Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma.

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Early prediction of end-stage kidney disease using electronic health record data: a machine learning approach with a 2-year horizon.

Objectives: In the United States, end-stage kidney disease (ESKD) is responsible for high mortality and significant healthcare costs, with the number of cases sharply increasing in the past 2 decades. In this study, we aimed to reduce these impacts by developing an ESKD model for predicting its occurrence in a 2-year period. Materials and Methods: We developed a machine learning (ML) pipeline to test different models for the prediction of ESKD. The electronic health record was used to capture several kidney disease-related variables. Various imputation methods, feature selection, and sampling approaches were tested. We compared the performance of multiple ML models using area under the ROC curve (AUCROC), area under the Precision-Recall curve (PR-AUC), and Brier scores for discrimination, precision, and calibration, respectively. Explainability methods were applied to the final model. Results: Our best model was a gradient-boosting machine with feature selection and imputation methods as additional components. The model exhibited an AUCROC of 0.97, a PR-AUC of 0.33, and a Brier score of 0.002 on a holdout test set. A chart review analysis by expert physicians indicated clinical utility. Discussion and Conclusion: An ESKD prediction model can identify individuals at risk for ESKD and has been successfully deployed within our health system.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Recent advances and future directions in etiopathogenesis and mechanisms of reactive oxygen species in cancer treatment.

A class of exceptionally bioactive molecules known as reactive oxygen species (ROS) have been widely studied in the context of cancer. They play a significant role in the etiopathogenesis for cancer. Implication of ROS in cancer biology is an evolving area, considering the recent advances; insights into their generation, role of genomic and epigenetic regulators for ROS, earlier thought to be a chemical process, with interrelations with cell death pathways- Apoptosis, ferroptosis, necroptosis and autophagy has been explored for newer targets that shift the balance of ROS towards cancer cell death. ROS are signal transducers that induce angiogenesis, invasion, cell migration, and proliferation at low to moderate concentrations and are considered normal by-products of a range of biological activities. Although ROS is known to exist in the oncology domain since time immemorial, its excessive quantities are known to damage organelles, membranes, lipids, proteins, and nucleic acids, resulting in cell death. In the last two decades, numerous studies have demonstrated immunotherapies and other anticancer treatments that modulate ROS levels have promising in vitro and in vivo effects. This review also explores recent targets for therapeutic interventions in cancer that are based on ROS generation or inhibition to disrupt the cell oxidative stress balance. Examples include-metabolic targets, targeted therapy with biomarkers, natural extracts and nutraceuticals and targets developed in the area of nano medicine. In this review, we present the molecular pathways which can be used to create therapy plans that target cancer by regulating ROS levels, particularly current developments and potential prospects for the effective implementation of ROS-mediated therapies in clinical settings. The recent advances in complex interaction with apoptosis especially ferroptosis and its role in epigenomics and modifications are a new paradigm, to just mechanical action of ROS, as highlighted in this review. Their inhibition by nutraceuticals and natural extracts has been a scientific challenging avenue that is explored. Also, the inhibition of generation of ROS by inhibitors, immune modulators and inhibitors of apoptosis and ferroptosis is explored in this review.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames, without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs accounts for proteins that are attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Analysis of Dielectric Parameters of Fe2O3-Doped Polyvinylidene Fluoride/Poly(methyl methacrylate) Blend Composites.

In this paper, we report the effect of metal oxide (Fe2O3) loading in different weight ratios (0.5%, 1%, 2%, and 4%) on the structural and electrical parameters, viz., the complex dielectric constant, electric modulus spectra, and the AC conductivity, of polymeric composites of PVDF/PMMA (30/70 weight ratio) blend. The structural and geometric measurements have been analyzed with the help of peak location, peak intensity, and peak shape obtained from XRD as well as from FTIR spectra. The electrical properties have been investigated using an impedance analyzer in the frequency range 100 Hz to 1 MHz. The real parts of the complex permittivity and the dielectric loss tangent of these materials are found to be frequency independent in the range from 20 KHz to 1 MHz, but they increase with the increase in the concentration of nano-Fe2O3. The conductivity also increases with an increased loading of Fe2O3 in PVDF/PMMA polymer blends. The electric modulus spectra were used to analyze the relaxation processes associated with the Maxwell-Wagner-Sillars mechanism and chain segmental motion in the polymer mix.

Lessons in clinical reasoning - pitfalls, myths, and pearls: a case of crushing, substernal chest pain.

OBJECTIVES: Diagnostic error is not uncommon and diagnostic accuracy can be improved with the use of problem representation, pre-test probability, and Bayesian analysis for improved clinical reasoning. CASE PRESENTATION: A 48-year-old female presented as a transfer from another Emergency Department (ED) to our ED with crushing, substernal pain associated with dyspnea, diaphoresis, nausea, and a tingling sensation down both arms with radiation to the back and neck. Troponins were elevated along with an abnormal electrocardiogram. A negative myocardial perfusion scan led to the patient's discharge. The patient presented to the ED 10 days later with an anterior ST-elevation myocardial infarction. CONCLUSIONS: An overemphasis on a single testing modality led to diagnostic error and a severe event. The use of pre-test probabilities guided by history-taking can lead to improved interpretation of test results, ultimately improving diagnostic accuracy and preventing serious medical errors.

Controlling release from encapsulated drug-loaded devices: insights from modeling the dissolution front propagation.

Dissolution of drug from its solid form to a dissolved form is an important consideration in the design and optimization of drug delivery devices, particularly owing to the abundance of emerging compounds that are extremely poorly soluble. When the solid dosage form is encapsulated, for example by the porous walls of an implant, the impact of the encapsulant drug transport properties is a further confounding issue. In such a case, dissolution and diffusion work in tandem to control the release of drug. However, the interplay between these two competing processes in the context of drug delivery is not as well understood as it is for other mass transfer problems, particularly for practical controlled-release considerations such as an encapsulant layer around the drug delivery device. To address this gap, this work presents a mathematical model that describes controlled release from a drug-loaded device surrounded by a passive porous layer. A solution for the drug concentration distribution is derived using the method of eigenfunction expansion. The model is able to track the dissolution front propagation, and predict the drug release curve during the dissolution process. The utility of the model is demonstrated through comparison against experimental data representing drug release from a cylindrical drug-loaded orthopedic fixation pin, where the model is shown to capture the data very well. Analysis presented here reveals how the various geometrical and physicochemical parameters influence drug dissolution and, ultimately, the drug release profile. It is found that the non-dimensional initial concentration plays a key role in determining whether the problem is diffusion-limited or dissolution-limited, whereas the nature of the problem is largely independent of other parameters including diffusion coefficient and encapsulant thickness. We expect the model will prove to be a useful tool for those designing encapsulated drug delivery devices, in terms of optimizing the design of the device to achieve a desired drug release profile.

What repeat expansion disorders can teach us about the Central Dogma.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.

Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs.

CAG trinucleotide repeat expansions cause several neurodegenerative diseases, including Huntington's disease and spinocerebellar ataxia. RNAs with expanded CAG repeats contribute to disease in two unusual ways. First, these repeat-containing RNAs may agglomerate in the nucleus as foci that sequester several RNA-binding proteins. Second, these RNAs may undergo aberrant repeat-associated non-AUG (RAN) translation in multiple frames and produce aggregation-prone proteins. The relationship between RAN translation and RNA foci, and their relative contributions to cellular dysfunction, are unclear. Here, we show that CAG repeat-containing RNAs that undergo RAN translation first accumulate at nuclear foci and, over time, are exported to the cytoplasm. In the cytoplasm, these RNAs are initially dispersed but, upon RAN translation, aggregate with the RAN translation products. These RNA-RAN protein agglomerates sequester various RNA-binding proteins and are associated with the disruption of nucleocytoplasmic transport and cell death. In contrast, RNA accumulation at nuclear foci alone does not produce discernable defects in nucleocytoplasmic transport or cell viability. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and alleviates cell toxicity. Our findings demonstrate that RAN translation-induced RNA-protein aggregation correlates with the key pathological hallmarks observed in disease and suggest that cytoplasmic RNA aggregation may be an underappreciated phenomenon in CAG trinucleotide repeat expansion disorders.

A highly stable terbium(III) metal-organic framework MOF-76(Tb) for hydrogen storage and humidity sensing.

The present study described the synthesis and characterization of MOF-76(Tb) for hydrogen storage and humidity sensing applications. The structure and morphology of as-synthesized material were studied using powder X-ray diffraction, scanning, and transmission electron microscopy. The crystal structure of MOF-76(Tb) consists of terbium(III) and benzene-1,3,5-tricarboxylate(-III) ions, one coordinated aqua ligand and one crystallization N,N´-dimethylformamide molecule. The polymeric framework of MOF-76(Tb) contains 1D sinusoidally shaped channels with sizes of 6.6 × 6.6 Å propagating along c crystallographic axis. The thermogravimetric analysis of the prepared material exhibited thermal stability up to 600 °C. At 77 K and pressure up to 20 bar; 0.6 wt.% hydrogen storage capacity for MOF-76(Tb) was observed. Finally, the humidity sensing measurements (water adsorption experiments) were performed, and the results indicate that MOF-76(Tb) is not a suitable material for moisture sensing applications.

Modeling Dual Drug Delivery from Eluting Stents: The Influence of Non-Linear Binding Competition and Non-Uniform Drug Loading.

OBJECTIVE: There is increasing interest in simultaneous endovascular delivery of more than one drug from a drug-loaded stent into a diseased artery. There may be an opportunity to obtain a therapeutically desirable uptake profile of the two drugs over time by appropriate design of the initial drug distribution in the stent. Due to the non-linear, coupled nature of diffusion and reversible specific/non-specific binding of both drugs as well as competition between the drugs for a fixed binding site density, a comprehensive numerical investigation of this problem is critically needed. METHODS: This paper presents numerical computation of dual drug delivery in a stent-artery system, accounting for diffusion as well as specific and non-specific reversible binding. The governing differential equations are discretized in space, followed by integration over time using a stiff numerical solver. Three different cases of initial dual drug distribution are considered. RESULTS: For the particular case of sirolimus and paclitaxel, results show that competition for a limited non-specific binding site density and the significant difference in the forward/backward reaction coefficients play a key role in determining the nature of drug uptake. The nature of initial distribution of the two drugs in the stent is also found to influence the binding process, which can potentially be used to engineer a desirable dual drug uptake profile. CONCLUSIONS: These results help improve the fundamental understanding of endovascular dual drug delivery. In addition, the numerical technique and results presented here may be helpful for designing and optimizing other drug delivery problems as well.