Single vs replicate Real-Time PCR SARS-CoV-2 testing: Lessons learned for effective pandemic management.

Coronavirus Disease 19 (COVID-19) caused by the SARS-CoV-2 virus remains a global pandemic having a serious impact on national economies and healthcare infrastructure. Accurate infection detection protocols are key to policy guidance and decision making. In this pilot study, we compared single versus replicate PCR testing for effective and accurate SARS-CoV-2 infection detection. One-Step Real-Time RT-PCR was employed for the detection of SARS-CoV-2 RNA isolated from individual nasopharyngeal swabs. A total of 10,014 swabs, sampled from the general public (hospital admissions, A&E, elective surgeries, cancer patients, care home residents and healthcare staff), were tested using standard replicate testing. Our analysis demonstrates that approximately 19% of SARS-CoV-2 infected individuals would have been reported as false negative if single sample Real-Time PCR testing was used. Therefore, two replicate tests can substantially decrease the risk of false negative reporting and reduce hospital and community infection rates. As the number of variants of concern increases, we believe that replicate testing is an essential consideration for effective SARS-CoV-2 infection detection and prevention of further outbreaks. A strategic approach limiting the number of missed infections is crucial in controlling the rise of new SARS-CoV-2 variants as well as the management of future pandemics.

The Significance of Metal Coordination in Imidazole-Functionalized Metal-Organic Frameworks for Carbon Dioxide Utilization.

The grafting of imidazole species onto coordinatively unsaturated sites within metal-organic framework MIL-101(Cr) enables enhanced CO2 capture in close proximity to catalytic sites. The subsequent combination of CO2 and epoxide binding sites, as shown through theoretical findings, significantly improves the rate of cyclic carbonate formation, producing a highly active CO2 utilization catalyst. An array of spectroscopic investigations, in combination with theoretical calculations reveal the nature of the active sites and associated catalytic mechanism which validates the careful design of the hybrid MIL-101(Cr).

PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown3,4. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined3,4. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

Tenogenic Differentiation of Human Embryonic Stem Cells.

Tendon healing is complex to manage because of the limited regeneration capacity of tendon tissue; stem cell-based tissue engineering approaches may provide alternative healing strategies. We sought to determine whether human embryonic stem cells (hESC) could be induced to differentiate into tendon-like cells by the addition of exogenous bone morphogenetic protein (BMP)12 (growth differentiation factor[GDF]7) and BMP13 (GDF6). hESC (SHEF-1) were maintained with or without BMP12/13 supplementation, or supplemented with BMP12/13 and the Smad signaling cascade blocking agent, dorsomorphin. Primary rat tenocytes were included as a positive control in immunocytochemistry analysis. A tenocyte-like elongated morphology was observed in hESC after 40-days continuous supplementation with BMP12/13 and ascorbic acid (AA). These cells displayed a tenomodulin expression pattern and morphology consistent with that of the primary tenocyte control. Analysis of tendon-linked gene transcription in BMP12/13 supplemented hESC demonstrated consistent expression of COL1A2, COL3A1, DCN, TNC, THBS4, and TNMD levels. Conversely, when hESCs were cultured in the presence of BMP12/13 and dorsomorphin COL3A1, DCN, and TNC gene expression and tendon matrix formation were inhibited. Taken together, we have demonstrated that hESCs are responsive to tenogenic induction via BMP12/13 in the presence of AA. The directed in vitro generation of tenocytes from pluripotent stem cells may facilitate the development of novel repair approaches for this difficult to heal tissue.

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE: Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.

Utilisation of gold nanoparticles on amine-functionalised UiO-66 (NH2-UiO-66) nanocrystals for selective tandem catalytic reactions.

Colloidal deposition of gold nanoparticles (Au NPs) onto NH2-UiO-66 nanocrystals has been demonstrated with the resulting hybrid catalyst proving robust and versatile for one-pot, heterogeneous conversions involving the selective oxidation of primary alcohols in tandem with Knoevenagel condensation reactions. Within these systems, structure-property correlations have been established to confirm that the active sites for the oxidation and condensation reactions are intrinsically correlated with the Au NPs and pendant amine groups respectively.

Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering.

Controlled production of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) nanoparticles for targeted and sustained drug delivery.

The ability to control the size and quality of nanoparticles (NPs) during production is critical for their success as a commercial product for clinical applications. Here, we employed a statistical design of experiment approach to identify the key process variables affecting the size of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) NPs during production via the solvent evaporation method. The number of sonication cycles had a standardzed effect on NP size of 55, with sonication power at 25, and PHBHHx concentration at 27 with a combination of these variables having a lower yet significant effect on NP size (p < 0.05). The PHBHHx NPs were stable for at least 7 days with an average polydispersity index of 0.18, a zeta potential of -10 to -40 mV, and an encapsulation efficiency of 63.5 ± 2%. These data were utilized to produce a prediction graph whereby particles could be produced with sizes ranging from 90 to 205 nm with a low mean curve prediction error of 1.96% for Haperzine-A-loaded NPs. Furthermore, a range of drug encapsulates NPs were produced and showed a sustained release of the encapsulated drug. This study demonstrates the ability to control the size of drug-loaded particles by manipulation of the production variables, which will allow targeted and controlled drug release to fit a variety of applications.

Observation of a physical matrix effect during cold vapour generation measurement of mercury in emissions samples.

The observation of a physical matrix effect during the cold vapour generation-atomic fluorescence measurement of mercury in emissions samples is reported. The effect is as a result of the different efficiencies of liberation of reduced mercury from solution as the matrix of the solution under test varies. The result of this is that peak area to peak height ratios decease as matrix concentration increases, passing through a minimum, before the ratio then increases as matrix concentration further increases. In the test matrices examined - acidified potassium dichromate and sodium chloride solutions - the possible biases caused by differences between the calibration standard matrix and the test sample matrix were as large as 2.8% (relative) representing peak area to peak height ratios for calibration standards and matrix samples of 45 and 43.75, respectively. For the system considered there is a good correlation between the density of the matrix and point of optimum liberation of dissolved mercury for both matrix types. Several methods employing matrix matching and mathematical correction to overcome the bias are presented and their relative merits discussed; the most promising being the use of peak area, rather than peak height, for quantification.

The application of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds for tendon repair in the rat model.

Tendon injuries and defects present a substantial burden to global healthcare economies. There are no synthetic/biosynthesised implants available which can restore full function or match the mechanical properties of native tendon. Therefore, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was investigated for its utility as a scaffold in a rat Achilles tendon repair model. Porous PHBHHx tubes and fibres were prepared with particle leaching and extrusion methods, respectively. Collagen gels reinforced by polymer fibres were inserted into the lumen of scaffold tubes to create the operational scaffold unit. Mechanical testing demonstrated that PHBHHx scaffolds had comparable mechanical properties to rat tendon, with maximal loads of 23.73 ± 1.08 N, compared to 17.35 ± 1.76 N in undamaged rat Achilles tendon. Sprague-Dawley (SD) rats were split into four experimental groups: control, PHBHHx scaffold only, PHBHHx scaffold and collagen, PHBHHx scaffold, collagen and tenocyte compositions for implantation to repair an induced Achilles tendon defect. No secondary immune response to PHBHHx was observed over a 40 days period of implantation. Movement was restored in PHBHHx scaffold-collagen-tenocyte recipient rats at an earlier time point than in other experimental groups, with complete load-bearing and function returning 20 days post-surgery as determined by the Achilles Functional Index. In vitro testing of tendon constructs after 40 days demonstrated reductions in PHBHHx molecular weight and polydispersity index accompanied by an increase in mean chain length indicating degradation of smaller polymer chain subunits. Similarly a reduction in PHBHHx tube ultimate tensile strength and elastic modulus was observed. Histological analysis provided evidence of tissue remodelling and cell alignment. In summary, PHBHHx scaffolds have been successfully applied in an in vivo tendon repair model raising promise for future utility in tissue engineering applications.

Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications.

The benefits associated with polyhydroxyalkanoates (PHA) in tissue engineering include high immunotolerance, low toxicity, and biodegradability. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a molecule from the PHA family of biopolymers, shares these features. In this study, the applicability of human embryonic stem cells (hESCs), spontaneously differentiated hESCs (SDhESCs), and mesenchymal stem cells (hMSCs) in conjunction with PHBHHx and collagen as a biocompatible replacement strategy for damaged tissues was exploited. Collagen gel contraction was monitored by seeding cells at controlled densities (0, 10(3), 10(4), and 10(5) cells/mL) and measuring length and diameter at regular time intervals thereafter when cultured in a complete medium. Cell viability was measured by trypan blue exclusion assay. Porous PHBHHx tube scaffolds were prepared using a dipping method followed by salt leaching. PHBHHx/collagen composites were generated via syringe injection of collagen/cell mixtures into sterile PHBHHx porous tubes. Reverse transcription polymerase chain reaction was used to determine the fate of cells within PHBHHx/collagen scaffolds with tendon, bone, cartilage, and fat-linked transcript expression being explored at days 0, 5 10, and 20. The capacity of PHBHHx/collagen scaffolds to support differentiation was explored using a medium specific for osteogenic, chondrogenic, and adipogenic lineage generation. Collagen gel tube contraction required initial seeding densities of ≥10(5) hMSCs or SDhESCs in 1.5 mg/mL collagen gel tubes. Gels with a collagen concentration of 3 mg/mL did not display contraction across the examined cell seeding densities. Cell viability was ∼50% for SDhESC and 90% for hMSCs at all cell densities tested in porous PHBHHx tube/3 mg/mL collagen hybrid scaffolds after 20 days in vitro culture. Undifferentiated hESCs did not contract collagen gel tubes and were unviable after 20 days culture. In the absence of additional stimuli, SOX9 was sporadically found, while RUNX2 was not present in both hMSC and SDhESC. Hybrid scaffolds were shown to promote retention of osteogenic, chondrogenic, and adipogenic differentiation by expression of RUNX2, SOX9, and PPARγ genes, respectively, following exposure to the appropriate induction medium. PHBHHx/collagen scaffolds have been successfully used to culture hMSC and SDhESC over an extended period supporting the potential of this scaffold combination in future tissue engineering applications.

Learned recognition of maternal signature odors mediates the first suckling episode in mice.

BACKGROUND: Soon after birth, all mammals must initiate milk suckling to survive. In rodents, this innate behavior is critically dependent on uncharacterized maternally derived chemosensory ligands. Recently, the first pheromone sufficient to initiate suckling was isolated from the rabbit. Identification of the olfactory cues that trigger first suckling in the mouse would provide the means to determine the neural mechanisms that generate innate behavior. RESULTS: Here we use behavioral analysis, metabolomics, and calcium imaging of primary sensory neurons and find no evidence of ligands with intrinsic bioactivity, such as pheromones, acting to promote first suckling in the mouse. Instead, we find that the initiation of suckling is dependent on variable blends of maternal "signature odors" that are learned and recognized prior to first suckling. CONCLUSIONS: As observed with pheromone-mediated behavior, the response to signature odors releases innate behavior. However, this mechanism tolerates variability in both the signaling ligands and sensory neurons, which may maximize the probability that this first essential behavior is successfully initiated. These results suggest that mammalian species have evolved multiple strategies to ensure the onset of this critical behavior.

Essential requirement for sphingosine kinase 2 in a sphingolipid apoptosis pathway activated by FTY720 analogues.

The clinical immunosuppressant FTY720 is a sphingosine analogue that, once phosphorylated by sphingosine kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for sphingosine 1-phosphate. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. Sphingosine itself is pro-apoptotic, and apoptosis induced with FTY720 or sphingosine is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including sphingosine. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered 1 mg/kg AAL(R), correlating with levels of AAL(R)-phosphate (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base phosphates and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.