Expiration of State Licensure Waivers and Out-of-State Telemedicine Relationships.

This cross-sectional study compares the use of telemedicine in states where COVID-19 pandemic­related licensure waivers expired vs states where waivers continued.

Effect of pre-analytical variables on Raman and FTIR spectral content of lymphocytes.

The use of Fourier transform infrared (FTIR) and Raman spectroscopy (RS) for the analysis of lymphocytes in clinical applications is increasing in the field of biomedicine. The pre-analytical phase, which is the most vulnerable stage of the testing process, is where most errors and sample variance occur; however, it is unclear how pre-analytical variables affect the FTIR and Raman spectra of lymphocytes. In this study, we evaluated how pre-analytical procedures undertaken before spectroscopic analysis influence the spectral integrity of lymphocytes purified from the peripheral blood of male volunteers (n = 3). Pre-analytical variables investigated were associated with (i) sample preparation, (blood collection systems, anticoagulant, needle gauges), (ii) sample storage (fresh or frozen), and (iii) sample processing (inter-operator variability, time to lymphocyte isolation). Although many of these procedural pre-analytical variables did not alter the spectral signature of the lymphocytes, evidence of spectral effects due to the freeze-thaw cycle, in vitro culture inter-operator variability and the time to lymphocyte isolation was observed. Although FTIR and RS possess clinical potential, their translation into a clinical environment is impeded by a lack of standardisation and harmonisation of protocols related to the preparation, storage, and processing of samples, which hinders uniform, accurate, and reproducible analysis. Therefore, further development of protocols is required to successfully integrate these techniques into current clinical workflows.

Emergent structural and functional properties of hippocampal multi-cellular aggregates.

Hippocampal neural networks are distinctly capable of integrating multi-modal sensory inputs to drive memory formation. Neuroscientific investigations using simplified in vitro models have greatly relied on planar (2D) neuronal cultures made from dissociated tissue. While these models have served as simple, cost-effective, and high-throughput tools for examining various morphological and electrophysiological characteristics of hippocampal networks, 2D cultures fail to reconstitute critical elements of the brain microenvironment that may be necessary for the emergence of sophisticated integrative network properties. To address this, we utilized a forced aggregation technique to generate high-density (>100,000 cells/mm3) multi-cellular three-dimensional aggregates using rodent embryonic hippocampal tissue. We contrasted the emergent structural and functional properties of aggregated (3D) and dissociated (2D) cultures over 28 days in vitro (DIV). Hippocampal aggregates displayed robust axonal fasciculation across large distances and significant neuronal polarization, i.e., spatial segregation of dendrites and axons, at earlier time points compared to dissociated cultures. Moreover, we found that astrocytes in aggregate cultures self-organized into non-overlapping quasi-domains and developed highly stellate morphologies resembling astrocyte structures in vivo. We maintained cultures on multi-electrode arrays (MEAs) to assess spontaneous electrophysiological activity for up to 28 DIV. We found that 3D networks of aggregated cultures developed highly synchronized networks and with high burstiness by 28 DIV. We also demonstrated that dual-aggregate networks became active by 7 DIV, in contrast to single-aggregate networks which became active and developed synchronous bursting activity with repeating motifs by 14 DIV. Taken together, our findings demonstrate that the high-density, multi-cellular, 3D microenvironment of hippocampal aggregates supports the recapitulation of emergent biofidelic morphological and functional properties. Our findings suggest that neural aggregates may be used as segregated, modular building blocks for the development of complex, multi-nodal neural network topologies.

Eastern oysters Crassostrea virginica settle near inlets in a lagoonal estuary: spatial and temporal distribution of recruitment in Mid-Atlantic Coastal Bays (Maryland, USA).

Background: Declines of the Eastern oyster, Crassostrea virginica, and its numerous ecological benefits have spurred oyster restoration initiatives. Successful restoration of a self-sustaining oyster population requires evaluating the temporal and spatial patterns of recruitment (settlement and survival) of oyster larvae in the target waterbody. Restoration of the Eastern oyster population in the Maryland Coastal Bays (MCBs), USA, a shallow lagoonal estuary, is of interest to federal, state, and non-governmental, but the location and timing of natural recruitment is not known. Methods: We assessed the spatial and temporal variation in oyster larval recruitment throughout the MCBs using horizontal ceramic tiles and PVC plates. Newly settled oyster larvae (recruits) were monitored biweekly from June to September 2019 and 2020 at 12 sites in the MCBs and a comparison site in Wachapreague, Virginia. Water quality measurements collected included temperature, salinity, dissolved oxygen, pH, and turbidity. The objectives of this study were to determine (1) the most effective substrate and design for monitoring oyster recruitment, (2) the spatial and temporal distribution of oyster larval recruitment in the MCBs, and (3) patterns in oyster larval recruitment that would be applicable to other lagoonal estuaries. Results: (1) Ceramic tiles were more effective than PVC plates for recruiting oyster larvae. (2) Peak settlement began during the period from late June through July, and oyster recruitment was greatest at sites closest to the Ocean City and Chincoteague inlets. (3) Areas near broodstock that have slow flushing rates to retain larvae may provide the best environments for recruitment of oysters to lagoonal estuaries. Discussion: As the first study on oyster larval recruitment in the MCBs, our results provide insight into their spatial and temporal distribution, methods that can serve as a foundation for future recruitment studies in other lagoonal estuaries, and baseline data that can be used to inform stakeholders and evaluate the success of oyster restoration projects in MCBs.

A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum.

Brown rot fungi are primary decomposers of wood and litter in northern forests. Relative to other microbes, these fungi have evolved distinct mechanisms that rapidly depolymerize and metabolize cellulose and hemicellulose without digesting the more recalcitrant lignin. Its efficient degradative system has therefore attracted considerable attention for the development of sustainable biomass conversion technologies. However, there has been a significant lack of genetic tools in brown rot species by which to manipulate genes for both mechanistic studies and engineering applications. To advance brown rot genetic studies, we provided a gene-reporting system that can facilitate genetic manipulations in a model fungus Gloeophyllum trabeum. We first optimized a transformation procedure in G. trabeum, and then transformed the fungus into a constitutive laccase producer with a well-studied white rot laccases gene (from Trametes versicolor). With this, we built a gene reporting system based on laccase gene's expression and its rapid assay using an 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) indicator dye. The laccase reporter system was validated robust enough to allow us to test the effects of donor DNA's formats, protoplast viability, and gene regulatory elements on transformation efficiencies. Going forward, we anticipate the toolset provided in this work would expedite phenotyping studies and genetic engineering of brown rot species. IMPORTANCE One of the most ubiquitous types of decomposers in nature, brown rot fungi, has lacked robust genetic tools by which to manipulate genes and understand its biology. Brown rot fungi are primary decomposers in northern forests helping recycle the encased carbons in trees back to ecosystem. Relative to other microbes, these fungi employ distinctive mechanisms to disrupt and consume the lignified polysaccharides in wood. Its decay mechanism allows fast, selective carbohydrate catabolization, but without digesting lignin-a barren component that produces least energy trade back for fungal metabolisms. Thus, its efficient degradative system provides a great platform for developing sustainable biotechnologies for biomass conversions. However, progress has been hampered by the lack genetic tools facilitating mechanistic studies and engineering applications. Here, the laccase reporter system provides a genetic toolset for genetic manipulations in brown rot species, which we expect would advance relevant genetic studies for discovering and harnessing the unique fungal degradative mechanisms.

Proteome of the Wood Decay Fungus Fomitopsis pinicola Is Altered by Substrate.

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Bioengineering applications for hearing restoration: emerging biologically inspired and biointegrated designs.

Cochlear implantation has become the standard of care for hearing loss not amenable to amplification by bypassing the structures of the cochlea and stimulating the spiral ganglion neurons directly. Since the first single channel electrodes were implanted, significant advancements have been made: multi-channel arrays are now standard, they are softer to avoid damage to the cochlea and pre-curved to better position the electrode array adjacent to the nerve, and surgical and stimulation techniques have helped to conform to the anatomy and physiology of the cochlea. However, even with these advances the experience does not approach that of normal hearing. In order to make significant advances in performance, the next generation of implants will require novel interface technology. Advances in regenerative techniques, optogenetics, piezoelectric materials, and bioengineered living scaffolds hold the promise for the next generation of implantable hearing devices, and hope for the restoration of natural hearing.

A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

Biopreservation of living tissue engineered nerve grafts.

Tissue engineered nerve grafts (TENGs) built from living neurons and aligned axon tracts offer a revolutionary new approach as "living scaffolds" to bridge major peripheral nerve defects. Clinical application, however, necessitates sufficient shelf-life to allow for shipping from manufacturing facility to clinic as well as storage until use. Here, hypothermic storage in commercially available hibernation media is explored as a potential biopreservation strategy for TENGs. After up to 28 days of refrigeration at 4℃, TENGs maintain viability and structure in vitro. Following transplantation into 1 cm rat sciatic defects, biopreserved TENGs routinely survive and persist for at least 2 weeks and recapitulate pro-regenerative mechanisms of fresh TENGs, including the ability to recruit regenerating host tissue into the graft and extend neurites beyond the margins of the graft. The protocols and timelines established here serve as important foundational work for the manufacturing, storage, and translation of other neuron-based tissue engineered therapeutics.

Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives.

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.

The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei.

BACKGROUND: On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes' hydrolytic strength towards the same substrates. RESULTS: Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. CONCLUSION: Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates-namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases-we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

Discrimination of immune cell activation using Raman micro-spectroscopy in an in-vitro & ex-vivo model.

Activation and proliferation of immune cells such as lymphocytes and monocytes are appropriate inflammatory responses to invading pathogens and are key to overcoming an infection. In contrast, uncontrolled and prolonged activation of these cellular signalling pathways can be deleterious to the body and result in the development of autoimmune conditions. The understanding of cellular activatory status therefore plays a significant role in disease diagnosis and progression. Conventional automated approaches such as enzyme linked immunosorbent assays (ELISA) and immune-labelling techniques are time-consuming and expensive, relying on a commercially available and specific antibody to identify cell activation. Developing a label-free method for assessing molecular changes would therefore offer a quick and cost-efficient alternative in biomedical research. Here Raman spectroscopy is presented as an effective spectroscopic method for the identification of activated immune cells using both cell lines and primary cells (including purified monocyte and lymphocyte subgroups and mixed peripheral blood mononuclear cell (PBMC) populations) obtained from healthy donors. All cell lines and primary cells were exposed to different stimulants and cellular responses confirmed by flow cytometry or ELISA. Machine learning models of cell discrimination using Raman spectra were developed and compared to reference flow-cytometry, with spectral discrimination levels comparing favourably with the reference method. Spectral signatures of molecular expression after activation were also extracted with results demonstrating alignment with expected profiles. High performance classification models constructed in these in-vitro and ex-vivo studies enabled identification of the spectroscopic discrimination of immune cell subtypes in their resting and activated state. Further spectral fitting analysis identified a number of potential spectral biomarkers that elucidate the spectral classification.

Vibrational spectroscopy of liquid biopsies for prostate cancer diagnosis.

BACKGROUND: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer. METHODS: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis. RESULTS: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer. CONCLUSIONS: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.

Monitoring Radiotherapeutic Response in Prostate Cancer Patients Using High Throughput FTIR Spectroscopy of Liquid Biopsies.

Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient's quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.

Characterizing the benthic community in Maryland's offshore wind energy areas using a towed camera sled: Developing a method to reduce the effort of image analysis and community description.

Offshore wind farms are a crucial component for the improvement of renewable energy in the United States. The Bureau of Ocean Energy Management (BOEM) designated ~170 km2 of shelf area for wind energy development off the coast of Maryland, USA. In order to understand potential environmental impacts of wind turbine installation on the benthic ecosystem within the designated area, we conducted a study to visually characterize bottom habitats and epibenthic communities in the Mid-Atlantic Outer Continental Shelf blocks of the Maryland wind energy area. Seven 5 km long transects were sampled using a towed camera sled with a downward-facing digital camera that captured images at 5 frames·s-1s. Additional small-mesh beam trawling was also conducted at selected locations complementary for species identification. Image data were analyzed using two image selection methods, random and systematic (i.e. video frames were selected at various intervals). For both methods, estimates of community diversity (Hill's N2) stabilized with sample sizes ranging from 316 to 398 frames. Our results allowed us to define distinct epibenthic communities and bottom habitats that are associated with offshore wind energy sites and to develop a sampling technique for digital images that can be applied to other research programs.