Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

Prime editing with genuine Cas9 nickases minimizes unwanted indels.

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Polymers based on thieno[3,4-c]pyrrole-4,6-dione and pyromellitic diimide by CH-CH arylation reaction for high-performance thin-film transistors.

Three homopolymers were successfully synthesized by direct CH-CH arylation polymerization of thieno[3,4-c]pyrrole-4,6-dione or pyromellitic diimide derivatives affording highly purified polymers with high molecular weights (43.0-174.7 K). Thieno[3,4-c]pyrrole-4,6-dione and pyromellitic diimide derivatives are considered as electron-withdrawing units. The synthesized homopolymers P1, P2, and P3 showed band gaps in the range of 2.13-2.08 eV, respectively. The electron mobilities of the three homopolymers have been investigated. The thin film transistor for P1 prepared by the eutectic-melt-assisted nanoimprinting method achieved an electron mobility of 2.11 × 10-3 cm2 s-1 V-1. Based on the obtained results, the synthesized polymers can be used as potential electron acceptors in solar cell applications.

Intermolecular interactions of an isoindigo-based organic semiconductor with various crosslinkers through hydrogen bonding.

Three crosslinkers (1,4-diaminobutane, 1,8-diaminooctane, and 1,6-hexanediol) were selected to produce hydrogen-bonded networks using a simple and effective method. The effects of these crosslinkers on the arrangement of crystalline structures were successfully studied using X-ray diffraction and high-voltage electron microscopy. The hydrogen-bonded isoindigo-based small molecules with 1,4-diaminobutane showed the best performance, with a crystal structure showing long-range order, due to the more suitable length of the 1,4-diaminobutane chain. The hole mobility estimated from hole-only devices based on isoindigo was enhanced from 1.24 × 10-6 cm2 V-1 s-1 to 7.28 × 10-4 cm2 V-1 s-1 as a result of the inclusion of this crosslinker, due to the formation of stronger interactions between the molecules.

Synthesis of Alternating Polyisocyanate Copolymers by Anionic Polymerization for Mimicking Amphiphilic Helical Peptides.

The amphiphilic conformation of α-helical peptides has important biological functions, such as ion transport, antifreeze, and innate immunity, which can be mimicked by alternating polyisocyanate copolymers. We synthesized poly(allyl isocyanate-alt-(S)-(-)-α-methylbenzyl isocyanate (P(AIC-alt-SMBIC)) and ammonium-containing P(AIC-alt-SMBIC) (N-P(AIC-alt-SMBIC)), ensuring the amphiphilic helical conformation. The benzyl group of SMBIC plays an important role in alternating copolymerization with its steric and electron-withdrawing effects, while AIC provides an alkene group capable of introducing a customized functional group. The P(AIC-alt-SMBIC) with predominantly alternating sequence was acquired at fSMBIC /fAIC =8 with a controlled molecular weight and narrow dispersity. N-P(AIC-alt-SMBIC)s were synthesized from thiol-ene radical addition with P(AIC-alt-SMBIC).

Self-assembly of POSS-Polystyrene Bottlebrush Block Copolymers on an Angle-Robust Selective Absorber for Enhancing the Purity of Reflective Structural Color.

A facile approach for improving color purity is explored by the introduction of an angle-robust selective absorber (ARSA) into bottlebrush block copolymer (BBCP)-based one-dimensional (1D) photonic crystals (PCs). The BBCPs of poly[(3-(12-(cis-5-norbornene-exo-2,3-dicarboximido)dodecanoylamino)propyl POSS)-block-(norbornene-graft-styrene)], Px (x = 1-4), with ultrahigh molecular weights (Mn ∼ 2260 kDa) and low dispersities (D̵ ∼ 1.07) are synthesized by ring-opening metathesis polymerization. The 1D PCs of the lamellar structure are fabricated by self-assembly of the BBCP with different periodicities for full color-generation (blue, green, and red). The optically tailored substrate (i.e., ARSA) is used to modulate the spectral line shape with selective absorption in the near-infrared range. Optical simulation proposes the optimized 1D PC structures on the ARSA, and it provides the reproducibility of the predictable color. The simulated structures are well matched with the experimental results, verifying the enhancement of color saturation even at various incident angles (0-70°).

Base editing in human cells with monomeric DddA-TALE fusion deaminases.

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

BMP4 signaling plays critical roles in self-renewal of R2i mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state under R2i culture conditions that inhibit the TGF-ß and ERK signaling pathways. BMP4 is another member of the TGF-ß family that plays a crucial role in maintaining the pluripotency state of mESCs. It has been reported that inhibition of BMP4 caused the death of R2i-grown cells. In this study, we used the loss-of-function approach to investigate the role of BMP4 signaling in mESC self-renewal. Inhibition of this pathway with Noggin and dorsomorphin, two bone morphogenetic protein (BMP) antagonists, elicited a quick death of the R2i-grown cells. We showed that the canonical pathway of BMP4 (BMP/SMAD) was dispensable for self-renewal and maintaining pluripotency of these cells. Transcriptome analysis of the BMPi-treated cells revealed that the p53 signaling and two adhesion (AD) and apoptotic mitochondrial change (MT) pathways could be involved in the cell death of the BMPi-treated cells. According to our results, inhibition of BMP4 signaling caused a decrease in cell adhesion and ECM detachment, which triggered anoikis in the R2i-grown cells. Altogether, these findings demonstrate that endogenous BMP signaling is required for the survival of mESCs under the R2i condition.

Synthesis and photophysical properties of N-alkyl dithieno[3,2-b:2',3'-d]pyrrole based donor/acceptor-π-conjugated copolymers for solar-cell application.

Two kinds of donor-acceptor π-conjugated copolymer based on poly{[N-hexyl-dithieno(3,2-b:2',3'-d)pyrrole-2,6-diyl]alt-[isoindigo]} (PDTP-IID) and poly{[N-hexyl-dithieno(3,2-b:2',3'-d)pyrrole-2,6-diyl]alt-[thiazol-2,5-diyl]} (PDTP-Thz) were investigated. These copolymers were synthesized via a Stille coupling reaction. The results showed the structure-property relationships of different donor-acceptor (D-A) combinations. The polymer structures and photophysical properties were characterized by 1H NMR, TGA, DSC, UV-vis absorption spectroscopy, AFM, CV, and XRD measurement. Through UV-vis absorption and cyclic voltammetry (CV) measurements, it showed that the copolymers exhibit not only a low bandgap of 1.29 eV and 1.51 eV but also a deep highest occupied molecular orbital (HOMO) of -5.49 and -5.11 eV. Moreover, photovoltaic properties in combination with the fullerene derivatives were investigated. The device based on the copolymers with PC71BM exhibited higher maximum power conversion efficiency and higher maximum short-circuit current density of 0.23% with 1.64 mA cm-2 of PDTP-IID:PC71BM and 0.13% with 1.11 mA cm-2 of PDTP-Thz:PC71BM than those of the copolymers with PC61BM. Measurements performed for N-hexyl-dithieno(3,2-b:2',3'-d)pyrrole-based copolymers proved the potential of these polymers to be applied in optoelectronic applications.

Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.

Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.

Brain-Derived Neurotrophic Factor Secreting Human Mesenchymal Stem Cells Improve Outcomes in Rett Syndrome Mouse Models.

Rett syndrome (RTT) is a severe X-linked dominant neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene; MeCP2 regulates the expression of brain-derived neurotrophic factor (BDNF) and increasing BDNF levels ameliorates RTT symptoms. However, the clinical application of BDNF is limited, because of its short half-life and low penetrance across the blood-brain barrier. In this study, we generated BDNF-secreting mesenchymal stem cells (MSCs) from the human umbilical cord cells, using CRISPR-Cas9. We studied the effects of BDNF-MSCs in MECP2 knockout and MECP2-deficient mice. BDNF-MSCs upregulated the expression of BDNF, pAKT, and pERK1/2 and downregulated that of pp38, both in vitro and in vivo. In our in vivo experiments, BDNF-MSCs increased the body and brain weights in mice. BDNF-MSCs increased the neuronal cell numbers in the hippocampus, cortex, and striatum; in addition, they increased the number of synapses. BDNF-MSCs upregulated BDNF and the activity of BDNF downstream effectors, such as pAKT and pERK 1/2; this upregulation was persistent. In conclusion, BDNF-MSCs generated using CRISPR-Cas9 could be a therapeutic strategy for treating RTT.

Clathrate Hydrate Inhibition by Polyisocyanate with Diethylammonium Group.

Polymers containing amide groups have been used as kinetic hydrate inhibitors (KHIs). The amide group has good performance for hydrate nucleus adsorption, resulting in inhibition of hydrate growth. Polyisocyanates composed of an amide backbone can be KHI candidates; however, the use of polyisocyanates as KHIs has not yet been reported. Herein, we prepared water-soluble poly[3-[[2-(diethylamino)ethyl]thio]-1-propyl isocyanate-ran-hexyl isocyanate] (P(DETPIC-ran-HIC)) to investigate the ability of polyisocyanates to inhibit hydrate formation. In the tetrahydrofuran (THF) clathrate hydrate crystal growth inhibition tests, P(DETPIC-ran-HIC) showed better performance than the polyamide, poly(N-vinylpyrrolidone) (PVP).

Preparation of a polymer nanocomposite via the polymerization of pyrrole : biphenyldisulfonic acid : pyrrole as a two-monomer-connected precursor on MoS2 for electrochemical energy storage.

We prepared a poly(pyrrole : biphenyldisulfonic acid : pyrrole (Py:BPDSA:Py)) nanocomposite of molybdenum disulfide (MoS2), P(Py:BPDSA:Py)-MoS2, with high crystallinity. The composite is synthesized by oxidative polymerization of Py:BPDSA:Py as a two-monomer-connected precursor (TMCP) linked by ionic bonding on a molybdenum disulfide (MoS2) monolayer. The chemical, structural and morphological characterization of this composite is confirmed by Raman spectroscopy, FT-IR, X-ray photoelectron spectroscopy (XPS), electron energy loss spectroscopy (EELS), and scanning electron microscopy (SEM). The crystal structure is analysed by X-ray diffraction (XRD) and high-voltage electron microscopy (HVEM), which shows a face-centered cubic (FCC) crystal structure for the composite. Nitrogen adsorption-desorption isotherms show an improved specific surface area (91.3 m2 g-1). The electrochemical properties of the composite with a unique crystal structure and a large specific surface area are analysed through cyclic voltammetry (CV), which shows a specific capacitance of 681 F g-1 demonstrating that the composite can be used as an efficient electrode active material for electrochemical energy storage systems.

Impact of N-Substituent and pKa of Azole Rings on Fuel Cell Performance and Phosphoric Acid Loss.

The influence of N-substituent and pKa of azole rings has been investigated for the performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFCs). Imidazole, benzimidazole, and triazole groups were functionalized on the side chains of poly(phenylene oxide), respectively. Each azole group is categorized by their N-substituent into two types: unsubstituted and methyl-substituted azoles. The membranes with methyl-substituted azoles showed higher phosphoric acid (PA) doping levels with an average increase of 20% compared to those with unsubstituted azoles in the full-doped states. However, unsubstituted azoles more effectively improved the proton conductivity and the membrane with unsubstituted imidazole (IMPPO-H) showed a high anhydrous proton conductivity of 153 mS/cm at 150 °C. In contrast, the membranes with methyl-substituted azoles showed a higher PA retention with an average increase of 81% compared to those with unsubstituted azoles. The higher PA retention of methyl-substituted azoles also led to the higher fuel cell performance with the maximum increase of 95% in the power density. It was also revealed that higher pKa of azoles enhanced the PA retention and the fuel cell performance. Based on the experimental results of PA retention and density functional theory calculations, the PA loss mechanism was also proposed.

Electrochemical properties of an activated carbon xerogel monolith from resorcinol-formaldehyde for supercapacitor electrode applications.

Activated carbon xerogel monoliths were prepared from resorcinol and formaldehyde via a catalyst-free and template-free hydrothermal polycondensation reaction, followed by pyrolysis and activation. The ratio of resorcinol (R) to distilled water (W) was varied to afford an interconnected pore structure with controlled pore size, while the pyrolysis temperature was optimized to give high specific surface area. Activation was carried out at 700 °C after soaking the samples in 6 M KOH aqueous solution. The same process, called "heat treatment", was also carried out without soaking in KOH for comparison. The weight loss upon pyrolysis, activation and heat treatment and the weight gain via KOH soaking were measured. Field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA) and an N2 sorption instrument were utilized for characterization. Additionally, electrochemical properties were evaluated using cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) and electrochemical impedance spectroscopy (EIS) with a 3-electrode system, while a 2-electrode system was also employed for selected samples. The highest specific capacitance of 323 F g-1 via GCD at 1 A g-1 was obtained at the R/W ratio of 45 and with 500 °C pyrolysis. In addition, this sample also exhibited 89.4% retention at 20 A g-1 in the current density variation and 100% retention in 5000 cycling tests.

Solution-State Long-Range Molecular Ordering in Poly(3-hexylthiophene).

A blend of poly(3-hexylthiophene) (P3HT) and poly(n-hexyl isocyanate-block-2-vinylpyridine) (PHIC-b-P2VP) in a common solvent shows the formation of long-range (micrometer-scale) nanowires of P3HT through hydrophobic interactions between the hexyl arms of P3HT and PHIC in a parallel way, which increase the planarity that leads to the generation of vibration bands with a lower free exciton bandwidth (W = 67 meV) in the solution state, which is further decreased to 9 meV after 48 h annealing of the blend film. The resulting nanowires of the P3HT show a 100-fold increase in current in comparison to pristine P3HT.

Effectiveness and Safety of Herbal Medicine for Atopic Dermatitis: An Overview of Systematic Reviews.

OBJECTIVES: Herbal medicine (HM) is attracting attention for treating atopic dermatitis (AD). This overview was conducted to summarize and critically evaluate the current systematic reviews (SRs) on HM for the treatment of AD. METHODS: Through comprehensive searches, all relevant SRs on HM for AD published until May 2020 were included. The quality of included SRs was assessed using the AMSTAR-2 tool. Moreover, original randomized controlled trials (RCTs) included in the SRs were resynthesized to investigate the efficacy and safety of oral HM for AD. The quality of evidence for the main findings was evaluated using the GRADE approach. RESULTS: Nine SRs were included in this overview. HM showed significantly better efficacy in terms of total effective rate (TER), itching and sleep symptom scores, quality of life, and the dose of topical treatment used compared with placebo. HM as a monotherapy and/or an adjunctive therapy to conventional medication (CM) showed significantly better results on the efficacy, symptom relief, and some laboratory parameters related to the inflammatory response. The methodological quality was generally low. When 58 original RCTs were reanalyzed, HM showed significantly lower SCORing Atopic Dermatitis (SCORAD) score and higher TER than the placebo or CM. In terms of the safety profile, HM was not significantly different from the placebo and was better than CM. The quality of evidence ranged from "moderate" to "very low." CONCLUSION: The results suggested that HM as a monotherapy or an adjunctive therapy is promising for the treatment of AD. However, due to low methodological quality and low quality of evidence, further rigorous, well-designed, high-quality SRs, and RCTs are needed to make clinical recommendations on HM use.

Risk Factors of Bacteremia following Multiple Traumas.

BACKGROUND: Bacteremia is a major nosocomial infection that frequently occurs in trauma patients, increasing morbidity and mortality. The aim of this study was to identify risk factors and to describe epidemiological patterns for early onset (EOB) and late onset (LOB) bacteremia after trauma. METHODS: We retrospectively reviewed medical records of all trauma patients admitted to the surgical intensive care unit and general ward between January 2011 and December 2015. The information was collected for each patient and recorded in a computer database: early onset bacteremia (EOB) was defined as when onset occurred within 7 days after trauma, and late onset bacteremia (LOB) was defined as when onset occurred after 7 days from trauma. RESULTS: Thirty-four patients of 859 (4%) developed bacteremia during their hospital stay: 4 (11.8%) developed EOB, 26 (76.4%) LOB, and 4 (11.8%) patients developed both of them. Sixty events of bacteremia happened to these patients: 9 (15.0%) EOB and 51 (85.0%) LOB. Gram-positive cocci were isolated more frequently than Gram-negative bacilli in both groups. Gram-positive cocci were more frequently isolated in EOB than in LOB; otherwise, there was no statistical significance (77.8% vs. 64.7%, p=0.683). Central line-associated blood stream infection (CLABSI) and surgical site infection (SSI) were the most common identified source for LOB. Presence of liver (OR: 2.66, p=0.035) and pelvic injury (OR: 2.25, p=0.038), gastrointestinal tract perforation (OR: 5.48, p=0.002), and massive transfusion (OR: 3.36, p=0.006) represented risk factors for bacteremia. CONCLUSIONS: Presence of pelvic and liver injury on arrival in emergency department, gastrointestinal tract perforation, and massive transfusion within the first 24 hours after trauma appears to be significant risk factors for bacteremia.

The size of pelvic hematoma can be a predictive factor for angioembolization in hemodynamically unstable pelvic trauma.

PURPOSE: Unstable pelvic fracture with bleeding can be fatal, with a mortality rate of up to 40%. Therefore, early detection and treatment are important in unstable pelvic trauma. We investigated the early predictive factors for possible embolization in patients with hemodynamically unstable pelvic trauma. METHODS: From January 2011 to December 2013, 46 patients with shock arrived at a single hospital within 24 hours after injury. Of them, 44 patients underwent CT scan after initial resuscitation, except for 2 who were dead on arrival. Nine patients with other organ injuries were excluded. Seventeen patients underwent embolization. A single radiologist measured the width (longest length in axial view) and length (longest length in coronal view) of pelvic hematoma on CT scans. Demographic, clinical, and radiological data were reviewed retrospectively. RESULTS: Among 35 patients with hemodynamically unstable pelvic fracture, 22 (62.9%) were men. Width (P = 0.002) and length (P = 0.006) of hematoma on CT scans were significantly different between the embolization and nonembolization groups. The predictors of embolization were width of pelvic hematoma (odds ratio [OR], 1.07; P = 0.028) and female sex (OR, 10.83; P = 0.031). The cutoff value was 3.35 cm. More embolization was performed (OR, 12.00; P = 0.003) and higher mortality was observed in patients with hematoma width >3.35 cm (OR, 4.96; P = 0.048). CONCLUSION: Patients with hemodynamically unstable pelvic trauma have a high mortality rate. CT is useful for the initial identification of the need for embolization among these patients. The width of pelvic hematoma can predict possible embolization in patients with unstable pelvic trauma.