tRNA engineering strategies for genetic code expansion.

The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques. This review explores diverse tRNA engineering approaches and provides illustrative examples in the context of GCE, offering insights into the user-friendly implementation of GCE technology.

Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

"Lighting up" fluoride: cellular imaging and zebrafish model interrogations using a simple ESIPT-based mycophenolic acid precursor-based probe.

The discovery and implementation of media that derive from bioinspired designs and bear optical readouts featuring large Stokes shifts are of continued interest to a wide variety of researchers and clinicians. Myco-F, a novel mycophenolic acid precursor-based probe features a cleavable tert-butyldimethylsiloxy group to allow for fluoride detection. Myco-F exhibits high selectivity and specificity towards F- (Stokes shift = 120 nm). All measurements were performed in complete aqueous media (LOD=0.38 µM). Myco-F enables detection of fluoride ions in living HEK293 cells and localizes in the eye region (among other regions) of the zebrafish. DFT calculations support the proposed ESIPT working photomechanism.

Engineered kirigami design of PVDF-Pt core-shell nanofiber network for flexible transparent electrode.

Nanofiber networks comprising polymer-metal core-shell structures exhibit several advantages, such as high uniformities and considerable flexibilities. Additionally, the flexibility of the nanofiber network may be further enhanced by engineering the network topology. Therefore, in this study, the topologies of polyvinylidene fluoride (PVDF)-Pt core-shell nanofiber (CS NF) networks were engineered, and their performances as flexible transparent electrodes were comprehensively evaluated. Three distinct topologies of nanofiber networks were induced using circular, square, and rectangular electrode collectors. A highly uniform nanofiber network was obtained using the square electrode collector, which generated a high density of nanofiber junctions (nodes). Consequently, this nanofiber network exhibited the smallest sheet resistance [Formula: see text] and lowest optical transmittance [Formula: see text] among the three CS NF networks. In contrast, nanofiber bundles were frequently formed in the randomly aligned CS NF network prepared using the circular electrode collector, reducing the node density. As a result, it simultaneously exhibited a very small [Formula: see text] and high [Formula: see text], generating the largest percolation figure of merit [Formula: see text]. Under certain strain directions, the CS NF network with the engineered topology exhibited a significantly enhanced mechanical durability. Finally, a flexible piezoelectric pressure sensor with CS NF network electrodes was fabricated and its sensing performance was excellent.

A Versatile Strategy for Screening Custom-Designed Warhead-Armed Cyclic Peptide Inhibitors.

Demand for peptide-based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide-based drug discovery, we developed a general scheme for cell-based screening of cyclic peptide inhibitors armed with a user-designed warhead. We combined unnatural amino acid incorporation and split intein-mediated peptide cyclization techniques and integrated a yeast-based colorimetric screening assay to generate a new scheme that we call the custom-designed warhead-armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5-6Q, that targets human histone deacetylase 8 (HDAC8) with a KD value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.

Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently.

One of the hallmarks of cancer cells is their ability to evade apoptosis, which confers survival advantages and resistance to anti-cancer drugs. Cancers often exhibit overexpression of anti-apoptotic BCL-2 proteins, the loss of which triggers apoptosis. In particular, the inhibition of both BCL-xL and MCL-1, but neither one individually, synergistically enhances apoptotic cell death. Here, we report computational design to produce a protein that inhibits both BCL-xL and MCL-1 simultaneously. To a reported artificial three-helix bundle whose second helix was designed to bind MCL-1, we added a fourth helix and designed it to bind BCL-xL. After structural validation of the design and further structure-based sequence design, we produced a dual-binding protein that interacts with both BCL-xL and MCL-1 with apparent dissociation constants of 820 pM and 196 pM, respectively. Expression of this dual binder in a subset of cancer cells induced apoptotic cell death at levels significantly higher than those induced by the pro-apoptotic BIM protein. With a genetic fusion of a mitochondria-targeting sequence or the BH3 sequence of BIM, the activity of the dual binder was enhanced even further. These data suggest that targeted delivery of this dual binder alone or as a part of a modular protein to cancers in the form of protein, mRNA, or DNA may be an effective way to induce cancer cell apoptosis.

Phosphinate-benzoindocyanin fluorescent probe for endogenous mitochondrial peroxynitrite detection in living cells and gallbladder access in inflammatory zebrafish animal models.

Potent oxidants such as peroxynitrite (ONOO-) play important roles in the regulation of different physiopathological processes; their overproduction is thought to potentially cause several diseases in living organisms. Hence, the precise and selective monitoring of ONOO- is imperative for elucidating its interplay and roles in pathological and physiological processes. Herein, we present a novel diphenyl phosphinate-masked benzoindocyanin "turn-on" fluorogenic probe to help detect mitochondrial ONOO- in living cells and zebrafish models. A pale yellow color solution of BICBzDP turns rose-red upon the addition of ONOO-, selectively, contrary to that of other competitive bioactive molecules. BICBzDP displays an ultra-sensitivity detection limit (47.8 nM) with outstanding selectivity and sensitivity towards mitochondrial ONOO- and possesses a notable 68-fold fluorescence enhancement involving a large redshift of 91 nm. Importantly, further biological experimental investigations with BICBzDP indicate specific sensitivity and reliability of the probe to track the ONOO- level, not only in live cells, but also demonstrates dynamic fluctuations in the inflammatory zebrafish animal models. Thus, BICBzDP could be employed as a future potential biological tool for exploiting the role of ONOO- in a variety of different physiological systems.

Predicting the Cochlear Dead Regions Using a Machine Learning-Based Approach with Oversampling Techniques.

Background and Objectives: Determining the presence or absence of cochlear dead regions (DRs) is essential in clinical practice. This study proposes a machine learning (ML)-based model that applies oversampling techniques for predicting DRs in patients. Materials and Methods: We used recursive partitioning and regression for classification tree (CT) and logistic regression (LR) as prediction models. To overcome the imbalanced nature of the dataset, oversampling techniques to duplicate examples in the minority class or to synthesize new examples from existing examples in the minority class were adopted, namely the synthetic minority oversampling technique (SMOTE). Results: The accuracy results of the 10-fold cross-validation of the LR and CT with the original data were 0.82 (±0.02) and 0.93 (±0.01), respectively. The accuracy results of the 10-fold cross-validation of the LR and CT with the oversampled data were 0.66 (±0.02) and 0.86 (±0.01), respectively. Conclusions: This study is the first to adopt the SMOTE method to assess the role of oversampling methods on audiological datasets and to develop an ML-based model. Considering that the SMOTE method did not improve the model's performance, a more flexible model or more clinical features may be needed.

Noncanonical Amino Acids in Synthetic Biosafety and Post-translational Modification Studies.

The incorporation of noncanonical amino acids (ncAAs) has been extensively studied because of its broad applicability. In the past decades, various in vitro and in vivo ncAA incorporation approaches have been developed to generate synthetic recombinant proteins. Herein, we discuss the methodologies for ncAA incorporation, and their use in diverse research areas, such as in synthetic biosafety and for studies of post-translational modifications.

The interaction between ubiquitin and yeast polymerase η C terminus does not require the UBZ domain.

Polymerase η (Polη) is one of the Y-family polymerases that is recruited by monoubiquitinated proliferating cell nuclear antigen (Ub-PCNA) to DNA damage sites during translesion synthesis (TLS). This interaction is mediated by an ubiquitin-binding zinc-finger (UBZ) domain and a PCNA-interacting protein (PIP) box in Polη, which binds to ubiquitin and PCNA, respectively. Here, we show that without the UBZ domain, the PIP box of yeast Polη has a novel binding function with ubiquitin. Furthermore, the UBZ domain and the PIP box share the same binding surfaces for ubiquitin. The interaction with ubiquitin via the PIP box stabilizes the Ub-PCNA/Polη complex. Moreover, the PIP residues I624 and L625 contribute to Polη function in TLS in vivo.

Glucose-dependent control of leucine metabolism by leucyl-tRNA synthetase 1.

Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.

Clinical characteristics and associated factors of canal switch in benign paroxysmal positional vertigo.

BACKGROUND: Canal switch benign paroxysmal positional vertigo (CS-BPPV) is a transition of BPPV involving one canal to another canal during or after canalith repositioning procedures (CRP). OBJECTIVE: To investigate the clinical characteristics of CS-BPPV and its associated factors. METHODS: The data of 2,303 patients with BPPV involving the lateral canal (LC) or posterior canal (PC) were retrospectively analyzed. Demographics, etiologies, and various clinical parameters related to CRP were compared between patients with and without CS-BPPV. RESULTS: Sixty-eight (2.95%) patients exhibited CS-BPPV. For patients with CS-BPPV from the PC to the LC, as well as those with CS-BPPV from the LC to the PC, the CRP number for the original canal in CS-BPPV was significantly greater than in non-CS-BPPV (P = 0.002). More CRP cycles were required to treat CS-BPPV than non-CS-BPPV involving the same canal. Multivariate analysis showed that CS-BPPV from the LC to the PC was significantly associated with multiple CRP cycles and use of the Gufoni maneuver (P = 0.038 and P < 0.001, respectively). CONCLUSIONS: The use of multiple cycles of CRP and the Gufoni maneuver were significantly associated with the onset of CS-BPPV. Furthermore, more CRP cycles were needed for the treatment of CS-BPPV than for non-CS-BPPV involving the same canal.

Predicting cochlear dead regions in patients with hearing loss through a machine learning-based approach: A preliminary study.

We propose a machine learning (ML)-based model for predicting cochlear dead regions (DRs) in patients with hearing loss of various etiologies. Five hundred and fifty-five ears from 380 patients (3,770 test samples) diagnosed with sensorineural hearing loss (SNHL) were analyzed. A threshold-equalizing noise (TEN) test was applied to detect the presence of DRs. Data were collected on sex, age, side of the affected ear, hearing loss etiology, word recognition scores (WRS), and pure-tone thresholds at each frequency. According to the cause of hearing loss as diagnosed by the physician, we categorized the patients into six groups: 1) SNHL with unknown etiology; 2) sudden sensorineural hearing loss (SSNHL); 3) vestibular schwannoma (VS); 4) Meniere's disease (MD); 5) noise-induced hearing loss (NIHL); or 6) presbycusis or age-related hearing loss (ARHL). To develop a predictive model, we performed recursive partitioning and regression for classification, logistic regression, and random forest. The overall prevalence of one or more DRs in test ears was 20.36% (113 ears). Among the 3,770 test samples, the overall frequency-specific prevalence of DR was 6.7%. WRS, pure-tone thresholds at each frequency, disease type (VS or MD), and frequency information were useful for predicting DRs. Sex and age were not associated with detecting DRs. Based on these results, we suggest possible predictive factors for determining the presence of DRs. To improve the predictive power of the model, a more flexible model or more clinical features, such as the duration of hearing loss or risk factors for developing DRs, may be needed.

Site-Specific Labeling of Proteins Using Unnatural Amino Acids.

Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. Over the last decade, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. In this review, we describe how the UAA incorporation technique has expanded our ability to achieve site-specific labeling and visualization of target proteins for functional analyses in live cells.

A comparison between wireless CROS/BiCROS and soft-band BAHA for patients with unilateral hearing loss.

This study directly compared the performance of a contralateral routing of signal (CROS)/bilateral routing of signal (BiCROS) and a soft-band bone-anchored hearing aid (BAHA) in patients with unilateral sensorineural hearing loss (SNHL) and assessed the relationship between hearing aid benefits and personal factors. Participants with unilateral SNHL were prospectively enrolled in the study and were tested under the following three conditions: unaided, with CROS/BiCROS, and with soft-band BAHA. Sound localization, consonant, hearing in noise, and psychoacoustic tests were performed. Pseudobinaural benefits (e.g., squelch, summation, and head shadow effect) were obtained in both the CROS/BiCROS and soft-band BAHA conditions and compared to the unaided condition. Sound localization ability was not improved in either the CROS/BiCROS condition or soft-band BAHA condition. Rather, sound localization ability was significantly decreased in the CROS/BiCROS setting. A CROS/BiCROS hearing aid and a soft-band BAHA provided additional benefit for speech-in-noise perception when target speech was directed to the impaired ear side. The CROS/BiCROS hearing aid was superior to the soft-band BAHA one in decreasing the head shadow effect, but it appeared to have a negative effect when the noise was delivered to the better ear. The positive and negative effects of CROS/BiCROS for localization and speech perception were significantly correlated with personal factors such as age, hearing threshold in the better ear, and unaided psychoacoustic performances. Despite the lack of device acclimatization, we believe that this study provides counseling information for hearing aid clinics to use in the context of patients with unilateral SNHL.

Risk stratification of ductal carcinoma in situ using whole-lesion histogram analysis of the apparent diffusion coefficient.

OBJECTIVES: To investigate the value of the whole-lesion histogram apparent diffusion coefficient (ADC) metrics for differentiating low-risk from non-low-risk ductal carcinoma in situ (DCIS). METHODS: The authors identified 93 women with pure DCIS who had undergone preoperative MR imaging and diffusion-weighted imaging from 2013 to 2016. Histogram analysis of pixel-based ADC data of the whole tumour volume was performed by two radiologists using a software tool. The results were compared between low-risk and non-low-risk DCIS. Associations between quantitative ADC metrics and low-risk DCIS were evaluated by receiver operating characteristics (ROC) curve and logistic regression analyses. RESULTS: In whole-lesion histogram analysis, mean ADC and 5th, 50th and 95th percentiles of ADC were significantly different between low-risk and non-low-risk DCIS (1.522, 1.207, 1.536 and 1.854 × 10-3 mm2/s versus 1.270, 0.917, 1.261 and 1.657 × 10-3 mm2/s, respectively; p = .004, p = .003, p = .004 and p = .024, respectively). ROC curve analysis for differentiating low-risk DCIS revealed that 5th percentile ADC yielded the largest area under the curve (0.786) among the metrics of whole-lesion histogram, and the optimal cut-off point was 1.078 × 10-3 mm2/s (sensitivity 80%, specificity 75.9%, p = .001). Multivariate regression analysis revealed that a high 5th percentile of ADC (> 1.078× 10-3 mm2/s; odds ratio [OR] = 10.494, p = .016), small tumour size (≤ 2 cm; OR = 12.692, p = .008) and low Ki-67 status (< 14%; OR = 10.879, p = .046) were significantly associated with low-risk DCIS. CONCLUSIONS: Assessment with whole-lesion histogram analysis of the ADC could be helpful for identifying patients with low-risk DCIS. KEY POINTS: • Whole-lesion histogram ADC metrics could be helpful for differentiating low-risk from non-low-risk DCIS. • A high 5th percentile ADC was a significant factor associated with low-risk DCIS. • Risk stratification of DCIS is important for their management.

Cyclic Peptides: Promising Scaffolds for Biopharmaceuticals.

To date, small molecules and macromolecules, including antibodies, have been the most pursued substances in drug screening and development efforts. Despite numerous favorable features as a drug, these molecules still have limitations and are not complementary in many regards. Recently, peptide-based chemical structures that lie between these two categories in terms of both structural and functional properties have gained increasing attention as potential alternatives. In particular, peptides in a circular form provide a promising scaffold for the development of a novel drug class owing to their adjustable and expandable ability to bind a wide range of target molecules. In this review, we discuss recent progress in methodologies for peptide cyclization and screening and use of bioactive cyclic peptides in various applications.

Overriding Phthalate Decomposition When Exploring Mycophenolic Acid Intermediates as Selenium-Based ROS Biological Probes.

Hypochlorous (OCl-) acid is the most well-known bacterial oxidant to be produced by neutrophils. Excess amounts of OCl- can cause various disorders in living systems. Herein, we have designed, synthesized, and characterized two novel organoselenium-based target molecules (Probe-1 and Probe-OCl) based on a synthetic intermediate of mycophenolic acid for the aqueous detection of OCl-. Probe 1 has been recently reported (Org. Lett. 2018, 20, 3557-3561); both probes show immediate "turn-on" fluorescence (<1 s) upon the addition of OCl-, display an increase in the fluorescence quantum yield (3.7-fold in Probe-1 and 11.6-fold in Probe-OCl), and are completely soluble in aqueous media without the help of any cosolvent. However, a decrease in the "turn-on" intensity with the oxidized version of Probe-1 in cell assays due to the anhydride/phthalate functionality suggests that probe degradation occurs based on hydrolytic action (a probe degradation half-life of ∼1500 s at 15 µM Probe-1 and 150 µM OCl). Thus, the change of "anhydride" to "methylamide" begets Probe-OCl, which possesses more stability without sacrificing its water solubility properties and responses at short times. Further studies suggest that Probe-OCl is highly stable within physiological pH (pH = 7.4). Surprisingly, in live cell experiments involving U-2 OS cells and HeLa cells, Probe-OCl accumulated and aggregated in lipid droplets and gives a "turn-on" fluorescence response. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays confirmed that Probe-OCl is not toxic. Cuvette aggregation studies were also performed (tetrahydrofuran/H2O) to demonstrate aggregation-induced fluorescence at longer times. Our current hypothesis is that the "turn-on" fluorescence effect is caused by the aggregation-induced emission mechanism available for Probe-OCl. In this case, in tandem, we reanalyzed the Mes-BOD-SePh derivative to compare and contrast cell localization as imaged by confocal microscopy; fluorescence emission occurs in the absence of, or prior to, Se oxidation.

MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression.

Mitogen- and stress-activated kinase 1 (MSK1) is a chromatin kinase that facilitates activator-dependent transcription by altering chromatin structure through histone H3 phosphorylation. The kinase activity of MSK1 is activated by intramolecular autophosphorylation, which is initially triggered by the activation of upstream mitogen-activated protein kinases (MAPKs), such as p38 and ERK1/2. MSK1 has been implicated in the expression of p21, a p53 target gene; however, the precise connection between MSK1 and p53 has not been clearly elucidated. Here, using in vitro and cell-based transcription assays, we show that MSK1 functions as a transcriptional coactivator of p53 in p21 expression, an action associated with MAPK-dependent phosphorylation of MSK1 and elevated kinase activity. Of special significance, we show that MSK1 directly interacts with p53 and is recruited to the p21 promoter, where it phosphorylates histone H3 in a p53-dependent manner. In addition, phosphomimetic mutant analysis demonstrated that negative charges in the hydrophobic motif are critical for serine 212 phosphorylation in the N-terminal kinase domain, which renders MSK1 competent for histone kinase activity. These studies suggest that MSK1 acts through a direct interaction with p53 to function as a transcriptional coactivator and that MSK1 activation by upstream MAPK signaling is important for efficient p21 gene expression.

A Hemicyanine-Embedded Diphenylselenide-Containing Probe "HemiSe" in which SePh2 Stays Reduced for Selective Detection of Superoxide in Living Cells.

A simple one-step synthesis of fluorescent probe HemiSe has been developed for the detection of superoxide (O2 .- ). The probe undergoes reaction specifically with O2 .- when in the presence of other competitive ROS/RNS/metal ions. The diphenylselenide was incorporated to completely quench the fluorescence of the hemicyanine unit through the action of a photoinduced electron transfer (PET) photomechanism. However, after the addition of O2 .- , the latent fluorophore regains its fluorescence owing to the reaction at the C=C bond of the hemicyanine with O2 .- through nucleophilic attack; the increase in blue emission is due to a reaction of the double bond within HemiSe followed by an increase in fluorescence quantum yield (Φ) up to 0.45; the limit of detection (LOD) is 11.9 nm. A time-dependent study shows that HemiSe can detect superoxide within 13 min with high sensitivity, high selectivity, over a wide pH range, and through confirmation with a xanthine/xanthine oxidase biochemical assay (λem =439 nm). A study in the RAW 264.7 macrophage living cells also shows that HemiSe is not toxic, cell permeable (experimental log P=2.11); confocal imaging results show that HemiSe can detect O2 .- in endogenous and exogeneous systems.