Heterozygous Seryl-tRNA Synthetase 1 Variants Cause Charcot-Marie-Tooth Disease.

OBJECTIVE: Despite the increasing number of genes associated with Charcot-Marie-Tooth (CMT) disease, many patients currently still lack appropriate genetic diagnosis for this disease. Autosomal dominant mutations in aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT. Here, we describe causal missense mutations in the gene encoding seryl-tRNA synthetase 1 (SerRS) for 3 families affected with CMT. METHODS: Whole-exome sequencing was performed in 16 patients and 14 unaffected members of 3 unrelated families. The functional impact of the genetic variants identified was investigated using bioinformatic prediction tools and confirmed using cellular and biochemical assays. RESULTS: Combined linkage analysis for the 3 families revealed significant linkage (Zmax LOD = 6.9) between the genomic co-ordinates on chromosome 1: 108681600-110300504. Within the linkage region, heterozygous SerRS missense variants segregated with the clinical phenotype in the 3 families. The mutant SerRS proteins exhibited reduced aminoacylation activity and abnormal SerRS dimerization, which suggests the impairment of total protein synthesis and induction of eIF2α phosphorylation. INTERPRETATION: Our findings suggest the heterozygous SerRS variants identified represent a novel cause for autosomal dominant CMT. Mutant SerRS proteins are known to impact various molecular and cellular functions. Our findings provide significant advances on the current understanding of the molecular mechanisms associated with ARS-related CMT. ANN NEUROL 2023;93:244-256.

[Analysis of a pedigree with autosomal dominant intermediate Charcot-Marie-Tooth disease type E and nephropathy].

OBJECTIVE: To explore phenotypic and mutational characteristics of a pedigree affected with autosomal dominant Charcot-Marie-Tooth disease (CMT) and nephropathy. METHODS: Clinical data of the proband and his family members was collected. Electrophysiology, renal biopsy and next-generation sequencing were carried out for the proband. RESULTS: The proband presented with distal lower limb weakness and proteinuria in childhood. His mother and brother had similar symptoms. Electrophysiological test of the proband revealed demyelination and axonal changes in both motor and sensory nerves. Renal biopsy suggested focal segmental glomerulosclerosis. Genetic testing revealed a heterozygous c.341G>A (p.G114D) mutation in exon 2 of the INF2 gene. CONCLUSION: The phenotypic feature of the pedigree is autosomal dominant intermediate CMT and focal segmental glomerulosclerosis, which may be attributed to the c.341G>A mutation of the INF2 gene.

Controlled fusion of synthetic lipid membrane vesicles.

Lipid membrane fusion is a fundamental noncovalent transformation as well as a central process in biology. The complex and highly controlled biological machinery of fusion has been the subject of intense investigation. In contrast, fewer synthetic approaches that demonstrate selective membrane fusion have been developed. Artificial recapitulation of membrane fusion is an informative pursuit in that fundamental biophysical concepts of biomembrane merger may be generally tested in a controlled reductionist system. A key concept that has emerged from extensive studies on lipid biophysics and biological membrane fusion is that selective membrane fusion derives from the coupling of surface recognition with local membrane disruption, or strain. These observations from native systems have guided the development of de novo-designed biomimetic membrane fusion systems that have unequivocally established the generality of these concepts in noncovalent chemistry. In this Account, we discuss the function and limitations of the artificial membrane fusion systems that have been constructed to date and the insights gained from their study by our group and others. Overall, the synthetic systems are highly reductionist and chemically selective, though there remain aspects of membrane fusion that are not sufficiently understood to permit designed function. In particular, membrane fusion with efficient retention of vesicular contents within the membrane-bound compartments remains a challenge. We discuss examples in which lipid mixing and some degree of vesicle-contents mixing is achieved, but the determinants of aqueous-compartment mixing remain unclear and therefore are difficult to generally implement. The ability to fully design membrane fusogenic function requires a deeper understanding of the biophysical underpinnings of membrane fusion, which has not yet been achieved. Thus, it is critical that biological and synthetic studies continue to further elucidate this biologically important process. Examination of lipid membrane fusion from a synthetic perspective can also reveal the governing noncovalent principles that drive chemically determined release and controlled mixing within nanometer-scale compartments. These are processes that figure prominently in numerous biotechnological and chemical applications. A rough guide to the construction of a functional membrane fusion system may already be assembled from the existing studies: surface-directed membrane apposition may generally be elaborated into selective fusion by coupling to a membrane-disruptive element, as observed over a range of systems that include small-molecule, DNA, or peptide fusogens. Membrane disruption may take different forms, and we briefly describe our investigation of the sequence determinants of fusion and lysis in membrane-active viral fusion peptide variants. These findings set the stage for further investigation of the critical elements that enable efficient, fully functional fusion of both membrane and aqueous compartments and the application of these principles to unite synthetic and biological membranes in a directed fashion. Controlled fusion of artificial and living membranes remains a chemical challenge that is biomimetic of native chemical transport and has a direct impact on drug delivery approaches.

Mass production and size control of lipid-polymer hybrid nanoparticles through controlled microvortices.

Lipid-polymer hybrid (LPH) nanoparticles can deliver a wide range of therapeutic compounds in a controlled manner. LPH nanoparticle syntheses using microfluidics improve the mixing process but are restricted by a low throughput. In this study, we present a pattern-tunable microvortex platform that allows mass production and size control of LPH nanoparticles with superior reproducibility and homogeneity. We demonstrate that by varying flow rates (i.e., Reynolds number (30-150)) we can control the nanoparticle size (30-170 nm) with high productivity (∼3 g/hour) and low polydispersity (∼0.1). Our approach may contribute to efficient development and optimization of a wide range of multicomponent nanoparticles for medical imaging and drug delivery.

BAG3 p.Pro209Ser mutation identified in a Chinese family with Charcot-Marie-Tooth disease.

Bcl2-associated athanogene 3 (BAG3) gene mutations cause dilated cardiomyopathy and myofibrillar myopathy. Recently, a novel c.625C>T (p.Pro209Ser) mutation in BAG3 was reported to cause axonal Charcot-Marie-Tooth (CMT) disease in three families. Here, we describe two patients with adult-onset and moderate CMT in a Chinese family. Nerve conduction velocity studies revealed an axonal sensorimotor neuropathy, which was supported by sural nerve biopsy. Lower limb magnetic resonance imaging (MRI) revealed fatty infiltration more severe in the soleus and deep posterior compartment muscles than in the medial gastrocnemius and anterior compartment muscles. Whole exome sequencing identified the same c.625C>T (p.Pro209Ser) mutation in BAG3, which co-segregated with the CMT disease in this family. This study further enforces the association between BAG3 gene and CMT disease, indicating that BAG3 should be considered in the genetic testing for CMT. The p.Pro209Ser mutation with different ethnic origins might be another hotspot mutation of BAG3. MRI is helpful to detect accurate extent of muscle involvement.

Intra- and intermembrane pairwise molecular recognition between synthetic hydrogen-bonding phospholipids.

Multivalency and preorganization are fundamental aspects of molecular recognition at the lipid membrane-water interface and can render weak monomeric binding interactions selective and robust; this concept is important throughout biology, biotechnology, and materials science. Though hydrogen bonding is typically weakened in water, intramembrane hydrogen bonding between native lipids has been well-studied and is thought to contribute to lipid bioactivity and membrane function. We hypothesized that avidity and preorganization effects at the lipid-water interface could overcome solvent competition and allow for selective hydrogen-bond recognition between small, unstructured components. We have found that electrostatically identical vesicular membranes composed of cyanuric acid and melamine functionalized phospholipids 1 and 2 undergo selective apposition, fusion and adhesion in suspension and on solid support, indicating that their well-known low-dielectric hydrogen bonding properties translate effectively to the lipid-water interface. This work is notable and of general interest given the few detailed studies of aqueous phase hydrogen-bonding systems; we have extensively characterized this system, gaining structural, functional, and thermodynamic data. Furthermore, we have found that the designed lipid-lipid headgroup interactions result in dramatic alteration of the lipid phase morphology, providing insight into the coupling of molecular interactions with assembly state. As such, this work contributes to our understanding of fundamental phenomena such as molecular recognition at the lipid-water interface membrane chemistry and further illustrates the general possibility of designing selective hydrogen-bonding adhesive interactions from simple starting materials at other polar-apolar interfaces; this could have numerous materials and biotechnological applications.

Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS.

An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5-10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (Cancer borealis pericardial organs (n = 4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as post-translational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.

Design and evaluation of mPEG-PLA micelles functionalized with drug-interactive domains as improved drug carriers for docetaxel delivery.

Polymeric micelles are very attractive drug delivery systems for hydrophobic agents, owing to their readily tailorable chemical structure and ease for scale-up preparation. However, the intrinsic poor stability of drug-loaded micelles presents one of the major challenges for most micellar systems in the translation to clinical applications. In this study, a simple, well-defined, and easy-to-scale up 9-Fluorenylmethoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) containing lysine dendronized mPEG-PLA (mPEG-PLA-Lys(FB)2) micellar formulation was designed and prepared for docetaxel (DTX) delivery, in an effort to improve the stability of the micelles, and its physicochemical properties, pharmacokinetics, and anti-tumor efficacy against SKOV-3 ovarian cancer were evaluated. MPEG-PLA-Lys(FB)2 was synthesized via a three-step synthetic route, and it actively interacted with DTX in aqueous media to form stable micelles with small particle sizes (~17-19 nm) and narrow size distribution (PI < 0.1), which can be lyophilized and easily reconstituted in saline without significant change in particle size distribution. In vitro drug-release study demonstrated that mPEG-PLA-Lys(FB)2 micelles achieved delayed and sustained release manner of DTX in comparison with mPEG-PLA micelles. Further in vivo xenograft tumor model in nude mice DTX/mPEG-PLA-Lys(FB)2 micelles demonstrated significantly higher inhibitory effect on tumor growth than the marketed formulation Taxotere. Thus, our system may hold promise as a simple and effective delivery system for DTX with a potential for translation into clinical study.

Stretchable polymeric multielectrode array for conformal neural interfacing.

A highly stretchable neural interface of concurrent robust electrical and mechanical properties is developed with a conducting polymer film as the sole conductor for both the electrodes and the leads. This neural interface offers the benefits of conducting polymer electrodes in a demanding stretchable format, including low electrode impedance and high chargeinjection capacity, due to the large electroactive surface area of the electrode.

Bio-inspired polymer composite actuator and generator driven by water gradients.

Here we describe the development of a water-responsive polymer film. Combining both a rigid matrix (polypyrrole) and a dynamic network (polyol-borate), strong and flexible polymer films were developed that can exchange water with the environment to induce film expansion and contraction, resulting in rapid and continuous locomotion. The film actuator can generate contractile stress up to 27 megapascals, lift objects 380 times heavier than itself, and transport cargo 10 times heavier than itself. We have assembled a generator by associating this actuator with a piezoelectric element. Driven by water gradients, this generator outputs alternating electricity at ~0.3 hertz, with a peak voltage of ~1.0 volt. The electrical energy is stored in capacitors that could power micro- and nanoelectronic devices.

Stabilization of vesicular and supported membranes by glycolipid oxime polymers.

We report herein new synthetic glycolipid dimers and polymers that provide unprecedented stability to both supported (SLBs) and vesicular lipid bilayers against dehydration and serum exposure. These novel physical properties will enable pharmaceutical delivery and development of SLB bioanalytical devices.