined as a post-exposure prophylaxis drug with no statistically significant difference in oral or intravenous routes of administration. More studies should be conducted on CCHF as the number of outbreaks and endemic areas seem to be on the rise. For the time being, supportive therapy along with adjuvant antivirals appear to be the main course of management of CCHF. However, the need for definitive therapeutic agents and guidelines is warranted.

Management of Self-Injurious Behavior, Aggression, and Psychogenic Non-Epileptic Seizures in Patients With Tuberous Sclerosis: A Case Report With a Review of Literature.

Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that affects multiple systems. TSC encompasses neurobehavioral abnormalities that are considered less sensitive and specific to the disease. Autism spectrum disorder, attention deficit disorder, anxiety, mood disorders, and self-injurious behavior (SIB) are neurobehavioral manifestations associated with tuberous sclerosis. Among them, SIB is poorly described and studied. We present a case report and a brief review of the literature, which offers us insight into the pathological mechanism that explains associated SIB in TSC patients and provides a possible multidisciplinary approach to handle this complicated association. The case details a 21-year-old female with tuberous sclerosis who went to the emergency department and then transfer to the psychiatric floor due to aggressive behavior and SIB. The patient had a history of infantile spasm in childhood and generalized tonic-clonic seizures (GTCS); the last episode was four years ago at the age of 17. During the hospital admission, the patient developed an apparent tonic-clonic seizure. Nevertheless, the electroencephalogram (EEG) shows no epileptiform pattern and because of the clinical presentation, it was concluded she had psychogenic nonepileptic seizures (PNES). The patient's CT scan showed a stable appearance of multiple calcified subependymal nodules and left frontal hypodensity. Mini-mental examination (MMSE) revealed mild cognitive impairment. Patients with TSC/SIB have higher frequencies of mental retardation, TSC2 mutations, history of infantile spasms, spike focus in the left frontal lobe. Also, TSC/SIB patients have a higher frequency of tubers in quadrants other than the left posterior neuroanatomical region in left occipital, parietal, and posterior temporal lobes. Our patient had four out of five of the risks factor for developing TSC/SIB. Almost all patients with tuberous sclerosis are expected to develop seizures. Nevertheless, our patient was seizure-free for two years and managed prophylactically with antiepileptic medication. PNES can also occur in patients with tuberous sclerosis. It is essential to be attentive to differentiate PNES from actual seizures due to their history of the high frequency of seizures in TSC. Given the multiple systems involved in the symptomatology of TSC, including the SIB and neurological concerns, multidisciplinary treatment strategies must be implemented. Treatment of TSC with SIB should include antiepileptic drugs covering seizures and managing the SIB's mood component. A neuroleptic could be added for patients who are difficult to manage.

Velcro-mimicking surface based on polymer loop brushes.

We herein report the fabrication of a Velcro-mimicking surface based on polymer brushes. Using poly(ε-caprolactone) (PCL) as the model polymer, polymer loop brushes (PLBs) and singly tethered polymer brushes (STPBs) with nearly identical tethering point density and brush heights were synthesized using a polymer single crystal (PSC)-assisted grafting-to method. Atomic force microscopy-based single molecular force spectroscopy (AFM-SMFS) and macroscale lap-shear experiments both demonstrated that the PLBs led to strong adhesion that is up to ∼10 times greater than the STPBs, which is attributed to the enriched chain entanglement between the probing polymer and the brushes. We envisage that our results will pave the way towards a new materials design for strong adhesives and nanocomposites.

3D printed capsules for quantitative regional absorption studies in the GI tract.

Drug development is a long process which requires careful evaluation of the drug substance (active pharmaceutical ingredient, API), drug product (tablet, capsule etc.) and the bioperformance (both pre-clinical and clinical) before testing the efficacy of the final dosage form. The earliest assessment of a new drug substance requires an understanding of the safety and clinical performance (Phase 1) wherein faster processes (like on-site formulation strategy) have been set in place for quick clinical read-outs. One key gap that exists in this early assessment is the ability to evaluate modified release drug products. Here, an additive manufacturing approach is used to prepare polyvinyl alcohol (PVA) capsule shells using 3D printing (3DP), where the shells can be filled with either a solid or a liquid vehicle containing the API. In this work we demonstrate how we can delay the release of the API from the printed capsules allowing us to evaluate regional absorption in pre-clinical studies. By using 3DP, a new method to provide a series of release profiles is demonstrated, where the induction time of a delayed burst release is controlled by the wall thicknesses of printed capsules. New hanging baskets were also designed and 3D printed for the dissolution tests to better understand the rupturing of these capsules in an USP 2 dissolution apparatus. By controlling the wall thickness of the capsule, the induction time of drug release can be controlled from 12 to 198 min. This wide range of induction times demonstrated with this 3DP strategy is not currently available in a commercially available oral drug product form. Varying the induction times to the drug release to interrogate different regions of the GI tract is exhibited in vivo (beagle dogs) and initial analysis suggested a good in vitro/in vivo relationship (IVIVR).

Pharmaceutical 3D printing: Design and qualification of a single step print and fill capsule.

Fused deposition modeling (FDM) 3D printing (3DP) has a potential to change how we envision manufacturing in the pharmaceutical industry. A more common utilization for FDM 3DP is to build upon existing hot melt extrusion (HME) technology where the drug is dispersed in the polymer matrix. However, reliable manufacturing of drug-containing filaments remains a challenge along with the limitation of active ingredients which can sustain the processing risks involved in the HME process. To circumvent this obstacle, a single step FDM 3DP process was developed to manufacture thin-walled drug-free capsules which can be filled with dry or liquid drug product formulations. Drug release from these systems is governed by the combined dissolution of the FDM capsule 'shell' and the dosage form encapsulated in these shells. To prepare the shells, the 3D printer files (extension '.gcode') were modified by creating discrete zones, so-called 'zoning process', with individual print parameters. Capsules printed without the zoning process resulted in macroscopic print defects and holes. X-ray computed tomography, finite element analysis and mechanical testing were used to guide the zoning process and printing parameters in order to manufacture consistent and robust capsule shell geometries. Additionally, dose consistencies of drug containing liquid formulations were investigated in this work.

Polyethylene nano crystalsomes formed at a curved liquid/liquid interface.

Crystallization is incommensurate with nanoscale curved space due to the lack of three dimensional translational symmetry of the latter. Herein, we report the formation of single-crystal-like, nanosized polyethylene (PE) capsules using a miniemulsion solution crystallization method. The miniemulsion was formed at elevated temperatures using PE organic solution as the oil phase and sodium dodecyl sulfate as the surfactant. Subsequently, cooling the system stepwisely for controlled crystallization led to the formation of hollow, nanosized PE crystalline capsules, which are named as crystalsomes since they mimic the classical self-assembled structures such as liposome, polymersome and colloidosome. We show that the formation of the nanosized PE crystalsomes is driven by controlled crystallization at the curved liquid/liquid interface of the miniemulson droplet. The morphology, structure and mechanical properties of the PE crystalsomes were characterized using scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and atomic force spectroscopy. Electron diffraction showed the single-crystal-like nature of the crystalsomes. The incommensurateness between the nanocurved interface and the crystalline packing led to reduced crystallinity and crystallite size of the PE crystalsome, as observed from the X-ray diffraction measurements. Moreover, directly quenching the emulsion below the spinodal line led to the formation of hierarchical porous PE crystalsomes due to the coupling of the PE crystallization and liquid/liquid phase separation. We anticipate that this unique crystalsome represents a new type of nanostructure that might be used as nanodrug carriers and ultrasound contrast agents.

Hybrid electrolytes with controlled network structures for lithium metal batteries.

Solid polymer electrolytes (SPEs) with tunable network structures are prepared by a facile one-pot reaction of polyhedral oligomeric silsesquioxane and poly(ethylene glycol). These SPEs, with high conductivity and high modulus, exhibit superior resistance to lithium dendrite growth even at high current densities. Measurements of lithium metal batteries with a LiFePO4 cathode show excellent cycling stability and rate capability.

Tuning ion conducting pathways using holographic polymerization.

Polymer electrolyte membranes (PEMs) with high and controlled ionic conductivity are important for energy-related applications, such as solid-state batteries and fuel cells. Herein we disclose a new strategy to fabricate long-range ordered PEMs with tunable ion conducting pathways using a holographic polymerization (HP) method. By incorporating polymer electrolyte into the carefully selected HP system, electrolyte layers/channels with length scales of a few tens of nanometers to micrometers can be formed with controlled orientation and anisotropy; ionic conductivity anisotropy as high as 37 has been achieved.