CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema.

BACKGROUND: Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose. METHODS: In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks. RESULTS: Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%. CONCLUSIONS: In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.).

Uracil-Selective Cross-Linking in RNA and Inhibition of miRNA Function by 2-Amino-6-vinyl-7-deazapurine Deoxynucleosides.

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group - which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP - a deoxy analog of ADpVP - through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.

Rapid Alkene-Alkene Photo-Cross-Linking on the Base-Flipping-Out Field in Duplex DNA.

Specific chemical reactions by enzymes acting on a nucleobase are realized by flipping the target base out of the helix. Similarly, artificial oligodeoxynucleotides (ODNs) can also induce the base flipping and a specific chemical reaction. We now report an easily prepared and unique structure-providing photo-cross-linking reaction by taking advantage of the base-flipping-out field formed by alkene-type base-flipping-inducing artificial bases. Two 3-arylethenyl-5-methyl-2-pyridone nucleosides with the Ph or An group were synthesized and incorporated into the ODNs. We found that the two Ph derivatives provided the cross-linked product in a high yield only by a 10 s photoirradiation when their alkenes overlap each other in the duplex DNA. The highly efficient reaction enabled forming a cross-linked product even when using the duplex with a low Tm value.

Hybridization-specific chemical reactions to create interstrand crosslinking and threaded structures of nucleic acids.

The interstrand crosslinking and threaded structures of nucleic acids have high potential in oligonucleotide therapeutics, chemical biology, and nanotechnology. For example, properly designed crosslinking structures provide high activity and nuclease resistance for anti-miRNAs. The noncovalent labeling and modification by the threaded structures are useful as new chemical biology tools. Photoreversible crosslinking creates smart materials, such as reversible photoresponsive gels and DNA origami objects. This review introduces the creation of interstrand crosslinking and threaded structures, such as catenanes and rotaxanes, based on hybridization-specific chemical reactions and their functions and perspectives.

Synthesis of crosslinked 2'-OMe RNA duplexes and their application for effective inhibition of miRNA function.

The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.

Shape-Pure, Nearly Monodispersed CsPbBr3 Nanocubes Prepared Using Secondary Aliphatic Amines.

Fully inorganic cesium lead halide perovskite (CsPbX3) nanocrystals (NCs) have been extensively studied due to their excellent optical properties, especially their high photoluminescence quantum yield (PLQY) and the ease with which the PL can be tuned across the visible spectrum. So far, most strategies for synthesizing CsPbX3 NCs are highly sensitive to the processing conditions and ligand combinations. For example, in the synthesis of nanocubes of different sizes, it is not uncommon to have samples that contain various other shapes, such as nanoplatelets and nanosheets. Here, we report a new colloidal synthesis method for preparing shape-pure and nearly monodispersed CsPbBr3 nanocubes using secondary amines. Regardless of the length of the alkyl chains, the oleic acid concentration, and the reaction temperature, only cube-shaped NCs were obtained. The shape purity and narrow size distribution of the nanocubes are evident from their sharp excitonic features and their ease of self-assembly in superlattices, reaching lateral dimensions of up to 50 µm. We attribute this excellent shape and phase purity to the inability of secondary amines to find the right steric conditions at the surface of the NCs, which consequently limits the formation of low-dimensional structures. Furthermore, no contamination from other phases was observed, not even from Cs4PbBr6, presumably due to the poor ability of secondary aliphatic amines to coordinate to PbBr2 and, hence, to provide a reaction environment that is depleted in Pb.

Engineering Interfacial Charge Transfer in CsPbBr3 Perovskite Nanocrystals by Heterovalent Doping.

Since compelling device efficiencies of perovskite solar cells have been achieved, investigative efforts have turned to understand other key challenges in these systems, such as engineering interfacial energy-level alignment and charge transfer (CT). However, these types of studies on perovskite thin-film devices are impeded by the morphological and compositional heterogeneity of the films and their ill-defined surfaces. Here, we use well-defined ligand-protected perovskite nanocrystals (NCs) as model systems to elucidate the role of heterovalent doping on charge-carrier dynamics and energy level alignment at the interface of perovskite NCs with molecular acceptors. More specifically, we develop an in situ doping approach for colloidal CsPbBr3 perovskite NCs with heterovalent Bi3+ ions by hot injection to precisely tune their band structure and excited-state dynamics. This synthetic method allowed us to map the impact of doping on CT from the NCs to different molecular acceptors. Using time-resolved spectroscopy with broadband capability, we clearly demonstrate that CT at the interface of NCs can be tuned and promoted by metal ion doping. We found that doping increases the energy difference between states of the molecular acceptor and the donor moieties, subsequently facilitating the interfacial CT process. This work highlights the key variable components not only for promoting interfacial CT in perovskites, but also for establishing a higher degree of precision and control over the surface and the interface of perovskite molecular acceptors.

High-Purity Hybrid Organolead Halide Perovskite Nanoparticles Obtained by Pulsed-Laser Irradiation in Liquid.

Nanoparticles of hybrid organic-inorganic perovskites have attracted a great deal of attention due to their variety of optoelectronic properties, their low cost, and their easier integration into devices with complex geometry, compared with microcrystalline, thin-film, or bulk metal halides. Here we present a novel one-step synthesis of organolead bromide perovskite nanocrystals based on pulsed-laser irradiation in a liquid environment (PLIL). Starting from a bulk CH3 NH3 PbBr3 crystal, our PLIL procedure does not involve the use of high-boiling-point polar solvents or templating agents, and runs at room temperature. The resulting nanoparticles are characterized by high crystallinity and are completely free of any microscopic product or organic coating layer. We also demonstrate the straightforward inclusion of laser-generated perovskite nanocrystals in a polymeric matrix to form a nanocomposite with single- and two-photon luminescence properties.

Efavirenz Inhibits the Human Ether-A-Go-Go Related Current (hERG) and Induces QT Interval Prolongation in CYP2B6*6*6 Allele Carriers.

BACKGROUND: Efavirenz (EFV) has been associated with torsade de pointes despite marginal QT interval lengthening. Since EFV is metabolized by the cytochrome P450 (CYP) 2B6 enzyme, we hypothesized that EFV would lengthen the rate-corrected QT (QTcF) interval in carriers of the CYP2B6*6 decreased functional allele. OBJECTIVE: The primary objective of this study was to evaluate EFV-associated QT interval changes with regard to CYP2B6 genotype and to explore mechanisms of QT interval lengthening. METHODS: EFV was administered to healthy volunteers (n = 57) as a single 600 mg dose followed by multiple doses to steady-state. Subjects were genotyped for known CYP2B6 alleles and ECGs and EFV plasma concentrations were obtained serially. Whole-cell, voltage-clamp experiments were performed on cells stably expressing hERG and exposed to EFV in the presence and absence of CYP2B6 expression. RESULTS: EFV demonstrated a gene-dose effect and exceeded the FDA criteria for QTcF interval prolongation in CYP2B6*6/*6 carriers. The largest mean time-matched differences ∆∆QTcF were observed at 6 hours (14 milliseconds; 95% CI [1; 27]), 12 hours (18 milliseconds; 95% CI [-4; 40]), and 18 hours (6 milliseconds; 95% CI [-1; 14]) in the CYP2B6*6/*6 genotype. EFV concentrations exceeding 0.4 µg/mL significantly inhibited outward hERG tail currents (P < 0.05). CONCLUSIONS: This study demonstrates that homozygous carriers of CYP2B6*6 allele may be at increased risk for EFV-induced QTcF interval prolongation via inhibition of hERG.

Anthropogenic-induced environmental and ecological changes in the Nile Delta over the past half-century.

Anthropogenic activities over the past half-century have had a negative impact on the wetland ecosystem in the Nile Delta, which provides essential provisioning and regulating services. Therefore, it is crucial to systematically investigate pollution levels and their ecological consequences at both spatial and temporal scales in order to promote sustainable development. In this study, data on metal pollution in the Manzala Lake were compiled through a systematic review of all published literature from 1968 to 2020. Additionally, agricultural data (including land use, pesticide and fertilizer usage, and discharge) and economic data for the same time period were collected to identify the main drivers of pollution. The results indicated an overall increasing trend in heavy metal concentrations during the study period. The average concentrations of metals, arranged in descending order, were as follows: Fe (15,115.5 µg/g) > Mn (722 µg/g) > Zn (115.4 µg/g) > Cu (65.9 µg/g) > Ni (62.5 µg/g) > Cr (58.1 µg/g) > Pb (54.1 µg/g) > Cd (4.7 µg/g) > Hg (0.1 µg/g). A linear regression model revealed that wastewater discharge, water reuse, and the use of pesticides and fertilizers are the main sources of heavy metal pollution in the Manzala Lake. Consequently, there has been a dramatic decrease in the biodiversity of fish and molluscan communities. The study also found a correlation between heavy metal pollution and socio-economic development, highlighting the urgent need for attention to the conservation, management, and sustainable development of the lake.

Defect Engineering and Emission Tuning of Wide-Bandgap MAPbCl3 Perovskite.

Lead-chloride perovskites are promising candidates for optoelectronic applications, such as visible-blind UV photodetection. It remains unclear how the deep defects in this wide-bandgap material impact the carrier recombination dynamics. In this work, we study the defect properties of MAPbCl3 (MA = CH3NH3) based on photoluminescence (PL) measurements. Our investigations show that apart from the intrinsic emission, four sub-bandgap emissions emerge, which are very likely to originate from the radiative recombination of excitons bound to several intrinsic vacancy and interstitial defects. The intensity of various emission features can be tuned by adjusting the type and ratio of precursors used during synthesis. Our study not only provides important insights into the defect property and carrier recombination mechanism in this class of material but also demonstrates efficient strategies for defect passivation and engineering, paving the way for further development of lead-chloride perovskite-based optoelectronic devices.

Clinical Pharmacology and Translational Considerations in the Development of CRISPR-Based Therapies.

Genome editing holds the potential for curative treatments of human disease, however, clinical realization has proven to be a challenging journey with incremental progress made up until recently. Over the last decade, advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have provided the necessary breakthrough for genome editing in the clinic. The progress of investigational CRISPR therapies from bench to bedside reflects the culmination of multiple advances occurring in parallel, several of which intersect with clinical pharmacology and translation. Directing the CRISPR therapy to the intended site of action has necessitated novel delivery platforms, and this has resulted in special considerations for the complete characterization of distribution, metabolism, and excretion, as well as immunogenicity. Once at the site of action, CRISPR therapies aim to make permanent alterations to the genome and achieve therapeutically relevant effects with a single dose. This fundamental aspect of the mechanism of action for CRISPR therapies results in new considerations for clinical translation and dose selection. Early advances in model-informed development of CRISPR therapies have incorporated key facets of the mechanism of action and have captured hallmark features of clinical pharmacokinetics and pharmacodynamics from phase I investigations. Given the recent emergence of CRISPR therapies in clinical development, the landscape continues to evolve rapidly with ample opportunity for continued innovation. Here, we provide a snapshot of selected topics in clinical pharmacology and translation that has supported the advance of systemically administered in vivo and ex vivo CRISPR-based investigational therapies in the clinic.

Patterns of Interstitial Lung Disease in Egyptian Patients with Systemic Sclerosis: Relation to Disease Parameters.

BACKGROUND: Pulmonary involvement is the most common leading cause of morbidity and mortality associated with systemic sclerosis. Therefore, identifying the various patterns of pulmonary affection is crucial in the clinical management of these patients. In the current study, we aim to investigate the patterns of interstitial lung disease (ILD) associated with SSc patients (SSc- ILD) and their relation to serologic markers and clinical parameters. METHODS: A cross-sectional study was undertaken on thirty-four adult SSc patients who met the 2013 ACR/EULAR criteria for SSc and Forty healthy controls of matched age and sex. The patients were subjected to history taking, clinical examination, skin assessment using the modified Rodnan Skin Score (mRSS), chest x-ray (CXR), pulmonary function test (PFTs), and high resolution computed tomography of the chest (HRCT). Routine laboratory tests were conducted in addition to immunologic tests and an enzyme-linked immunosorbent assay (ELISA) to determine the IL-33 level. RESULTS: ILD was found in 23 SSc patients (67.6%); 20 patients had diffuse type while 3 patients had limited type. Non-specific interstitial pneumonia (NSIP) was found in 56.5%, usual interstitial pneumonia (UIP) was found in 21.7%, pleuroparenchymal fibroelastosis (PPFE) was found in 8.7%, and organizing pneumonia (OP) with the mixed pattern was found in 13% of SSc patients. Additionally, the mean IL-33 level in SSc patients was 98±12.7 compared to 66.2±10.6 in the control group (p < 0.001), with ILD patients having a significantly higher level (101.7±13.4) than those without (90.4±6.2), and a strong positive correlation with mRSS. CONCLUSION: Even in asymptomatic patients with SSc, ILD is prevalent, with NSIP being the most common pattern. IL-33 could be considered a potential biomarker for predicting the presence of ILD in SSc patients.

Mixed agricultural, industrial, and domestic drainage water discharge poses a massive strain on freshwater ecosystems: a case from the Nile River in Upper Egypt.

Heavy metal and pesticide pollution of freshwater ecosystems, i.e., rivers, raises significant concerns worldwide, where practical solutions to reduce the threats become urgent need. Heavy metals and pesticides are top of the list of environmental toxicants endangering nature; therefore, pesticides and heavy metals were measured at 10 stations along the Al-Zennar agricultural drain and the Nile River at Assiut city in Upper Egypt, to assess potential negative impact on the water/sediment's quality. The sediment of the streambed is a sink for pesticides and heavy metals, where both water and sediments have higher contamination factor (CF) for Cd, Pb, Cr, Cu, and Zn. In addition, the Nile water is highly contaminated by PCBs. The distance to the point source and hydrodynamics (flow rate and stream gradient) has major influences in pollutant concentrations as indicated by regression models. Dilution effect and rapid sedimentation may comment on the lower concentrations of the pollutants in the Nile comparatively to the drain and on the water comparatively to the sediments. The physiochemistry of the stations has minor effect on the metal/pesticide concentration, where the variable importance of projection (VIP) of the partial least square model indicated that total dissolved solids (TDS), total suspended solids (TSS), SO42-, and BOD/TOC/COD are the main contributors to the metal/pesticide concentration. Concentrations were not correlated between water and sediment suggesting a historical accumulation in sediments and temporal variation in the pollution load in the Al-Zennar drain. Bray-Curtis clustering confirmed that heavy metals have the same anthropogenic source in contrast to natural source of both Mn and Fe.

Historical changes in fish landings indicate a significant shift in fish catch composition and biodiversity loss in the Nile-Delta lakes.

To monitor the changes in fish biodiversity and to elucidate the factors responsible for these changes, the landings composition (LC) over the past 30 years in the Nile-Delta lakes was quantitatively analyzed. The LC data indicates a shift in target species towards demersal opportunistic species. A significant difference (P < 0.001) between two main intervals highlighted in both PERMANOVA and PCA plot; the first interval (1991-2002) is dominated by Tilapia and rare species, while the second interval (2003-2020) is dominated by the opportunistic catfish and mullet species. Noticeable declines in species richness and landings of rare species were observed, where rare taxa either have been overexploited or they may be positively affected by increasing pollution levels than do other dominated species such as Tilapia. In contrast, opportunistic fish species such as catfish and mullet, have flourished in such polluted water due to their ability to tolerate seasonal pollution-related hypoxia.

Surface Engineering of Methylammonium Lead Bromide Perovskite Crystals for Enhanced X-ray Detection.

The surface quality of lead halide perovskite crystals can extremely influence their optoelectronic properties and device performance. Here, we report a surface engineering crystallization technique in which we in situ grow a polycrystalline methylammonium lead tribromide (MAPbBr3) film on top of bulk mm-sized single crystals. Such MAPbBr3 crystals with a MAPbBr3 passivating film display intense green emission under UV light. X-ray photoelectron spectroscopy demonstrates that these crystals with emissive surfaces are compositionally different from typical MAPbBr3 crystals that show no emission under UV light. Time-resolved photoluminescence and electrical measurements indicate that the MAPbBr3 film/MAPbBr3 crystals possess less surface defects compared to the bare MAPbBr3 crystals. Therefore, X-ray detectors fabricated using the surface-engineered MAPbBr3 crystals provide an almost 5 times improved sensitivity to X-rays and a more stable baseline drift with respect to the typical MAPbBr3 crystals.

Important Factors to Expect the Outcome After Intracerebral Hemorrhage: An Observational Study From a University Hospital in Saudi Arabia.

BACKGROUND: Intracerebral hemorrhage (ICH) has worse clinical outcomes than other stroke types. The risk factors contributing to ICH outcomes are not entirely understood, and published literature from Saudi Arabia on ICH outcomes is limited. Our goal was to study the specific clinical and imaging determinants of ICH outcomes. METHODS: We retrospectively retrieved all patients with spontaneous ICH (SICH) from a prospective King Fahd Hospital University registry between 2017 and 2019. The clinical characteristics of ICH events and data on clinical outcomes (6 to 12 mo) were recorded. Groups of patients with a favorable modified Rankin Scale of 0 to 2 and nonfavorable outcomes of a modified Rankin Scale of 3 to 6 were investigated. The relationship between the clinical characteristics of the SICH event and its outcomes was assessed using linear and logistic regression analyses. RESULTS: A total of 148 patients with a mean age of 60.3 years (±15.2) and a median follow-up of 9 months were included. Unfavorable outcomes were reported in 98 patients (66.2%). The ICH event variables associated with unfavorable outcomes were impaired renal function, Glasgow Coma Score <8, hematoma volume, hematoma expansion, and intraventricular extension (IVE). CONCLUSIONS: Our study demonstrated important clinical and radiologic features in patients with ICH that may affect their clinical long-term functional outcomes. A larger multicenter study is required to validate our results and evaluate the methods to improve health care in patients with SICH.

Mixed Organic Cations Promote Ambient Light-Induced Formation of Metallic Lead in Lead Halide Perovskite Crystals.

One major concern toward the performance and stability of halide perovskite-based optoelectronic devices is the formation of metallic lead that promotes nonradiative recombination of charge carriers. The origin of metallic lead formation is being disputed whether it occurs during the perovskite synthesis or only after light, electron, or X-ray beam irradiation or thermal annealing. Here, we show that the quantity of metallic lead detected in perovskite crystals depends on the concentration and composition of the precursor solution. Through a controlled crystallization process, we grew black-colored mixed dimethylammonium (DMA)/methylammonium (MA) lead tribromide crystals. The black color is suggested to be due to the presence of small lead clusters. Despite the unexpected black coloring, the crystals show higher crystallinity and less defect density with respect to the standard yellow-colored DMA/MAPbBr3 crystals, as indicated by X-ray rocking curve and dark current measurements, respectively. While the formation of metallic lead could still be induced by external factors, the precursor solution composition and concentration can facilitate the formation of metallic lead during the crystallization process. Our results indicate that additional research is required to fully understand the perovskite precursor solution chemistry.

Structural properties of defective (CH3NH3)2Cu(Cl1-xBrx)4 compounds.

The crystal structures of (CH3NH3)2Cu(Cl1-xBrx)4 compounds have been investigated by means of synchrotron powder X-ray diffraction and pair distribution function analysis at room temperature. As a result, new insights are gained about the structural properties of these compounds, suggesting a monoclinic symmetry (space group No. 14: P21/c - C_{2h}^{5}) induced by the co-operative orbital ordering produced by the Jahn-Teller distortion characterizing the 3d9 Cu2+ ion. In contrast to previous studies, a significant amount of vacancies is found at halogen positions, a feature that can be likely ascribed to the synthesis technique adopted in the present study. Br atoms preferentially occupy axial positions, likely on account of reduced steric hindrance at these sites.