Signal Sequence Research Articles

SaRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern

Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

Ocular motor nerve palsy in patients with diabetes: High-resolution MR imaging of nerve enhancement

To evaluate the extent of signal abnormality in impaired ocular motor nerves using high signal and spatial resolution MRI sequences and to discuss the involvement of inflammatory or microvascular impairment in patients with diabetic ophthalmoplegia. We conducted a retrospective study of 10 patients referred for acute ocular motor nerve palsy in the context of diabetes mellitus from September 15th, 2021 to April 24th, 2022. 3T MRI evaluation included diffusion, 3D TOF, FLAIR, coronal STIR and post-injection 3D T1 SPACE DANTE sequences. Ten patients were included: 9 males and 1 female aged from 46 to 79 years. Five patients presented with cranial nerve (CN) III palsy, and 5 presented with CN VI palsy. Third nerve palsy was pupil-sparing in 4 patients and pupil-involved in 1 patient. Pain was associated in all patients with CN III deficiencies and in 2 patients CN VI deficiencies. In all patients, MRI sequences ruled out mass effect and vascular pathology, such as acute stroke or aneurysm. Eight patients presented with STIR hypersignals, some with enlargement of the involved nerve. The diagnosis was confirmed through a post-injection 3D T1 SPACE DANTE sequence, which showed extended enhancement along the abnormal portion of the nerve. High-resolution MRI evaluation of diplopia in diabetic patients is used to rule out a diagnosis of acute stroke and contributes to the positive diagnosis of ocular motor nerve impairment, possibly combining the influences of inflammatory and microvascular phenomena. Dedicated MR imaging should be included in the initial diagnosis and longitudinal follow-up of patients with diabetic ophthalmoplegia.

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Extracellular protein production is primarily preferred to facilitate the downstream processes in recombinant protein production. Secretion of recombinant proteins is mediated by the processing of signal peptides in their N-terminal portion by the secretory mechanism of host expression systems. These molecular elements involved in secretion are functionally interchangeable between different species and secretion sequence screening is one of the widely used approaches to improve extracellular protein production. In this study, α-mating and protein internal repeats (PIR) secretion sequences isolated from different yeasts (Kluyveromyces lactis, Kluyveromyces marxianus and Hansenula polymorpha) were tested in Pichia pastoris for the production of two different proteins (α-amylase and xylanase) and compared with the well-known secretory signals, S. cerevisiae α-mating factor (Sc-MF) and P. pastoris protein internal repeats PIR (PpPIR). The results obtained showed the potential of signal sequences tested. Among the tested peptides, the highest yields were achieved with H. polymorpha protein internal repeats (HpPIR) and K. lactis α-mating factor (Kl-MF) for xylanase and K. marxianus protein internal repeats (KmPIR) and K. lactis α-mating factor (Kl-MF) for amylase. In further studies, these sequences can be evaluated as alternatives in the production of different proteins in P. pastoris and in the production of recombinant proteins in different expression systems.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Improved Support Vector Machine for Voiceprint Diagnosis of Typical Faults in Power Transformers

The traditional power transformer diagnosis method relies on a lot of experience knowledge and a complex sampling process, which brings great difficulties to the fault diagnosis work. To solve this problem, a fault feature extraction method based on fully adaptive noise set empirical mode decomposition (CEEMDAN) is proposed, and the hunter–prey optimization (HPO) algorithm is used to optimize the support vector machine (SVM) to identify and classify the voice print faults of power transformers. Firstly, the CEEMDAN algorithm is used to decompose the voicemarks into several IMF components. IMF components containing fault information are selected according to the envelope kurtosis index and reconstructed to generate new signal sequences. PCA dimensionality reduction is performed on the reconstructed signal, and the principal components are extracted with a high cumulative contribution rate as input to SVM. Then, the HPO-SVM algorithm is used to classify and identify transformer faults. Apply the proposed method to the diagnosis of typical faults in power transformers. The results show that the accuracy of this method in identifying various fault states of power transformers can reach 98.5%, and it has better classification performance than other similar methods.

Reduced realization for switched linear systems with known mode sequence

We consider switched linear systems with mode-dependent state-dimensions and/or state jumps and propose a method to obtain a switched system of reduced size with identical input–output behavior. A key feature of our method is that only the mode sequence of the switching signal needs to be known and not the exact switching times. Since simple examples show that a minimal realization will depend on the switching times, our algorithm cannot result in a minimal realization in general, but we conjecture that it results in a minimal realization for almost all switching times. Our approach is based on considering time-dependent reachability and unobservability spaces as well as suitable extended reachability and restricted unobservability spaces together with the notion of a weak Kalman decomposition.

Extracellular production of a thermostable Cellvibrio endolytic β-agarase in Escherichia coli for agarose liquefaction

Four GH16 family β-agarases (GH16A, GH16B, GH16C, and GH16D), originated from an agarolytic bacterium Cellvibrio sp. KY-GH-1, were expressed in an Escherichia coli system and their activities were compared. Only GH16B (597 amino acids, 63.8 kDa), with N-terminal 22-amino acid signal sequence, was secreted into the culture supernatant and demonstrated a robust endolytic agarose hydrolyzing activity for producing neoagarotetraose (NA4) and neoagarohexaose (NA6) as end products. The optimal temperature and pH for the enzyme activity were 50 °C and 7.0, respectively. The enzyme was stable up to 50 °C and over a pH range of 5.0–8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B β-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3 s−1, and 4.80 × 106 s−1 M−1, respectively. The addition of 1 mM MnCl2 and 15 mM tris(2-carboxyethyl)phosphine enhanced the enzymatic activity. When agarose or neoagaro-oligosaccharides were used as substrates, the end products of enzymatic catalysis were NA4 and NA6, whereas agaropentaose was produced along with NA4 and NA6 when agaro-oligosaccharides were used as substrates. Treatment of 9%[w/v] melted agarose with the enzyme (1.6 µg/mL) under continuous magnetic stirring at 50 °C for 14 h resulted in efficient agarose liquefaction into NA4 and NA6. Purification of NA4 and NA6 from the enzymatic hydrolysate (9%[w/v] agarose, 20 mL) via Sephadex G-15 column chromatography yielded ~ 650 mg NA4/~ 900 mg NA6 (i.e., ~ 85.3% of the theoretical maximum yield). These findings suggest that the recombinant thermostable GH16B β-agarase is useful for agarose liquefaction to produce NA4 and NA6.Graphical

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / β reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10−8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10−8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Monovalent Ionic Atmosphere Modulates the Selection of Suboptimal RNA Sequences by Splicing Factors’ RNA Recognition Motifs

The U2AF2 splicing factor is involved in the RNA recognition of the pre-mRNA poly-pyrimidine signaling sequence. This protein contains two RRM domains connected by a flexible linker, which ensure the preferential selection of a poly-uridine sequence over a poly-cytosine one. In this work, all-atom simulations provide insights into the U2AF2 recognition mechanism and on the features underlying its selectivity. Our outcomes show that U2AF2's RNA recognition is driven by cooperative events modulated by RNA-protein and RNA-ion interactions. Stunningly, monovalent ions contribute to mediating the binding of the weakly binding polyC strand, thus contributing to the selection of suboptimal poly-pyrimidine tracts. This finding broadens our understanding of the diverse traits tuning splicing factors' selectivity and adaptability to precisely handle and process diverse pre-mRNA sequences.

Identification and characterization of pectin remodeling gene families in Withania somnifera and their interaction during biotic stress

The Pectin methylesterase (PME), Pectin methylesterase inhibitor (PMEI), and Subtilase (SBT) are pectin remodeling enzymes that play an important role in plant growth and development as well as response to various biotic and abiotic stress conditions. Withania somnifera is an important medicinal plant that gained attention for its phytochemical properties, but relatively little in-depth genomic exploration has been available, including PME, PMEI, and SBT genes. In the present study, 54 PMEs, 19 PMEIs, and 51 SBTs genes were identified from the W. somnifera transcriptome. A detailed phylogeny was performed, followed by a conserved sequence signature analysis, which revealed the major phylogenetic division with Solanum lycopersicum. Insect infestation can cause a significant increase in the functions of these genes, which were identified using PME activity, methanol content, and emission, often by several orders of magnitude. WsPME28, WsPME32, and WsPME50 are putative genes that are significantly upregulated up to six fold during insect infestation, according to gene expression profile analysis. To study specific relative interactions for a PME-SBT pair, molecular docking analysis was performed and found that the serine residue of WsSBT37 interacts with the processing motif (RKLL) of WsPME28 and releases mature PME. As per our best knowledge, this is the first investigation into the identification and characterization of the PME, PMEI, and SBT gene families in W. somnifera. This study will lay the groundwork for future studies targeting these genes, thus giving a better understanding of the stress tolerance mechanism in W. somnifera, and will serve as a reference for determining the most specific SBT-PME pairs in development and stress conditions.

Updating the definition of V(D)J recombination signal sequences with high-throughput recombination assays

Abstract V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Antigen receptor diversity is required for adaptive immunity in all jawed vertebrates. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence, and the molecular mechanism for semi-selective V(D)J recombination specificity is unknown. Here, we developed a cell-based, high-throughput V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs in parallel. Specifically, we focused our study on the RSS heptamer and adjoining spacer region because it undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally selected purine/pyrimidine motifs instead of specific bases. Molecular dynamics simulations of RSSs showed that DNA sequences preferred by RAG1/2 had dynamic structural features that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by dynamic DNA properties dependent on purine/pyrimidine pattern. Further investigation of RAG1/2 specificity will help elucidate the genetic instructions guiding V(D)J recombination to antigen receptor gene segments. National Institutes of Health (AI128137, AI15635); Oklahoma Center for Advancement in Science and Technology (OCAST) [HR21-142]; Presbyterian Health Foundation of Oklahoma City; National Institute of Allergy and Infectious Diseases Immunology training grant [T32 AI7633-16]

Fault-Diagnosis and Fault-Recovery System of Hall Sensors in Brushless DC Motor Based on Neural Networks.

This paper proposes a neural-network-based framework using Convolutional Neural Network and Long-Short Term Memory (CNN-LSTM) for detecting faults and recovering signals from Hall sensors in brushless DC motors. Hall sensors are critical components in determining the position and speed of motors, and faults in these sensors can disrupt their normal operation. Traditional fault-diagnosis methods, such as state-sensitive and transition-sensitive approaches, and fault-recovery methods, such as vector tracking observer, have been widely used in the industry but can be inflexible when applied to different models. The proposed fault diagnosis using the CNN-LSTM model was trained on the signal sequences of Hall sensors and can effectively distinguish between normal and faulty signals, achieving an accuracy of the fault-diagnosis system of around 99.3% for identifying the type of fault. Additionally, the proposed fault recovery using the CNN-LSTM model was trained on the signal sequences of Hall sensors and the output of the fault-detection system, achieving an efficiency of determining the position of the phase in the sequence of the Hall sensor signal at around 97%. This work has three main contributions: (1) a CNN-LSTM neural network structure is proposed to be implemented in both the fault-diagnosis and fault-recovery systems for efficient learning and feature extraction from the Hall sensor data. (2) The proposed fault-diagnosis system is equipped with a sensitive and accurate fault-diagnosis system that can achieve an accuracy exceeding 98%. (3) The proposed fault-recovery system is capable of recovering the position in the sequence states of the Hall sensors, achieving an accuracy of 95% or higher.

Release systems based on self-assembling RADA16-I hydrogels with a signal sequence which improves wound healing processes

Self-assembling peptides can be used for the regeneration of severely damaged skin. They can act as scaffolds for skin cells and as a reservoir of active compounds, to accelerate scarless wound healing. To overcome repeated administration of peptides which accelerate healing, we report development of three new peptide biomaterials based on the RADA16-I hydrogel functionalized with a sequence (AAPV) cleaved by human neutrophil elastase and short biologically active peptide motifs, namely GHK, KGHK and RDKVYR. The peptide hybrids were investigated for their structural aspects using circular dichroism, thioflavin T assay, transmission electron microscopy, and atomic force microscopy, as well as their rheological properties and stability in different fluids such as water or plasma, and their susceptibility to digestion by enzymes present in the wound environment. In addition, the morphology of the RADA-peptide hydrogels was examined with a unique technique called scanning electron cryomicroscopy. These experiments enabled us to verify if the designed peptides increased the bioactivity of the gel without disturbing its gelling processes. We demonstrate that the physicochemical properties of the designed hybrids were similar to those of the original RADA16-I. The materials behaved as expected, leaving the active motif free when treated with elastase. XTT and LDH tests on fibroblasts and keratinocytes were performed to assess the cytotoxicity of the RADA16-I hybrids, while the viability of cells treated with RADA16-I hybrids was evaluated in a model of human dermal fibroblasts. The hybrid peptides revealed no cytotoxicity; the cells grew and proliferated better than after treatment with RADA16-I alone. Improved wound healing following topical delivery of RADA-GHK and RADA-KGHK was demonstrated using a model of dorsal skin injury in mice and histological analyses. The presented results indicate further research is warranted into the engineered peptides as scaffolds for wound healing and tissue engineering.

Global asymptotic stability of switched boolean networks with missing data

This paper investigates the global asymptotic stability of a switched Boolean network (SBN) with missing data. Due to the physical constraints of the communication media, data loss often occurs during the transmission. In this paper, the data loss is described as a Bernoulli distribution sequence, and the switching signal sequence is determined by a logical system. Using a semi-tensor product, the dynamics of a SBN with missing data are converted to an algebraic form. Based on this, a necessary and sufficient condition for global asymptotic stability of SBNs with missing data and auxiliary theorems are provided. Two illustrative examples are presented to show the applicability of the developed theorems.

Secretory expression of recombinant small laccase genes in Gram-positive bacteria

BackgroundLaccases are multicopper enzymes that oxidize a wide range of aromatic and non-aromatic compounds in the presence of oxygen. The majority of industrially relevant laccases are derived from fungi and are produced in eukaryotic expression systems such as Pichia pastoris and Saccharomyces cerevisiae. Bacterial laccases for research purposes are mostly produced intracellularly in Escherichia coli, but secretory expression systems are needed for future applications. Bacterial laccases from Streptomyces spp. are of interest for potential industrial applications because of their lignin degrading activities.ResultsIn this study, we expressed small laccases genes from Streptomyces coelicolor, Streptomyces viridosporus and Amycolatopsis 75iv2 with their native signal sequences in Gram-positive Bacillus subtilis and Streptomyces lividans host organisms. The extracellular activities of ScLac, SvLac and AmLac expressed in S. lividans reached 1950 ± 99 U/l, 812 ± 57 U/l and 12 ± 1 U/l in the presence of copper supplementation. The secretion of the small laccases was irrespective of the copper supplementation; however, activities upon reconstitution with copper after expression were significantly lower, indicating the importance of copper during laccase production. The production of small laccases in B. subtilis resulted in extracellular activity that was significantly lower than in S. lividans. Unexpectedly, AmLac and ScLac were secreted without their native signal sequences in B. subtilis, indicating that B. subtilis secretes some heterologous proteins via an unknown pathway.ConclusionsSmall laccases from S. coelicolor, S. viridosporus and Amycolatopsis 75iv2 were secreted in both Gram-positive expression hosts B. subtilis and S. lividans, but the extracellular activities were significantly higher in the latter.

Tracking Control Under Round-Robin Scheduling: Handling Impulsive Transmission Outliers.

In this article, the tracking control problem is investigated for a type of linear networked systems subject to the round-Robin (RR) protocol scheduling and impulsive transmission outliers (ITOs). The communication between the controller and sensors is implemented through a shared network, on which the signal transmissions are scheduled by the RR protocol. The considered ITOs are modeled by a sequence of impulsive signals whose amplitudes (i.e., the norms of all impulsive signals) and interval lengths (i.e., the duration between all adjacent impulsive signals) are greater than two known thresholds, respectively. The occurrence moment for each ITO is first examined by using a certain outlier detection approach, and then a novel parameter-dependent tracking controller is proposed to protect the tracking performance from ITOs by removing the "harmful" signals (i.e., the transmitted signals contaminated by ITOs). Sufficient conditions are presented to ensure the exponentially ultimate boundedness of the resulted tracking error, and the controller gain matrices are subsequently designed by solving a constrained optimization problem. Finally, a simulation example is provided to demonstrate the effectiveness of our developed outlier-resistant tracking control scheme.

Simulation Study of Improved DDSRF-PLL under Distorted Grid Voltage

When the grid voltage is distorted and harmonic and DC components are mixed, the phase locking accuracy of decoupled double synchronous reference frame for phase-locked loop (DDSRF-PLL) will be greatly reduced. This paper proposes an improved DDSRF-PLL(IDDSRF-PLL) structure based on Frequency Adaptive Sliding Discrete Fourier Transform (FASDFT) filtering. A cascade SDFT structure is adopted for FASDFT filtering. The SDFT with fixed sliding window width at the front stage extracts the fundamental wave signal of power grid voltage to eliminate the influence of harmonics and DC components. The latter SDFT detects the sliding window width of power grid frequency update in real time through a nonlinear tracking differentiator (TD) to suppress spectrum leakage. Then DDSRFT-PLL is used to separate the positive and negative sequence of the fundamental signal and lock the phase. Finally, the simulation results in Matlab/Simulink verify the correctness and effectiveness of the proposed method.

Molecular Biomarkers of Disease Outcomes and Mechanisms of Acquired Resistance to First-Line Osimertinib in Advanced EGFR-Mutant Lung Cancers.

Preferred first-line treatment for patients with metastatic EGFR-mutant lung cancer is osimertinib, yet it is not known whether patient outcomes may be improved by identifying and intervening on molecular markers associated with therapeutic resistance. All patients with metastatic EGFR-mutant lung cancer treated with first-line osimertinib at the Memorial Sloan Kettering Cancer Center (n= 327) were identified. Available pretreatment and postprogression tumor samples underwent targeted gene panel sequencing and mutational signature analysis using SigMA algorithm. Progression-free survival (PFS) and overall survival were estimated using the Kaplan-Meier method. Using multivariate analysis, baseline atypical EGFR (median PFS= 5.8 mo, p < 0.001) and concurrent TP53/RB1 alterations (median PFS= 10.5 mo, p= 0.015) were associated with shorter PFS on first-line osimertinib. Of 95 patients with postprogression biopsies, acquired resistance mechanisms were identified in 52% (off-target, n= 24; histologic transformation, n= 14; on-target, n= 12), with MET amplification (n= 9), small cell lung transformation (n= 7), and acquired EGFR amplification (n= 7), the most frequently identified mechanisms. Although there was no difference in postprogression survival on the basis of identified resistance (p= 0.07), patients with subsequent second-line therapy tailored to postprogression biopsy results had improved postprogression survival (hazard ratio= 0.09, p= 0.006). The paired postprogression tumors had higher tumor mutational burden (p= 0.008) and further dominant APOBEC mutational signatures (p= 0.07) compared with the pretreatment samples. Patients with EGFR-mutant lung cancer treated with first-line osimertinib have improved survival with treatment adaptation on the basis of identified mechanisms of resistance at time of progression using tissue-based genomic analysis. Further survival gains may be achieved using risk-based treatment adaptation of pretreatment genomic alterations.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-β, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.