DNA barcoding of fresh seafood in Australian markets reveals misleading labelling and sale of endangered species.

Flake and shark samples were purchased from outlets in several coastal Australian regions and genetically barcoded using the cytochrome oxidase subunit 1 (CO1) gene to investigate labelling reliability and species-specific sources of ambiguously labelled fillets. Of the 41 shark fillet samples obtained, 23 yielded high-quality CO1 sequences, out of which 57% (n = 13) were labelled ambiguously (misleading) and 35% (n = 8) incorrectly. In contrast, barramundi fillets, which are widely available and sought after in Australian markets, were shown to be accurately labelled. Species identified from shark samples, including the shortfin mako (n = 3) and the scalloped hammerhead (n = 1), are assessed by the IUCN as endangered and critically endangered, respectively, with several others classified as vulnerable and near threatened.

Sustained interleukin-10 delivery reduces inflammation and improves motor function after spinal cord injury.

BACKGROUND: The anti-inflammatory cytokine interleukin-10 (IL-10) has been explored previously as a treatment method for spinal cord injury (SCI) due to its ability to attenuate pro-inflammatory cytokines and reduce apoptosis. Primary limitations when using systemic injections of IL-10 are that it is rapidly cleared from the injury site and that it does not cross the blood-spinal cord barrier. OBJECTIVE: Here, mineral-coated microparticles (MCMs) were used to obtain a local sustained delivery of IL-10 directly into the injury site after SCI. METHODS: Female Sprague-Dawley rats were contused at T10 and treated with either an intraperitoneal injection of IL-10, an intramedullary injection of IL-10, or MCMs bound with IL-10 (MCMs+IL-10). After treatment, cytokine levels were measured in the spinal cord, functional testing and electrophysiology were performed, axon tracers were injected into the brainstem and motor cortex, macrophage levels were counted using flow cytometry and immunohistochemistry, and lesion size was measured. RESULTS: When treated with MCMs+IL-10, IL-10 was significantly elevated in the injury site and inflammatory cytokines were significantly suppressed, prompting significantly less cells expressing antigens characteristic of inflammatory macrophages and significantly more cells expressing antigens characteristic of earlier stage anti-inflammatory macrophages. Significantly more axons were preserved within the rubrospinal and reticulospinal tracts through the injury site when treated with MCMs+IL-10; however, there was no significant difference in corticospinal tract axons preserved, regardless of treatment group. The rats treated with MCMs+IL-10 were the only group with a significantly higher functional score compared to injured controls 28 days post-contusion. CONCLUSION: These data demonstrate that MCMs can effectively deliver biologically active IL-10 for an extended period of time altering macrophage phenotype and aiding in functional recovery after SCI.

Buccal injection of articaine to anesthetize the palatal mucosa.

Buccal and palatal injections are required for administration of anesthetic agents before maxillary tooth extractions, but palatal injections are painful for patients. Studies suggest that the palatal injection can be eliminated when articaine is delivered as a local anesthetic agent via buccal injection, but the anatomical mechanism for this effectiveness remains unclear. The objective of this study was to explore the potential mechanism by which buccal infiltration results in palatal anesthesia. The study approach included examining cadaveric specimens and investigating the pharmacologic properties of articaine. Twenty-eight formalin-fixed cadaveric hemimaxillae were dissected and sectioned into anterior, premolar, and molar regions. The maxillary sections were measured in 3 planes: inferior, middle, and superior. Buccal cortical plate (BCP), palatal cortical plate (PCP), and total buccopalatal (TBP) thickness were independently evaluated by 2 measurers using standard digital calipers. Statistical analysis of regional maxillary thickness measurements was achieved via 2-way analysis of variance. Measurements of BCP and PCP thickness revealed no statistically significant differences along the maxillae (P > 0.05). Both the BCP and PCP mean values were significantly less than the TBP measurement (P < 0.0001). In all 3 regions, the mean TBP thickness in the superior plane was significantly greater than that of the inferior plane (P < 0.05). The mean TBP thickness was significantly greater in the molar and premolar regions than in the anterior region (P < 0.05). The mean BCP measurements were significantly lesser in the maxillary premolar and molar regions than in the corresponding mandibular regions (P < 0.0001). The pharmacologic properties of articaine, which is capable of diffusing greater distances than other local anesthetics, coupled with the uniformly thin, cancellous maxillary bone, provide a plausible explanation for the success of palatal anesthesia achieved through buccal infiltration of articaine, obviating the need for a palatal injection.

Interplay of Intermolecular and Intramolecular Hydrogen Bonds on Complex Formation: The 3-Aminopropanol-Water van der Waals Complex.

This combined experimental and theoretical study answers the question whether the intramolecular hydrogen-bond strength in amino alcohols is dependent on the ring size. For this purpose, the rotational spectrum of the 3-aminopropanol-H2O van der Waals complex was recorded using Fourier-transform microwave spectroscopy and fit to the rotational, quadrupole coupling, and centrifugal distortion constants of the Watson A-reduction Hamiltonian. The experimental results are consistent with an ab initio conformation calculated at the MP2/6-311++G(d,p) level that involves the lowest energy 3-aminopropanol monomer and consists of a hydrogen bonding network. The calculated global minimum ab initio complex however comprises a higher energy monomer conformation of 3-aminopropanol. Upon complex formation with water, the O-H····N intramolecular hydrogen bond and OCCN backbone conformation of the lower energy monomer remain unchanged, in contrast to 2-aminoethanol. This behavior is consistent with the increasing strength of the intramolecular hydrogen bond of linear amino alcohols as a function of increasing chain length.

Multilayered Inorganic Microparticles for Tunable Dual Growth Factor Delivery.

There is an increasing need to control the type, quantity, and timing of growth factors released during tissue healing. Sophisticated delivery systems offering the ability to deliver multiple growth factors with independently tunable kinetics are highly desirable. Here, a multilayered, mineral coated micro-particle (MCMs) platform that can serve as an adaptable dual growth factor delivery system is developed. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are bound to the mineral coatings with high binding efficiencies of up to 80%. BMP-2 is firstly bound onto a 1st mineral coating layer; then VEGF is bound onto a 2nd mineral coating layer. The release of BMP-2 is sustained over a period of 50 days while the release of VEGF is a typical two-phase release with rapid release in the first 14 days and more sustained release for the following 36 days. Notably, the release behaviors of both growth factors can be independently tailored by changing the intrinsic properties of the mineral coatings. Furthermore, the release of BMP-2 can be tuned by changing the thickness of the 2nd layer. This injectable microparticle based delivery platform with tunable growth factor release has immense potential for applications in tissue engineering and regenerative medicine.

Serum-dependence of affinity-mediated VEGF release from biomimetic microspheres.

Vascular endothelial growth factor (VEGF) activity is highly regulated via sequestering within the ECM and cell-demanded proteolysis to release the sequestered VEGF. Numerous studies have demonstrated that VEGF activity mediates cellular events leading to angiogenesis and capillary formation in vivo. This has motivated the study of biomaterials to sustain VEGF release, and in many cases, the materials are inspired by the structure and function of the native ECM. However, there remains a need for materials that can bind to VEGF with high specificity, as the in vivo environment is rich in a variety of growth factors (GFs) and GF-binding moieties. Here we describe a strategy to control VEGF release using hydrogel microspheres with tethered peptides derived from VEGF receptor 2 (VEGFR2). Using biomaterials covalently modified with varying concentrations of two distinct VEGFR2-derived peptides with varying serum stability, we analyzed both biomaterial and environmental variables that influence VEGF release and activity. The presence of tethered VEGF-binding peptides (VBPs) resulted in significantly extended VEGF release relative to control conditions, and the resulting released VEGF significantly increased the expansion of human umbilical vein endothelial cells in culture. VEGF release rates were also strongly influenced by the concentration of serum. The presence of Feline McDonough Sarcoma-like tyrosine kinase 1 (sFlt-1), a serum-borne receptor fragment derived from VEGF receptor 1, increased VEGF release rates, although sFlt-1 was not sufficient to recapitulate the release profile of VEGF in serum. Further, the influence of serum on VEGF release was not due to protease activity or nonspecific VEGF interactions in the presence of serum-borne heparin. VEGF release kinetics correlated well with a generalizable mathematical model describing affinity-mediated release of VEGF from hydrogel microspheres in defined conditions. Modeling results suggest a potential mechanism whereby competition between VEGF and multiple VEGF-binding serum proteins including sFlt-1, soluble kinase insert domain receptor (sKDR), and α2-macroglobulin (α2-M) likely influenced VEGF release from microspheres. The materials and mathematical model described in this approach may be useful in a range of applications in which sustained, biologically active GF release of a specific GF is desirable.

Minimization and complete loss of general transcription factor proteins in the intracellular parasite Encephalitozoon cuniculi.

Genome compaction is a common evolutionary feature of parasites. The unicellular, obligate intracellular parasite Encephalitozoon cuniculi has one of smallest known eukaryotic genomes, and is nearly four times smaller than its distant fungi relative, the budding yeast Saccharomyces cerevisiae. Comparison of the proteins encoded by compacted genomes to those encoded by larger genomes can reveal the most highly conserved features of the encoded proteins. In this study, we identified the proteins comprising the RNA polymerases and their corresponding general transcription factors by using several bioinformatic approaches to compare the transcription machinery of E. cuniculi and S. cerevisiae. Surprisingly, our analyses revealed an overall reduction in the size of the proteins comprising transcription machinery of E. cuniculi, which includes the loss of entire regions or functional domains from proteins, as well as the loss of entire proteins and complexes. Unexpectedly, we found that the E. cuniculi ortholog of Rpc37 (a RNA Polymerase III subunit) more closely resembles the H. sapiens ortholog of Rpc37 than the S. cerevisiae ortholog of Rpc37, in both size and structure. Overall, our findings provide new insight into the minimal core eukaryotic transcription machinery and help define the most critical features of Pol components and general transcription factors.

Tunable Conductive Hydrogel Scaffolds for Neural Cell Differentiation.

Multielectrode arrays would benefit from intimate engagement with neural cells, but typical arrays do not present a physical environment that mimics that of neural tissues. It is hypothesized that a porous, conductive hydrogel scaffold with appropriate mechanical and conductive properties could support neural cells in 3D, while tunable electrical and mechanical properties could modulate the growth and differentiation of the cellular networks. By incorporating carbon nanomaterials into an alginate hydrogel matrix, and then freeze-drying the formulations, scaffolds which mimic neural tissue properties are formed. Neural progenitor cells (NPCs) incorporated in the scaffolds form neurite networks which span the material in 3D and differentiate into astrocytes and myelinating oligodendrocytes. Viscoelastic and more conductive scaffolds produce more dense neurite networks, with an increased percentage of astrocytes and higher myelination. Application of exogenous electrical stimulation to the scaffolds increases the percentage of astrocytes and the supporting cells localize differently with the surrounding neurons. The tunable biomaterial scaffolds can support neural cocultures for over 12 weeks, and enable a physiologically mimicking in vitro platform to study the formation of neuronal networks. As these materials have sufficient electrical properties to be used as electrodes in implantable arrays, they may allow for the creation of biohybrid neural interfaces and living electrodes.

SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection.

Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events 1 . This prothrombotic state is considered a key factor in the increased risk of stroke, which has been observed clinically during both acute infection and long after symptoms have cleared 2 . Here we developed a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly smooth muscle cells (SMCs), are a specifically susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterized the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, and endothelial cells (ECs). We observed that infected human SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we showed human ECs exposed to the secretome of infected SMCs produce hemostatic factors that can contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.

Fragile X Syndrome Patient-Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes.

BACKGROUND: Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. METHODS: To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. RESULTS: The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons. CONCLUSIONS: This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.

Development of a physiological insulin resistance model in human stem cell-derived adipocytes.

Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell-derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell-derived adipocytes.

Subcategories of Fibromyalgia: A New Concept.

Fibromyalgia has previously been categorized as primary, secondary, and juvenile fibromyalgia. However, these definitions do not adequately explain the etiopathology of disease, nor do they help direct new specific therapies. Herein, we review the previously known categorizations of fibromyalgia. Based on common patient characteristics and previously studied pathophysiologies, we propose new subcategorizations of fibromyalgia that we have self-narrated, including hormonal fibromyalgia, neuroendocrine fibromyalgia, psychologic fibromyalgia, inflammatory fibromyalgia, and lastly, neuropathic fibromyalgia. Future research needs to be done to verify, add to, and fully describe these self-narrated categories of fibromyalgia that we have proposed.

SARS-CoV-2 infection of human pluripotent stem cell-derived liver organoids reveals potential mechanisms of liver pathology.

Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infection can also damage other organs, including the brain, gut, and liver. Symptoms of liver damage are observed in nearly half of patients that succumb to severe SARS-CoV-2 infection. Here we use human-induced pluripotent stem cell-derived liver organoids (HLOs) to recapitulate and characterize liver pathology following virus exposure. Utilizing single-cell sequencing technology, we identified robust transcriptomic changes that occur in SARS-CoV-2 infected liver cells as well as uninfected bystander cells. Our results show a significant induction of many inflammatory pathways, including IFN-α, INF-γ, and IL-6 signaling. Our results further identify IL-6 signaling as a potential mechanism for liver-mediated activation of circulating macrophages.

A Localized Materials-Based Strategy to Non-Virally Deliver Chondroitinase ABC mRNA Improves Hindlimb Function in a Rat Spinal Cord Injury Model.

Spinal cord injury often results in devastating consequences for those afflicted, with very few therapeutic options. A central element of spinal cord injuries is astrogliosis, which forms a glial scar that inhibits neuronal regeneration post-injury. Chondroitinase ABC (ChABC) is an enzyme capable of degrading chondroitin sulfate proteoglycan (CSPG), the predominant extracellular matrix component of the glial scar. However, poor protein stability remains a challenge in its therapeutic use. Messenger RNA (mRNA) delivery is an emerging gene therapy technology for in vivo production of difficult-to-produce therapeutic proteins. Here, mineral-coated microparticles as an efficient, non-viral mRNA delivery vehicles to produce exogenous ChABC in situ within a spinal cord lesion are used. ChABC production reduces the deposition of CSPGs in an in vitro model of astrogliosis, and direct injection of these microparticles within a glial scar forces local overexpression of ChABC and improves recovery of motor function seven weeks post-injury.

FRET-enabled optical modulation for high sensitivity fluorescence imaging.

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Engineered tissues and strategies to overcome challenges in drug development.

Current preclinical studies in drug development utilize high-throughput in vitro screens to identify drug leads, followed by both in vitro and in vivo models to predict lead candidates' pharmacokinetic and pharmacodynamic properties. The goal of these studies is to reduce the number of lead drug candidates down to the most likely to succeed in later human clinical trials. However, only 1 in 10 drug candidates that emerge from preclinical studies will succeed and become an approved therapeutic. Lack of efficacy or undetected toxicity represents roughly 75% of the causes for these failures, despite these parameters being the primary exclusion criteria in preclinical studies. Recently, advances in both biology and engineering have created new tools for constructing new preclinical models. These models can complement those used in current preclinical studies by helping to create more realistic representations of human tissues in vitro and in vivo. In this review, we describe current preclinical models to identify their value and limitations and then discuss select areas of research where improvements in preclinical models are particularly needed to advance drug development. Following this, we discuss design considerations for constructing preclinical models and then highlight recent advances in these efforts. Taken together, we aim to review the advances as of 2020 surrounding the prospect of biological and engineering tools for adding enhanced biological relevance to preclinical studies to aid in the challenges of failed drug candidates and the burden this poses on the drug development enterprise and thus healthcare.

Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion.

Translation of human pluripotent stem cell (hPSC)-derived therapies to the clinic demands scalable, cost-effective methods for cell expansion. Culture media currently used for hPSC expansion rely on high concentrations and frequent supplementation of recombinant growth factors due to their short half-life at physiological temperatures. Here, we developed a biomaterial strategy using mineral-coated microparticles (MCMs) to sustain delivery of basic fibroblast growth factor (bFGF), a thermolabile protein critical for hPSC pluripotency and proliferation. We show that the MCMs stabilize bFGF against thermally induced activity loss and provide more efficient sustained release of active growth factor compared to polymeric carriers commonly used for growth factor delivery. Using a statistically driven optimization approach called Design of Experiments, we generated a bFGF-loaded MCM formulation that supported hPSC expansion over 25 passages without the need for additional bFGF supplementation to the media, resulting in greater than 80% reduction in bFGF usage compared to standard approaches. This materials-based strategy to stabilize and sustain delivery of a thermolabile growth factor has broad potential to reduce costs associated with recombinant protein supplements in scalable biomanufacturing of emerging cell therapies.

SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome.

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.

Single-dose mRNA therapy via biomaterial-mediated sequestration of overexpressed proteins.

Nonviral mRNA delivery is an attractive therapeutic gene delivery strategy, as it achieves efficient protein overexpression in vivo and has a desirable safety profile. However, mRNA's short cytoplasmic half-life limits its utility to therapeutic applications amenable to repeated dosing or short-term overexpression. Here, we describe a biomaterial that enables a durable in vivo response to a single mRNA dose via an "overexpress and sequester" mechanism, whereby mRNA-transfected cells locally overexpress a growth factor that is then sequestered within the biomaterial to sustain the biologic response over time. In a murine diabetic wound model, this strategy demonstrated improved wound healing compared to delivery of a single mRNA dose alone or recombinant protein. In addition, codelivery of anti-inflammatory proteins using this biomaterial eliminated the need for mRNA chemical modification for in vivo therapeutic efficacy. The results support an approach that may be broadly applicable for single-dose delivery of mRNA without chemical modification.

Teriparatide Treatment for Hypercalcemia Associated With Adynamic Bone Disease.

Hypercalcemia most often results from primary hyperparathyroidism and malignancy. Adynamic bone disease (ABD) is a form of renal osteodystrophy characterized by reduced bone turnover, which can limit the ability of bone to release or store calcium, potentially leading to low, normal, or high serum calcium levels. We describe a 51-year-old dialysis-dependent female with hypercalcemia after parathyroidectomy. A demeclocycline-labeled bone biopsy confirmed adynamic bone disease. Teriparatide, a recombinant form of parathyroid hormone (PTH) used to treat postmenopausal osteoporosis, was prescribed for 12 months and normalized serum calcium levels. Although previous case reports and series have described favorable changes in spine bone mineral density when teriparatide was prescribed for ABD, ours is the first documented case in which teriparatide resolved hypercalcemia due to ABD. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.