Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions

Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells

We investigated the ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) in Ewing sarcoma cells, in the perspective of in vivo anti-cancer nucleic acid drug delivery. siRNA was adsorbed onto NDs previously coated with cationic polymer. Cell uptake of NDs has been demonstrated by taking advantage of NDs intrinsic fluorescence coming from embedded color center defects. Cell toxicity of these coated NDs was shown to be low. Consistent with the internalization efficacy, we have shown a specific inhibition of EWS/Fli-1 gene expression at the mRNA and protein level by the ND vectorized siRNA in a serum containing medium

Can artificial intelligence improve the management of pneumonia

The use of artificial intelligence (AI) to support clinical medical decisions is a rather promising concept. There are two important factors that have driven these advances: the availability of data from electronic health records (EHR) and progress made in computational performance. These two concepts are interrelated with respect to complex mathematical functions such as machine learning (ML) or neural networks (NN). Indeed, some published articles have already demonstrated the potential of these approaches in medicine. When considering the diagnosis and management of pneumonia, the use of AI and chest X-ray (CXR) images primarily have been indicative of early diagnosis, prompt antimicrobial therapy, and ultimately, better prognosis. Coupled with this is the growing research involving empirical therapy and mortality prediction, too. Maximizing the power of NN, the majority of studies have reported high accuracy rates in their predictions. As AI can handle large amounts of data and execute mathematical functions such as machine learning and neural networks, AI can be revolutionary in supporting the clinical decision-making processes. In this review, we describe and discuss the most relevant studies of AI in pneumonia

Haplotyping the human leukocyte antigen system from single chromosomes

We describe a method for determining the parental HLA haplotypes of a single individual without recourse to conventional segregation genetics. Blood samples were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 amplification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function

A CX3CRI Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.Peer reviewe

Chain length affects pancreatic lipase activity and the extent and pH–time profile of triglyceride lipolysis

Triglycerides (TG) are one of the most common excipients used in oral lipid-based formulations. The chain length of the TG plays an important role in the oral bioavailability of the co-administered drug. Fatty acid (FA) chain-length specificity of porcine pancreatic lipase was studied by means of an in vitro lipolysis model under bio-relevant conditions at pH 6.80. In order to determine the total extent of lipolysis, back-titration experiments at pH 11.50 were performed. Results suggest that there is a specific chain length range (C2–C8) for which pancreatic lipase shows higher activity. This specificity could result from a combination of physicochemical properties of TGs, 2-monoglycerides (2-MGs) and FAs, namely the droplet size of the TGs, the solubility of 2-MGs within mixed micelles, and the relative stability of the FAs as leaving groups in the hydrolysis reaction. During experimentation, it was evident that an optimisation of lipolysis conditions was needed for tighter control over pH levels so as to better mimic in vivo conditions. 1 M NaOH, 3.5 mL/min maximum dosing rate, and 3 μL/min minimum dosing rate were the optimised set of conditions that allowed better pH control, as well as the differentiation of the lipolysis of different lipid loads

Correlating gastric emptying of amphotericin B and paracetamol solid lipid nanoparticles with changes in particle surface chemistry

Oral delivery of pharmaceuticals requires that they retain their physical and chemical attributes during transit within the gastrointestinal (GI) tract, for the manifestation of desired therapeutic profiles. Solid lipid nanoparticles (SLNs) are used as carriers to improve the absorption of hydrophobic drugs. In this study, we examine the stability of amphotericin B (AmB) and paracetamol (PAR) SLNs in simulated GI fluids during gastric emptying. On contact with the simulated fluids, the particles increased in size due to ingress of the dissolution media into the particles. Simulated gastric emptying revealed that the formulations had mean sizes <350 nm and neutral surface charges, both of which are optimal for intestinal absorption of SLNs. There was ingress of the fluids into the SLNs, followed by diffusion of the dissolved drug, whose rate depended on the solubility of the loaded-drug in the particular medium. Time-of-flight secondary ion mass spectrometry analyses indicated that drug loading followed the core-shell model and that the AmB SLNs have a more drug-enriched core than the PAR SLNs do. The AmB SLNs are therefore a very suitable carrier of AmB for oral delivery. The stability of the SLNs in the simulated GI media indicates their suitability for oral delivery

Emergence of Progressive Mutations in SARS-CoV-2 From a Hematologic Patient With Prolonged Viral Replication

We documented a hematologic patient with prolonged SARS-CoV-2 viral replication in whom emergence of viral mutations was documented after the consecutive use of antivirals and convalescent plasma. The virus detected in the last of 12 clinical samples (day 237) had accumulated 22 changes in amino acids and 29 in nucleotides. Some of these changes, such as the E484Q, were mutations of concern as defined by WHO. This finding represents an enormous epidemiological threat and poses a major clinical challenge. Combined antiviral strategies, as well as specific strategies related to the diagnostic approach of prolonged infections for this specific population, may be needed.This work has been financed by funds for research ad hoc COVID-19 from patronage provided by citizens and organizations to Hospital Clínic de Barcelona-Fundació Clínic per a la Recerca Biomèdica. This work received support from FONDO-COVID19 (ISCIII Grant number: COV20-00679), Instituto de Salud Carlos III (PI21/01640) and by European Region (ERDF, “A way to make Europe”). PP-A [JR20/00012 and PI21/00498], NG-P [FI19/00133], have also received research grants from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III. The funders had neither a specific role in study design or collection of data, nor in writing of the paper or decision to submit.S

Melanocortin receptors in GtoPdb v.2021.3

Melanocortin receptors (provisional nomenclature as recommended by NC-IUPHAR [41]) are activated by members of the melanocortin family (&#945;-MSH, &#946;-MSH and &#947;-MSH forms; &#948; form is not found in mammals) and adrenocorticotrophin (ACTH). Endogenous antagonists include agouti and agouti-related protein. ACTH(1-24) was approved by the US FDA as a diagnostic agent for adrenal function test, whilst NDP-MSH was approved by EMA for the treatment of erythropoietic protoporphyria. Several synthetic melanocortin receptor agonists are under clinical development