Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Leveraging open hardware to alleviate the burden of COVID-19 on global health systems.

With the current rapid spread of COVID-19, global health systems are increasingly overburdened by the sheer number of people that need diagnosis, isolation and treatment. Shortcomings are evident across the board, from staffing, facilities for rapid and reliable testing to availability of hospital beds and key medical-grade equipment. The scale and breadth of the problem calls for an equally substantive response not only from frontline workers such as medical staff and scientists, but from skilled members of the public who have the time, facilities and knowledge to meaningfully contribute to a consolidated global response. Here, we summarise community-driven approaches based on Free and Open Source scientific and medical Hardware (FOSH) as well as personal protective equipment (PPE) currently being developed and deployed to support the global response for COVID-19 prevention, patient treatment and diagnostics.

Substituent Effects in Carbon-Nanotube-Supported Copper Phenolato Complexes for Oxygen Reduction Reaction.

Unprotected mononuclear pyrene-modified (bispyridylaminomethyl)methylphenol copper complexes were designed to be immobilized at multiwalled carbon nanotube (MWCNT) electrodes and form dinuclear bis(µ-phenolato) complexes on the surface. These complexes exhibit a high oxygen reduction reaction activity of 12.7 mA cm-2 and an onset potential of 0.78 V versus reversible hydrogen electrode. The higher activity of these complexes compared to that of mononuclear complexes with bulkier groups is induced by the favorable early formation of a dinuclear catalytic species on MWCNT.

Pediatric oncology social workers' experience of compassion fatigue.

Pediatric oncology social workers play an important role in supporting cancer patients and their families as they learn to talk about and cope with the physical and psychological impacts of cancer. As a result, social workers are particularly vulnerable to compassion fatigue and the associated psychological and physical impacts. The purpose of this qualitative study was to understand the experience of compassion fatigue among 27 pediatric oncology social workers. Four main themes emerged throughout the five focus groups: Conditions that contribute to compassion fatigue; the influence of compassion fatigue; coping strategies to alleviate compassion fatigue; and desire for systematic support to prevent compassion fatigue. Our study findings emphasize the importance of developing programs, policies and research geared toward the prevention of compassion fatigue, in addition to coping with symptoms. Further, this study brings attention to the importance of including pediatric oncology social workers in efforts to develop and implement systemic supports.

A Structurally Characterized CuIII Complex Supported by a Bis(anilido) Ligand and Its Oxidative Catalytic Activity.

Three copper(II) complexes of the (R,R)-N,N'-bis(3,5-di-tert-butyl-2-aminobenzylidene)-1,2-diaminocyclohexane ligand, namely [Cu(N L)], [Cu(N LH)]+ and [Cu(N LH2 )]2+ , were prepared and structurally characterized. In [Cu(N LH2 )]2+ the copper ion lies in an octahedral geometry with the aniline groups coordinated in equatorial positions. In [Cu(N L)] the anilines are deprotonated (anilido moieties) and coordinated to an almost square-planar metal ion. Complex [Cu(N L)] displays two oxidation waves at E1/2ox, 1 =-0.14 V and E1/2ox, 2 =0.36 V vs. Fc+ /Fc in CH2 Cl2 . Complex [Cu(N LH2 )]2+ displays an irreversible oxidation wave at high potential (1.21 V), but shows a readily accessible and reversible metal-centered reduction at E1/2red =-0.67 V (CuII /CuI redox couple). Oxidation of [Cu(N L)] by AgSbF6 produces [Cu(N L)](SbF6 ), which was isolated as single crystals. X-ray structure analysis discloses a contraction of the coordination sphere by 0.05 Šupon oxidation, supporting a metal-centered process. Complex [Cu(N L)](SbF6 ) displays an intense NIR band at 1260 nm corresponding to an anilido-to-copper(III) charge transfer transition. This compound slowly evolves in CH2 Cl2 solution towards [Cu(N LH)](SbF6 ), which is a copper(II) complex comprised of both anilido and aniline groups coordinated to the metal center. The copper(III) complex [Cu(N L)](SbF6 ) is an efficient catalyst for benzyl alcohol oxidation, with 236 TON in 24 h at 298 K, without additives other than oxygen and a base.

Wolbachia Modulates Lipid Metabolism in Aedes albopictus Mosquito Cells.

UNLABELLED: Certain strains of the intracellular endosymbiont Wolbachia can strongly inhibit or block the transmission of viruses such as dengue virus (DENV) by Aedes mosquitoes, and the mechanisms responsible are still not well understood. Direct infusion and liquid chromatography-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry-based lipidomics analyses were conducted using Aedes albopictus Aa23 cells that were infected with the wMel and wMelPop strains of Wolbachia in comparison to uninfected Aa23-T cells. Substantial shifts in the cellular lipid profile were apparent in the presence of Wolbachia Most significantly, almost all sphingolipid classes were depleted, and some reductions in diacylglycerols and phosphatidylcholines were also observed. These lipid classes have previously been shown to be selectively enriched in DENV-infected mosquito cells, suggesting that Wolbachia may produce a cellular lipid environment that is antagonistic to viral replication. The data improve our understanding of the intracellular interactions between Wolbachia and mosquitoes. IMPORTANCE: Mosquitoes transmit a variety of important viruses to humans, such as dengue virus and Zika virus. Certain strains of the intracellular bacterial genus called Wolbachia found in or introduced into mosquitoes can block the transmission of viruses, including dengue virus, but the mechanisms responsible are not well understood. We found substantial shifts in the cellular lipid profiles in the presence of these bacteria. Some lipid classes previously shown to be enriched in dengue virus-infected mosquito cells were depleted in the presence of Wolbachia, suggesting that Wolbachia may produce a cellular lipid environment that inhibits mosquito-borne viruses.

Moral distress in the resuscitation of extremely premature infants.

OBJECTIVE: To increase our understanding of moral distress experienced by neonatal registered nurses when directly or indirectly involved in the decision-making process of resuscitating infants who are born extremely premature. DESIGN: A secondary qualitative analysis was conducted on a portion of the data collected from an earlier study which explored the ethical decision-making process among health professionals and parents concerning resuscitation of extremely premature infants. SETTING: A regional, tertiary academic referral hospital in Ontario offering a perinatal program. PARTICIPANTS: A total of 15 registered nurses were directly or indirectly involved in the resuscitation of extremely premature infants. METHODS: Interview transcripts of nurses from the original study were purposefully selected from the original 42 transcripts of health professionals. Inductive content analysis was conducted to identify themes describing factors and situations contributing to moral distress experienced by nurses regarding resuscitation of extremely premature infants. ETHICAL CONSIDERATIONS: Ethical approval was obtained from the research ethics review board for both the initial study and this secondary data analysis. RESULTS: Five themes, uncertainty, questioning of informed consent, differing perspectives, perceptions of harm and suffering, and being with the family, contribute to the moral distress felt by nurses when exposed to neonatal resuscitation of extremely premature infants. An interesting finding was the nurses' perceived lack of power and influence in the neonatal resuscitation decision-making process. CONCLUSION: Moral distress continues to be a significant issue for nursing practice, particularly among neonatal nurses. Strategies are needed to help mediate the moral distress experienced by nurses, such as debriefing sessions, effective communication, role clarification, and interprofessional education and collaboration.

Bispidines for dual imaging.

The efficient transformation of the hexadentate bispidinol 1 into carbamate derivatives yields functional bispidines enabling convenient functionalization for targeted imaging. The BODIPY-substituted bispidine 3 combines a coordination site for metal ions, such as radioactive (64) Cu(II) , with a fluorescent unit. Product 3 was thoroughly characterized by standard analytical methods, single crystal X-ray diffraction, radiolabeling, and photophysical analysis. The luminescence of ligand 3 was found to be strongly dependent on metal ion coordination: Cu(II) quenches the BODIPY fluorescence, whereas Ni(II) and Zn(II) ions do not affect it. It follows that, in imaging applications with the positron emitter (64) Cu(II) , residues of its origin from enriched (64) Ni and the decay products (64) Ni(II) and (64) Zn(II) , efficiently restore the fluorescence of the ligand. This allows for monitoring of the emitted radiation as well as the fluorescence signal. The stability of the (64) Cu(II) 3 complex is investigated by transmetalation experiments with Zn(II) and Ni(II) , using fluorescence and radioactivity detection, and the results confirm the high stability of (64) Cu(II) 3. In addition, metal complexes of ligand 3 with the lanthanide ions Tb(III) , Eu(III) , and Nd(III) are shown to exhibit emission of the BODIPY ligand and the lanthanide ion, thus enabling dual emission detection.

Lanthanide-functionalized nanoparticles as MRI and luminescent probes for sensing and/or imaging applications.

The combination of lanthanides and nanoparticles to develop new hybrid nanomaterials has become a highly topical area of research in the field of sensing, biomedical imaging, drug delivery, etc. However, these novel nanomaterials have to be carefully designed to ensure that the unique properties conveyed by each component, i.e., lanthanide ions and nanoparticles, are maximized and not negatively affected by one another. In this Forum Article, the main advances in the design of lanthanide-based nanoparticles will be discussed, with the first part focusing on the design of gadolinium(III)-based nanoparticles and their use as magnetic resonance imaging agents. The second part will then describe the main and most recent designs of luminescent lanthanide-based nanoparticles and their applications as sensors or imaging agents, with a special emphasis on our contribution to this area.

Thiourea derived Tröger's bases as molecular cleft receptors and colorimetric sensors for anions.

Thiourea-functionalized Tröger's base receptors 1 and 2 have been synthesized and evaluated as novel for the recognition of anions. Receptor 2 gave rise to significant changes in the absorption spectrum upon titration with AcO(-) and H2PO4(-) and acted as a colorimetric sensor for F(-), the interaction of which was also evaluated using (1)H NMR spectroscopy.

A strongly emitting liquid-crystalline derivative of Y(3)N@C(80): bright and long-lived near-IR luminescence from a charge transfer state.

Great balls of fire: C60 and Y3 N@C80 were connected to the same oligo(phenyleneethynylene) unit to investigate their structural and photophysical properties. NMR investigations revealed a fulleroid structure for the Y3 N@C80 derivative, and both dyads gave rise to columnar phases with core-shell cylinders. The black and gray spheres represent the fullerene core units of the Y3 N@C80 derivative, which is an ideal candidate to be involved in energy and electron transfer processes.

Synthetic biology regulation in Europe: containment, release and beyond.

While synthetic biology is hoped to hold promise and potential to address pressing global challenges, the issue of regulation is an under-appreciated challenge. Particularly in Europe, the regulatory frameworks involved are rooted in historical concepts based on containment and release. Through a series of case studies including a field-use biosensor intended to detect arsenic in well water in Nepal and Bangladesh, and insects engineered for sterility, we explore the implications that this regulatory and conceptual divide has had on the deployment of synthetic biology projects in different national contexts. We then consider some of the broader impacts that regulation can have on the development of synthetic biology as a field, not only in Europe but also globally, with a particular emphasis on low- and middle-income countries. We propose that future regulatory adaptability would be increased by moving away from a containment and release dichotomy and toward a more comprehensive assessment that accounts for the possibility of varying degrees of 'contained release'. Graphical Abstract.

Synthesis of luminescent homo-dinuclear cationic lanthanide cyclen complexes bearing amide pendant arms through the use of copper catalysed (1,3-Huisgen, CuAAC) click chemistry.

The design and synthesis of dinuclear-lanthanide complexes possessing triazole-based bridges, formed by using copper catalysed 1,3-cycloaddition reactions between heptadentate alkyne functionalised cyclen europium or terbium complexes and di-azides (CuAAC reactions), are described. While this click reaction worked well for the formation of the homo-Eu(III) and Tb(III) bis-tri-arm cyclen N,N-dimethyl acetamide complexes, 2Eu and 2Tb, and for the homo-Eu(III) chiral N-methylnaphthalene based complexes 3Eu (S,S,S) and 4Eu (R,R,R), the formation of the Eu(III) complex of the primary amide analogue of 2, namely 1Eu, was not successful, clearly demonstrating the effect that the nature of the pendant arms has on this reaction. Furthermore, the click reactions between the free alkyne cyclen bis-derivatives (5-8) and the di-azide were unsuccessful, most likely due to the high affinity of the cyclen macrocycles for Cu(II). The Eu(III) complexes of 2-4 and 2Tb all gave rise to sensitised metal ion centred emission upon excitation of the triazole or the naphthalene antennae in methanol solution, and their hydration states were determined, which showed that while the Eu(III) mono-nuclear complexes had q ∼ 2, the click products all had q ∼ 1. In the case of 3Eu (S,S,S) and 4Eu (R,R,R), the circular polarised emission (CPL) was also observed for both, demonstrating the chiral environment of the lanthanide centres.

Lanthanide Complexes (GdIII and EuIII ) Based on a DOTA-TEMPO Platform for Redox Monitoring via Relaxivity.

Three lanthanide complexes (Ln=Gd, Eu) based on a DO3 A ([Ln(L1 )]) or DO2 A ([Ln(L2-3 )]+ ) platform appended by a redox active TEMPO-based arm were prepared. Complex [Ln(L2 )]+ shows an alkyne arm, offering the possibility of postfunctionalization by click reaction to yield [Ln(L3 )]+ . The complexes demonstrate a redox response whereby the hydroxylamine, nitroxide and oxoammonium forms of the arm can be obtained in turn. Luminescence measurements on the europium complexes support an octadentate (L1 , L3 ) or heptadentate (L2 ) chelation by the ligand, with one water molecule in the inner coordination sphere. The relaxivity was determined from 20 kHz to 30 MHz by fast-field cycling NMR. The three GdIII complexes under their hydroxylamine form [Gd(L1 )] and [Gd(L2-3 )]+ show r1 values of 7.0, 5.1 and 5.0 mM-1 s-1 (30 KHz), which increase to 8.8, 5.5 and 6.1 mM-1 s-1 in the nitroxide form. The radical complexes are not toxic against M21 cell lines, at least up to 40 µM. By using EPR spectroscopy we establish that they do not penetrate the cells with the exception of [Eu(L2 )]+ .

Symmetric CEST-active lanthanide complexes for redox monitoring.

Two symmetric ligands harbouring two TEMPO radicals and two functionalized acetamide arms (R = OMe (L1), CF3 (L2)) were prepared and chelated to lanthanide ions (EuIII, YbIII for both L1 and L2, DyIII for L1). Luminescence measurements on the europium complexes support the coordination of a single water molecule. The TEMPO arms are magnetically interacting in L1 (and its complexes) but not in L2. The TEMPO moieties can be reversibly oxidized into an oxoammonium (0.33-0.36 V vs. Fc+/Fc) or reduced into a hydroxylamine (ill-defined redox wave, reduction by ascorbate), which are both diamagnetic. The europium complexes [Eu(L1)]3+ and [Eu(L2)]3+ in their hydroxylamine form exhibit a temperature dependent CEST effect, which is maximal at 25 °C (30%) and 37 °C (12%), respectively. The CEST activity is dramatically reduced in the corresponding nitroxide forms due to the paramagnetism of the ligand. The europium complexes show no cytotoxicity against M21 cell lines over long incubation times (72 h) at high concentration (40 µM).

Homebrew reagents for low-cost RT-LAMP.

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has gained popularity for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The high specificity, sensitivity, simple protocols, and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents, and their distribution under cold chain shipping limit RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits, and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range, and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs; thus, we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low-cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Homebrew reagents for low cost RT-LAMP.

RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Luminescent pro-nitroxide lanthanide complexes for the detection of reactive oxygen species.

The DOTA-based ligand H3L (5) appended with a pro-nitroxide moiety has been synthesized. The europium and ytterbium complexes 5Ln show metal-centred luminescence. They react with ROS in aqueous media to give a transient iminonitroxide and a stable nitronylnitroxide radical authenticated by EPR, with change in luminescence.

An open toolkit for tracking open science partnership implementation and impact.

Serious concerns about the way research is organized collectively are increasingly being raised. They include the escalating costs of research and lower research productivity, low public trust in researchers to report the truth, lack of diversity, poor community engagement, ethical concerns over research practices, and irreproducibility. Open science (OS) collaborations comprise of a set of practices including open access publication, open data sharing and the absence of restrictive intellectual property rights with which institutions, firms, governments and communities are experimenting in order to overcome these concerns. We gathered two groups of international representatives from a large variety of stakeholders to construct a toolkit to guide and facilitate data collection about OS and non-OS collaborations. Ultimately, the toolkit will be used to assess and study the impact of OS collaborations on research and innovation. The toolkit contains the following four elements: 1) an annual report form of quantitative data to be completed by OS partnership administrators; 2) a series of semi-structured interview guides of stakeholders; 3) a survey form of participants in OS collaborations; and 4) a set of other quantitative measures best collected by other organizations, such as research foundations and governmental or intergovernmental agencies. We opened our toolkit to community comment and input. We present the resulting toolkit for use by government and philanthropic grantors, institutions, researchers and community organizations with the aim of measuring the implementation and impact of OS partnership across these organizations. We invite these and other stakeholders to not only measure, but to share the resulting data so that social scientists and policy makers can analyse the data across projects.