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.

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.

Osmotic Stress Triggers Phase Separation.

In this issue of Molecular Cell, Jalihal et al. (2020) show that cell volume changes upon osmotic stress result in rapid and reversible condensation of numerous multivalent proteins.

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.

Aberrant baseline cytokine profile in patients with newly diagnosed acquired aplastic anaemia correlates with disease severity and the treatment response.

BACKGROUND: Immune dysregulation is crucial in the pathogenesis of acquired aplastic anaemia (aAA). There is paucity of data regarding correlation of baseline cytokine profile with treatment response in aAA. OBJECTIVE: Present prospective case-control study aimed to correlate the baseline cytokines in patients with aAA with the treatment response. METHODS: Fifty-one patients with newly-diagnosed aAA > 13 years of either sex were enrolled over 1.5 years. Twenty age-and sex-matched healthy controls (HC) were also included. The cytokine profile (IL-2, 4, 6, 8, 10, 17, IFN-γ and TNF-α) in the peripheral blood plasma of aAA patients was performed at the baseline using cytometric bead analysis. The cytokine levels were compared with HC and correlated with response to immunosuppressive therapy (IST) at 3-months. RESULTS: The median age of cases was 29 years (range,13-74). The cases had higher mean levels of IL2 (p = 0.326), IL4 (p = 0.038), IL6 (p = 0.000), IL10 (p = 0.002), TNF-α (p = 0.302), IFN-γ (p = 0.569) and IL-17 (p = 0.284) than the HC. The baseline levels of all the cytokines were higher (statistically non-significant) among responders (n = 13) than the non-responders (n = 14) to IST. CONCLUSIONS: Baseline cytokine profile in patients with aAA might predict response to the IST. Larger studies are needed to validate our results.

Laplace Transform Based Modeling of Drug Delivery with Reversible Drug Binding in a Multilayer Tissue.

OBJECTIVE: Drug delivery from a drug-loaded device into an adjacent tissue is a complicated process involving drug transport through diffusion and advection, coupled with drug binding kinetics responsible for drug uptake in the tissue. This work presents a theoretical model to predict drug delivery from a device into a multilayer tissue, assuming linear reversible drug binding in the tissue layers. METHODS: The governing mass conservation equations based on diffusion, advection and drug binding in a multilayer cylindrical geometry are written, and solved using Laplace transformation. The model is used to understand the impact of various non-dimensional parameters on the amounts of bound and unbound drug concentrations as functions of time. RESULTS: Good agreement for special cases considered in past work is demonstrated. The effect of forward and reverse binding reaction rates on the multilayer drug binding process is studied in detail. The effect of the nature of the external boundary condition on drug binding and drug loss is also studied. For typical parameter values, results indicate that only a small fraction of drug delivered binds in the tissue. Additionally, the amount of bound drug rises rapidly with time due to early dominance of the forward reaction, reaches a maxima and then decays due to the reverse reaction. CONCLUSIONS: The general model presented here can account for other possible effects such as drug absorption within the device. Besides generalizing past work on drug delivery modeling, this work also offers analytical tools to understand and optimize practical drug delivery devices.

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.

RNA phase transitions in repeat expansion disorders.

Expansions of short nucleotide repeats produce several neurological and neuromuscular disorders including Huntington disease, muscular dystrophy, and amyotrophic lateral sclerosis. A common pathological feature of these diseases is the accumulation of the repeat-containing transcripts into aberrant foci in the nucleus. RNA foci, as well as the disease symptoms, only manifest above a critical number of nucleotide repeats, but the molecular mechanism governing foci formation above this characteristic threshold remains unresolved. Here we show that repeat expansions create templates for multivalent base-pairing, which causes purified RNA to undergo a sol-gel transition in vitro at a similar critical repeat number as observed in the diseases. In human cells, RNA foci form by phase separation of the repeat-containing RNA and can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

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.

Optimization of Initial Drug Distribution in Spherical Capsules for Personalized Release.

OBJECTIVE: Customization of the rate of drug delivered based on individual patient requirements is of paramount importance in the design of drug delivery devices. Advances in manufacturing may enable multilayer drug delivery devices with different initial drug distributions in each layer. However, a robust mathematical understanding of how to optimize such capabilities is critically needed. The objective of this work is to determine the initial drug distribution needed in a spherical drug delivery device such as a capsule in order to obtain a desired drug release profile. METHODS: This optimization problem is posed as an inverse mass transfer problem, and optimization is carried out using the solution of the forward problem. Both non-erodible and erodible multilayer spheres are analyzed. Cases with polynomial forms of initial drug distribution are also analyzed. Optimization is also carried out for a case where an initial burst in drug release rate is desired, followed by a constant drug release rate. RESULTS: More than 60% reduction in root-mean-square deviation of the actual drug release rate from the ideal constant drug release rate is reported. Typically, the optimized initial drug distribution in these cases prevents or minimizes large drug release rate at early times, leading to a much more uniform drug release overall. CONCLUSIONS: Results demonstrate potential for obtaining a desired drug delivery profile over time by carefully engineering the drug distribution in the drug delivery device. These results may help engineer devices that offer customized drug delivery by combining advanced manufacturing with mathematical optimization.

Pseudo-Binary Phase Diagram of LiNH2-MH (M = Na, K) Eutectic Mixture.

The hunt for a cleaner energy carrier leads us to consider a source that produces no toxic byproducts. One of the targeted alternatives in this approach is hydrogen energy, which, unfortunately, suffers from a lack of efficient storage media. Solid-state hydrogen absorption systems, such as lithium amide (LiNH2) systems, may store up to 6.5 weight percent hydrogen. However, the temperature of hydrogenation and dehydrogenation is too high for practical use. Various molar ratios of LiNH2 with sodium hydride (NaH) and potassium hydride (KH) have been explored in this paper. The temperature of hydrogenation for LiNH2 combined with KH and NaH was found to be substantially lower than the temperature of individual LiNH2. This lower temperature operation of both LiNH2-NaH and LiNH2-KH systems was investigated in depth, and the eutectic melting phenomenon was observed. Systematic thermal studies of this amide-hydride system in different compositions were carried out, which enabled the plotting of a pseudo-binary phase diagram. The occurrence of eutectic interaction increased atomic mobility, which resulted in the kinetic modification followed by an increase in the reactivity of two materials. For these eutectic compositions, i.e., 0.15LiNH2-0.85NaH and 0.25LiNH2-0.75KH, the lowest melting temperature was found to be 307 °C and 235 °C, respectively. Morphological studies were used to investigate and present the detailed mechanism linked with this phenomenon.

Demise of the stethoscope.

Medical practice has undergone a massive drift over the past several years. With the advent of modernization and technical advancements in both diagnostic and therapeutic fields, bedside clinical skills have suffered a setback. Increasing patient load in the hospitals, administrative commitments of the physicians, and need to get superspecialty fellowships are some of the factors which preclude resident teaching, which, in-turn hamper their long-term clinical skills and influence the patient-doctor relationship. In this perspective, I narrate my own experience regarding changing attitude of the younger generation of doctors toward patient care and also describe the role of a mentor in shaping the believes and practices of his medical students. Using real-world examples, I further discuss the obstacles which hamper good medical practice and suggest possible ways to overcome some of them.

Aberrant expression of cytokines in polycythemia vera correlate with the risk of thrombosis.

BACKGROUND: Thrombo-hemorrhagic complications cause significant morbidity and mortality in patients with polycythemia vera (PV). OBJECTIVES: To assay and correlate inflammatory cytokines with the thrombotic risk in PV patients. METHODS: This prospective observational study was carried out at Postgraduate Institute of Medical Education and Research, Chandigarh, India over 18-months. The study enrolled 52 patients with PV (newly diagnosed = 28, follow-up = 24), and 20 age/sex-matched controls. Cytokine analysis for IL 1ß, IL2, IL4, IL6, IL8, IL10, IL11, IL12/23p40, TNFα, and IFN-γ was performed on the peripheral blood (before treatment initiation for newly diagnosed cases, and after 7 days of stopping drugs for follow-up cases) by flow cytometry-based cytokine bead analysis (CBA) using CBA kits (BD™ biosciences, USA). Results were analyzed using SPSS Statistics 22.0. RESULTS: The mean age of patients was 51.9 ± 13 years. Levels of IL-6, IL-1ß, IL-8, IL-11, IL-12/23p40 were significantly raised, however, TNF-α, and IFN-γ levels were significantly lower in the PV population as compared to controls. A significant correlation between the levels of IL-6, IL-2, and IL-8 with the overall risk of thrombosis in PV patients was observed. CONCLUSIONS: PV patients display an aberrant pattern of plasma cytokine expression, the levels of which correlate with the thrombotic risk.

Variables affecting the presence of mesenchymal stromal cells in peripheral blood and their relationship with apheresis products.

Mesenchymal stromal cells (MSCs) demonstrate the properties of self-renewal, multipotentiality, and immunosuppression, which are responsible for their widespread clinical applications in tissue repair and regeneration. MSCs have been isolated from bone marrow, adipose tissue, and cord blood using culture in specialised media. Their presence in peripheral blood (PB) is debatable. We studied the presence of MSCs at baseline (PB) and following mobilisation with growth factors [PB and apheresis product (AP)] in patients undergoing autologous stem cell transplantation and healthy donors using flow cytometry. We conclude that both mobilised PB and AP are potential sources of MSCs. Given their small numbers in PB/AP, clinical use is feasible following ex-vivo expansion. Variables affecting the presence of MSCs in PB and AP are also discussed.

Randomized controlled trial of twice-daily versus alternate-day oral iron therapy in the treatment of iron-deficiency anemia.

Recent studies in iron-depleted women have challenged the current approach of treating iron-deficiency anemia (IDA) with oral iron in divided daily doses. Alternate day dosing leads to more fractional absorption of iron. In this randomized controlled trial, we looked at the efficacy and safety of alternate-day (AD) versus twice-daily (BD) oral iron in all severity of IDA. Total of 62 patients were randomized, 31 patients in BD arm received 60 mg elemental iron twice daily while 31 patients in AD arm received 120 mg iron on alternate days. The primary endpoint of 2 g/dl rise in hemoglobin was met in significantly more patients in the BD arm at 3 weeks (32.3% vs. 6.5%, p < 0.0001) and 6 weeks (58% vs. 35.5%, p = 0.001). There was a significant rise in the median hemoglobin at 3 (1.6 vs. 1.1, p = 0.02) and 6 weeks (2.9 vs. 2.0 g/dl, p = 0.03) in the BD arm. However, the median hemoglobin rise in the AD arm at 6 weeks was not significantly different than the BD arm at 3 weeks. Alternate-day dosing for 6 weeks and twice-daily dosing for 3 weeks resulted in the provision of the same total amount of iron. There were more reports of nausea in the BD arm (p = 0.03). In conclusion, the choice of twice-daily or alternate-day oral iron therapy should depend on the severity of anemia, the rapidity of response desired, and patient preference to either regimen due to adverse events. Trial Registration: CTRI reg. no. CTRI/2018/07/015106 http://ctri.nic.in/Clinicaltrials/login.php.

CRISPR-Cas9-based treatment of myocilin-associated glaucoma.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9-mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.

Implementation of Bismuth Chalcogenides as an Efficient Anode: A Journey from Conventional Liquid Electrolyte to an All-Solid-State Li-Ion Battery.

Bismuth chalcogenide (Bi2X3; X = sulfur (S), selenium (Se), and tellurium (Te)) materials are considered as promising materials for diverse applications due to their unique properties. Their narrow bandgap, good thermal conductivity, and environmental friendliness make them suitable candidates for thermoelectric applications, photodetector, sensors along with a wide array of energy storage applications. More specifically, their unique layered structure allows them to intercalate Li+ ions and further provide conducting channels for transport. This property makes these suitable anodes for Li-ion batteries. However, low conductivity and high-volume expansion cause the poor electrochemical cyclability, thus creating a bottleneck to the implementation of these for practical use. Tremendous endeavors have been devoted towards the enhancement of cyclability of these materials, including nanostructuring and the incorporation of a carbon framework matrix to immobilize the nanostructures to prevent agglomeration. Apart from all these techniques to improve the anode properties of Bi2X3 materials, a step towards all-solid-state lithium-ion batteries using Bi2X3-based anodes has also been proven as a key approach for next-generation batteries. This review article highlights the main issues and recent advances associated with Bi2X3 anodes using both solid and liquid electrolytes.

Transplantation of iPSC-derived TM cells rescues glaucoma phenotypes in vivo.

Glaucoma is a common cause of vision loss or blindness and reduction of intraocular pressure (IOP) has been proven beneficial in a large fraction of glaucoma patients. The IOP is maintained by the trabecular meshwork (TM) and the elevation of IOP in open-angle glaucoma is associated with dysfunction and loss of the postmitotic cells residing within this tissue. To determine if IOP control can be maintained by replacing lost TM cells, we transplanted TM-like cells derived from induced pluripotent stem cells into the anterior chamber of a transgenic mouse model of glaucoma. Transplantation led to significantly reduced IOP and improved aqueous humor outflow facility, which was sustained for at least 9 wk. The ability to maintain normal IOP engendered survival of retinal ganglion cells, whose loss is ultimately the cause for reduced vision in glaucoma. In vivo and in vitro analyses demonstrated higher TM cellularity in treated mice compared with littermate controls and indicated that this increase is primarily because of a proliferative response of endogenous TM cells. Thus, our study provides in vivo demonstration that regeneration of the glaucomatous TM is possible and points toward novel approaches in the treatment of this disease.

Eutectic Phenomenon of LiNH2-KH Composite in MH-NH3 Hydrogen Storage System.

Hydrogenation of a lithium-potassium (double-cation) amide (LiK(NH2)2), which is generated as a product by ammonolysis of litium hydride and potassium hydride (LiH-KH) composite, is investigated in details. As a result, lithium amide (LiNH2) and KH are generated after hydrogenation at 160 °C as an intermediate. It is noteworthy that the mixture of LiH and KNH2 has a much lower melting point than that of the individual melting points of LiNH2 and KH, which is recognized as a eutectic phenomenon. The hydrogenation temperature of LiNH2 in the mixture is found to be significantly lower than that of LiNH2 itself. This improvement of reactivity must be due to kinetic modification, induced by the enhanced atomic mobility due to the eutectic interaction.