Plasmonic Fiberoptic Absorbance Biosensor (P-FAB) for Rapid Detection of SARS-CoV-2 Nucleocapsid Protein.

SARS-CoV-2 nucleocapsid protein-based COVID-19 diagnosis is a promising alternative to the high-priced, time-consuming, and labor-intensive RT-PCR tests. Here, we developed a rapid, dip-type, wash-free plasmonic fiber optic absorbance biosensor (P-FAB) strategy for the point-of-care detection of SARS-CoV-2 N-protein, expressed abundantly during the infection. P-FAB involves a sandwich assay with plasmonic labels on the surface of a U-bent fiber optic sensor probe with a high evanescent wave absorbance (EWA) sensitivity. The SARS-CoV-2 N-protein is quantified in terms of the change in the intensity of the light propagating through the U-bent sensor probe coupled to a green LED and a photodetector. Firstly, the optical fiber material (silica vs. polymeric optical fiber), was evaluated to realize a sensitive sensor platform. The optimal size of AuNP labels (20, 40, and 60 nm) to achieve high sensitivity and a lower limit of detection (LoD) was investigated. Following the P-FAB strategy, fused silica/glass optical fiber (GOF) U-bent senor probe and citrate-capped AuNP labels (size ~40 nm) gave rise to an LoD down to ~2.5 ng/mL within 10 mins of read-out time. Further, studies on development and validation of a point of care (PoC) read-out device, and preclinical studies are in progress.

Intermediaries of branched chain amino acid metabolism induce fetal hemoglobin, and repress SOX6 and BCL11A, in definitive erythroid cells.

High levels of fetal hemoglobin (HbF) can ameliorate human ß-globin gene disorders. The short chain fatty acid butyrate is the paradigmatic metabolic intermediary that induces HbF. Inherited disorders of branched-chain amino acid (BCAA) metabolism have been associated with supranormal HbF levels beyond infancy, e.g., propionic acidemia (PA) and methylmalonic acidemia (MMA). We tested intermediaries of BCAA metabolism for their effects on definitive erythropoiesis. Like butyrate, the elevated BCAA intermediaries isovalerate, isobutyrate, and propionate, induce fetal globin gene expression in murine EryD in vitro, are associated with bulk histone H3 hyperacylation, and repress the transcription of key gamma globin regulatory factors, notably BCL11A and SOX6. Metabolic intermediaries that are elevated in Maple Syrup Urine Disease (MSUD) affect none of these processes. Percent HbF and gamma (γ) chain isoforms were also measured in non-anemic, therapeutically optimized subjects with MSUD (Group I, n=6) or with Isovaleric Acidemia (IVA), MMA, or PA (Group II, n=5). Mean HbF was 0.24 ± 0.15% in Group I and 0.87 ± 0.13% in Group II (p=.01); only the Gγ isoform was detected. We conclude that a family of biochemically related intermediaries of branched chain amino acid metabolism induces fetal hemoglobin during definitive erythropoiesis, with mechanisms that mirror those so far identified for butyrate.

A Comparative Study of Cationic Copper(I) Reagents Supported by Bipodal Tetramethylguanidinyl-Containing Ligands as Nitrene-Transfer Catalysts.

The bipodal compounds [(TMG2biphenN-R)CuI-NCMe](PF6) (R = Me, Ar (4-CF3Ph-)) and [(TMG2biphenN-Me)CuI-I] have been synthesized with ligands that feature a diarylmethyl- and triaryl-amine framework and superbasic tetramethylguanidinyl residues (TMG). The cationic Cu(I) sites mediate catalytic nitrene-transfer reactions between the imidoiodinane PhI = NTs (Ts = tosyl) and a panel of styrenes in MeCN, to afford aziridines, demonstrating comparable reactivity profiles. The copper reagents have been further explored to execute C-H amination reactions with a variety of aliphatic and aromatic hydrocarbons and two distinct nitrene sources PhI = NTs and PhI = NTces (Tces = 2,2,2-trichloroethylsulfamate) in benzene/HFIP (10:2 v/v). Good yields have been obtained for sec-benzylic and tert-C-H bonds of various substrates, especially with the more electron-deficient catalyst [(TMG2biphenN-Ar)CuI-NCMe](PF6). In conjunction with earlier studies, the order of reactivity of these bipodal cationic reagents as a function of the metal employed is established as Cu > Fe > Co ≥ Mn. However, as opposed to the base-metal analogues, the bipodal Cu reagents are less reactive than a similar tripodal Cu catalyst. The observed fluorophilicity of the bipodal Cu compounds may provide a deactivation pathway.

C-H amination chemistry mediated by trinuclear Cu(I) sites supported by a ligand scaffold featuring an arene platform and tetramethylguanidinyl residues.

Tripodal ligands that can encapsulate single or multiple metal sites in C3-symmetric geometric configurations constitute valuable targets for novel catalysts. Of particular interest in ligand development are efforts toward incorporating apical elements that exhibit little if any electron donicity, to enhance the electrophilic nature of a trans positioned active oxidant (e.g., metal-oxo, -nitrene). The tripodal ligand TMG3trphen-Arene has been synthesized, featuring an arene platform 1,3,5-substituted with phenylene arms possessing tetramethylguanidinyl (TMG) residues. Compound [(TMG3trphen-Arene)Cu3(µ-Cl)3] has been subsequently synthesized by extracting a Cu3(µ-Cl)3 cluster from anhydrous CuCl and shown to encapsulate a crown-shaped Cu3(µ-Cl)3 fragment, supported by Cu-NTMG bonds and modest Cu3⋯arene long-range contacts. Energy decomposition analysis (EDA) indicates that electrostatic contributions to the total interaction energy far exceed those due to orbital interactions. The latter involve orbital pairings largely associated with the NTMG stabilization of the Cu3(µ-Cl)3 cluster. The independent gradient model based on the Hirshfeld partition (IGMH) corroborates that contacts between the arene platform and the Cu3 triangle are noncovalent in nature. Catalyst [(TMG3trphen-Arene)Cu3(µ-Cl)3] enables amination of sec-benzylic and tert-C-H bonds of a panel of substrates by pre-synthesized PhINTces in solvent matrices that incorporate small amounts of HFIP. The involvement of an electrophilic aminating agent is evidenced by the better yields obtained for electron-rich benzylic sites and is further supported by Hammett analysis that reveals the development of a small positive charge during C-H bond activation. A rather modest KIE effect (2.1) is obtained from intramolecular H(D) competition in the amination of ethylbenzene, at the borderline of reported values for concerted and stepwise C-H amination systems. DFT analysis of the putative copper-nitrene oxidant indicates that the nitrene N atom is bridging between two copper sites in closely spaced triplet (ground state) and broken-symmetry singlet electronic configurations.

Maintenance of feeder free anchorage independent cultures of ES and iPS cells by retinol/vitamin A.

Pluripotent embryonic stem (ES) cells derived from mammalian blastocyst and the adult fibroblast derived induced pluripotent stem (iPS cells) exhibit complete potential to form cells representing all the primary germ layers such as mesoderm, endoderm and ectoderm. These cells are usually co-cultured with mouse embryonic fibroblast feeders to prevent spontaneous differentiation. Feeder free cultures can provide substantial advantage to improve the efficiency and consistency of the culture conditions. In these studies, we demonstrate that a small dietary compound retinol, the alcohol form of vitamin A has capacity to regulate the pluripotency of pluripotent stem cells and maintain highly enriched population of pluripotent ES and iPS cells in feeder free suspension cultures. Retinol maintains long-term cultures of undifferentiated cells via elevated expression of stem cell specific transcription factors Nanog and Oct4. The studies provide evidence that retinol regulates the self-renewal of pluripotent stem cells via the over expression of insulin like growth factor II (IGFII) that engages PI3 kinase signaling pathway via IGF1 receptor tyrosine kinase. The ES cells retain capacity to generate high degree germline competent chimeric animals after microinjection into blastocysts. The studies offer a convenient system for long term maintenance of pluripotent stem cells via the activation of intracellular machinery for self-renewal by a physiologically relevant compound for large-scale production of high quality pluripotent stem cells.

Emerging trends in point-of-care sensors for illicit drugs analysis.

Consumption of illicit narcotic drugs and fatal or criminal activities under their influence has become an utmost concern worldwide. These drugs influence an individual's feelings, perceptions, and emotions by altering the state of consciousness and thus can result in serious safety breaches at critical workplaces. Point-of-care drug-testing devices have become the need-of-the-hour for many sections such as the law enforcement agencies, the workplaces, etc. for safety and security. This review focuses on the recent progress on various electrochemical and optical nanosensors developed for the analysis of the most common illicit drugs (or their metabolites) such as tetrahydrocannabinol (THC), cocaine (COC), opioids (OPs), amphetamines & methamphetamine, and benzodiazepine (BZDs). The paper also highlights the sensitivity and selectivity of various sensing modalities along with evolving parameters such as real-time monitoring and measurement via a smart user interface. An overall outlook of recent technological advances in point of care (POC) devices and guided insights and directions for future research is presented.

Induction of fetal/embryonic globin gene expression depends on intact cell signaling in definitive erythroid cells.

OBJECTIVE: The induction of fetal hemoglobin during definitive erythropoiesis is a major therapeutic goal in ß-globin gene disorders. Butyrate induces fetal hemoglobin, and p38 phosphorylation has been implicated in this process. We studied p38 and the effect of its inhibitors in a physiologic primary cell model of fetal/embryonic globin gene induction during definitive erythropoiesis. METHODS: p38 phosphorylation was evaluated in a short-term culture of definitive erythroid precursor (EryD) cells following butyrate induction, absent prolonged exposure to cytokines. The impact of p38 inhibitors on embryonic/fetal globin gene induction by butyrate and on normal erythroid processes, including proliferation, differentiation, cell cycle occupancy, and RNA transcription, was also examined. RESULTS: p38 phosphorylation, maximal at harvest of murine fetal liver-derived EryD (FL EryD), when minimal embryonic/fetal globin gene expression is seen, is suppressed by EPO (as reported by others). Butyrate initially delays EPO-mediated suppression of p38 phosphorylation, but p38 phosphorylation thereafter, at 30 minutes to 48 hours, is equivalent and at low levels in EPO-treated FL EryD, with or without butyrate. Inhibitors of p38, at 10-50 µM, prevent butyrate-mediated induction of embryonic/fetal globin gene expression. We found that p38 inhibitors, which also disrupt non-p38 signaling pathways, perturb cell division, erythroid differentiation, transit through the cell cycle, and RNA transcription in primary EryD. CONCLUSION: p38 inhibitors interrupt normal erythropoiesis and the capacity for embryonic/fetal globin gene induction. However, p38 signaling is maximal in primary EryD at harvest, when embryonic globin genes are minimally expressed, and diminishes thereafter. We conclude that p38 inhibitors disrupt cellular pathways that are essential to butyrate-induced embryonic/fetal globin gene expression. However, levels of p38 phosphorylation are not coordinate with embryonic/fetal globin gene expression in EryD, and increased signaling through p38 may not be the sine qua non for embryonic/fetal globin gene induction.

Short-chain fatty acid-mediated effects on erythropoiesis in primary definitive erythroid cells.

Short-chain fatty acids (SCFAs; butyrate and propionate) up-regulate embryonic/fetal globin gene expression through unclear mechanisms. In a murine model of definitive erythropoiesis, SCFAs increased embryonic beta-type globin gene expression in primary erythroid fetal liver cells (eFLCs) after 72 hours in culture, from 1.7% (+/- 1.2%) of total beta-globin gene expression at day 0 to 4.9% (+/- 2.2%) in propionate and 5.4% (+/- 3.4%) in butyrate; this effect was greater in butyrate plus insulin/erythropoietin (BIE), at 19.5% (+/- 8.3%) compared with 0.1% (+/- 0.1%) in ins/EPO alone (P < .05). Fetal gamma-globin gene expression was increased in human transgene-containing eFLCs, to 35.9% (+/- 7.0%) in BIE compared with 4.4% (+/- 4.2%) in ins/EPO only (P < .05). Embryonic globin gene expression was detectable in 11 of 15 single eFLCs treated with BIE, but in0 of 15 ins/EPO-only treated cells. Butyrate-treated [65.5% (+/- 9.9%)] and 77.5% (+/- 4.0%) propionate-treated eFLCs were highly differentiated in culture, compared with 21.5% (+/- 3.5%) in ins/EPO (P < .005). Importantly, signaling intermediaries, previously implicated in induced embryonic/fetal globin gene expression (STAT5, p42/44, and p38), were not differentially activated by SCFAs in eFLCs; but increased bulk histone (H3) acetylation was seen in SCFA-treated eFLCs. SCFAs induce embryonic globin gene expression in eFLCS, which are a useful short-term and physiologic primary cell model of embryonic/fetal globin gene induction during definitive erythropoiesis.

P-FAB: A Fiber-Optic Biosensor Device for Rapid Detection of COVID-19.

Rapid and low-cost diagnosis of COVID-19 is essential to identify the infected subjects, particularly the asymptomatic cases, primarily to arrest the spread of the disease through local transmission. Antibody-based chromatographic serological tests, as an alternative to RT-PCR, offer only limited help due to high false positives. We propose to exploit our field-deployable/portable plasmonic fiber-optic absorbance biosensor (P-FAB) platform for one-step, wash-free detection of SARS-CoV-2 virus particles directly in saliva sample with minimal sample pre-processing.

PHYSICIAN ADVISERS ADD UP FOR SOME HOSPITALS.

A team of health care experts crunches the numbers sand reaches the conclusion that creating - or expanding - a physician adviser program is a good return on investment for any organization that's considering it.