Bulk brain tissue cell-type deconvolution with bias correction for single-nuclei RNA sequencing data using DeTREM.

BACKGROUND: Quantifying cell-type abundance in bulk tissue RNA-sequencing enables researchers to better understand complex systems. Newer deconvolution methodologies, such as MuSiC, use cell-type signatures derived from single-cell RNA-sequencing (scRNA-seq) data to make these calculations. Single-nuclei RNA-sequencing (snRNA-seq) reference data can be used instead of scRNA-seq data for tissues such as human brain where single-cell data are difficult to obtain, but accuracy suffers due to sequencing differences between the technologies. RESULTS: We propose a modification to MuSiC entitled 'DeTREM' which compensates for sequencing differences between the cell-type signature and bulk RNA-seq datasets in order to better predict cell-type fractions. We show DeTREM to be more accurate than MuSiC in simulated and real human brain bulk RNA-sequencing datasets with various cell-type abundance estimates. We also compare DeTREM to SCDC and CIBERSORTx, two recent deconvolution methods that use scRNA-seq cell-type signatures. We find that they perform well in simulated data but produce less accurate results than DeTREM when used to deconvolute human brain data. CONCLUSION: DeTREM improves the deconvolution accuracy of MuSiC and outperforms other deconvolution methods when applied to snRNA-seq data. DeTREM enables accurate cell-type deconvolution in situations where scRNA-seq data are not available. This modification improves characterization cell-type specific effects in brain tissue and identification of cell-type abundance differences under various conditions.

Dispersant-First Dispersive Liquid-Liquid Microextraction (DF-DLLME), a Novel Sample Preparation Procedure for NDMA Determination in Metformin Products.

Since December 2019, global batch recalls of metformin pharmaceutical products have highlighted an urgent need to control N-nitrosodimethylamine (NDMA) contamination to demonstrate patient safety and maintain supply of this essential medicine. Due to their formulation, the metformin extended-release products present difficult analytical challenges for conventional sample preparation procedures, such as artefactual (in-situ) NDMA formation, gelling, and precipitation. To overcome these challenges, a new version of dispersive liquid-liquid microextraction (DLLME) termed dispersant-first DLLME (DF-DLLME) was developed and optimized for the analysis of NDMA in metformin extended-release products using a detailed Design of Experiments (DoE) to optimize sample preparation. Gas chromatography-high resolution accurate mass-mass spectrometry (GC-HRAM-MS) combined with automated DF-DLLME were successfully applied to monitor the NDMA levels of two different metformin extended-release AstraZeneca products to ultra-trace levels (parts per billion). The additional benefits associated with DF-DLLME, which include automation, time/costs saving, and greener sample preparation, make this novel technique easier to transfer from a development to Quality Control (QC) environment. In addition, this also offers an attractive candidate for the wider platform analysis of N-nitrosamines in pharmaceutical drug products.

Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model.

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte assembloids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 is used to address the putative dysfunctional HSP chaperone system and produces a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

Molecular Quantitative Trait Locus Mapping in Human Complex Diseases.

Mapping quantitative trait loci (QTLs) for molecular traits from chromatin to metabolites (i.e., xQTLs) provides insight into the locations and effect modes of genetic variants that influence these molecular phenotypes and the propagation of functional consequences of each variant. xQTL studies indirectly interrogate the functional landscape of the molecular basis of complex diseases, including the impact of non-coding regulatory variants, the tissue specificity of regulatory elements, and their contribution to disease by integrating with genome-wide association studies (GWAS). We summarize a variety of molecular xQTL studies in human tissues and cells. In addition, using the Alzheimer's Disease Sequencing Project (ADSP) as an example, we describe the ADSP xQTL project, a collaborative effort across the ADSP Functional Genomics Consortium (ADSP-FGC). The project's ultimate goal is a reference map of Alzheimer's-related QTLs using existing datasets from multiple omics layers to help us study the consequences of genetic variants identified in the ADSP. xQTL studies enable the identification of the causal genes and pathways in GWAS loci, which will likely aid in the discovery of novel biomarkers and therapeutic targets for complex diseases. © 2022 Wiley Periodicals LLC.

NDMA analytics in metformin products: Comparison of methods and pitfalls.

BACKGROUND: For nearly three years, the concerns regarding trace levels of N-nitrosamines in pharmaceuticals and the associated cancer risk have significantly expanded and are a major issue facing the global pharmaceutical industry. N-nitrosodimethylamine (NDMA) found in formulations of the popular anti-diabetic drug metformin is a prominent example. This has resulted in product recalls raising the profile within the media. Issues of method robustness, sample preparation and several unexpected sources of nitrosamine contamination have been highlighted as false positive risks. It has become apparent that the identification of the root causes of artefactual formation of nitrosamines must be identified to mitigate risk associated with the analysis. METHODS: A comparison study between four laboratories, across three companies was designed, employing orthogonal mass spectrometric methods for the quantification of NDMA in two metformin immediate release (IR) formulations and one extended release (XR) formulation. These were 2x LC-MS/MS, GC-MS/MS and GC-HRMS. RESULTS: Good agreement of results was obtained for the IR formulations. However, we measured higher concentrations of NDMA in the XR formulation using GC-MS/MS compared to LC-MS/MS. We could show that this was due to artefactual (in situ) formation of NDMA when samples were extracted with dichloromethane. Removal of dimethylamine (DMA) and nitrite from the extracted sample or the addition of a nitrosation scavenger are shown to be effective remedies. NDMA in situ formation was not observed in 10% MeOH or acetonitrile. CONCLUSION: Metformin pharmaceuticals contain traces of the API impurity DMA as well as inorganic nitrite from excipients. This can lead to artefactual formation of NDMA and hence false positive results if DCM is used for sample extraction. Similar artefacts are likely also in other pharmaceuticals if these contain the secondary amine precursor of the respective nitrosamine analyte.

Phenotypic Screening Following Transcriptomic Deconvolution to Identify Transcription Factors Mediating Axon Growth Induced by a Kinase Inhibitor.

After injury to the central nervous system (CNS), both neuron-intrinsic limitations on regenerative responses and inhibitory factors in the injured CNS environment restrict regenerative axon growth. Instances of successful axon regrowth offer opportunities to identify features that differentiate these situations from that of the normal adult CNS. One such opportunity is provided by the kinase inhibitor RO48, which dramatically enhances neurite outgrowth of neurons in vitro and substantially increased contralateral sprouting of corticospinal tract neurons when infused intraventricularly following unilateral pyramidotomy. The authors present here a transcriptomic deconvolution of RO48-associated axon growth, with the goal of identifying transcriptional regulators associated with axon growth in the CNS. Through the use of RNA sequencing (RNA-seq) and transcription factor binding site enrichment analysis, the authors identified a list of transcription factors putatively driving differential gene expression during RO48-induced neurite outgrowth of rat hippocampal neurons in vitro. The 82 transcription factor motifs identified in this way included some with known association to axon growth regulation, such as Jun, Klf4, Myc, Atf4, Stat3, and Nfatc2, and many with no known association to axon growth. A phenotypic loss-of-function screen was carried out to evaluate these transcription factors for their roles in neurite outgrowth; this screen identified several potential outgrowth regulators. Subsequent validation suggests that the Forkhead box (Fox) family transcription factor Foxp2 restricts neurite outgrowth, while FoxO subfamily members Foxo1 and Foxo3a promote neurite outgrowth. The authors' combined transcriptomic-phenotypic screening strategy therefore allowed identification of novel transcriptional regulators of neurite outgrowth downstream of a multitarget kinase inhibitor.

A modified total equivalent warming impact analysis: Addressing direct and indirect emissions due to corrosion.

A global rise in HVAC-R utilization requires a deeper understanding of the industry's effect on electricity consumptions and greenhouse gas emissions. The Total Equivalent Warming Impact (TEWI) methodology was designed to analyze emissions from direct release of refrigerant and indirect emissions through electricity consumption of HVAC-R systems to increase the understanding of system design on emissions, and to guide refrigerant replacement. However, the original TEWI calculation neglects the system degradation due to corrosion. This paper studies on the impact of corrosion and highlights how the original TEWI method underrepresents the lifetime emissions due to energy efficiency decrease and refrigerant release. Corrosion impacts direct emissions by increasing refrigerant leakage rates over time and indirect emissions through heat exchanger efficiency degradation and suboptimal refrigerant level. A modified TEWI equation is proposed to capture the dynamic corrosion impacts over the lifetime of HVAC operations. Three scenarios (low corrosivity, conservative and moderate corrosivity) are examined to analyze different corrosion environments. This analysis indicates 6%-27% increase in TEWI emissions based on a typical residential air conditioner (AC), when the impacts of corrosion are included, with the greatest emissions increase from reduced electrical efficiency. The impact of several current and future corrosion protection scenarios on TEWI are also included. Appropriate corrosion mitigation can reduce total lifecycle emissions of systems by 6% ~ 10%. The proposed modified TEWI method is expected to provide a more accurate emission estimation for AC sustainability and policy making.

Midline Longitudinal Sacral Fracture in an Anterior-Posterior Compression Pelvic Injury -A Surgical Decision-making and Outcome.

INTRODUCTION: Anterior-posterior compression pelvic injuries occur from high-energy blunt trauma and can result in devastating outcomes. Often, widening of the pubic symphysis occurs with subsequent disruption of posterior pelvic ligamentous structures. Associated sacral fractures may increase the likelihood of neurologic injury and pelvic ring instability. The most common sacral fracture in this injury is an avulsion fracture of the sacral ala. Midline longitudinal sacral fractures into the spinal canal are an extremely rare variant of sacral injuries in conjunction with pelvic trauma. In contrast to traditional anterior-posterior pelvic ring injuries, those associated with this unique fracture type carry a decreased risk of neurologic injury. Only a small number of these cases exist in English literature. Given its rarity, further, investigation of the fracture mechanism and treatment protocol is warranted. CASE REPORT: A 67-year-old Caucasian male presented with an anterior-posterior compression pelvic ring injury after he was struck by an oncoming vehicle on his motorcycle. Radiographs revealed pubic symphyseal widening of 4.7 cm. A pelvic binder was placed as a temporary measure to minimize intrapelvic hemorrhage. Advanced imaging revealed a complete, midline sagittal sacral fracture through the posterior sacral elements. During his hospital admission, the patient required multiple procedures for sustained upper extremity fractures and subsequently underwent surgical fixation of his anterior and posterior pelvic ring injuries 2 days after admission. Our patient to date has achieved sacral fracture union, minimal residual pubic diastasis, and has no evidence of neurologic injury. He has some persistent impotence and is ambulating without assistance. Despite his significant injuries, his outcome to date has been quite impressive. CONCLUSION: It is critical to always examine the posterior pelvis and sacrum when examining anterior-posterior compression injuries. While most cases have reported fixation of only the anterior pelvis, we explain the need for additional posterior pelvic fixation to address the inherent instability of these unique sacral fractures and ultimately decrease patient morbidity. The surgical fixation technique of this patient's pelvic injuries is described, as well as his post-operative course and outcome to date.

Extensive Recovery of Embryonic Enhancer and Gene Memory Stored in Hypomethylated Enhancer DNA.

Developing and adult tissues use different cis-regulatory elements. Although DNA at some decommissioned embryonic enhancers is hypomethylated in adult cells, it is unknown whether this putative epigenetic memory is complete and recoverable. We find that, in adult mouse cells, hypomethylated CpG dinucleotides preserve a nearly complete archive of tissue-specific developmental enhancers. Sites that carry the active histone mark H3K4me1, and are therefore considered "primed," are mainly cis elements that act late in organogenesis. In contrast, sites decommissioned early in development retain hypomethylated DNA as a singular property. In adult intestinal and blood cells, sustained absence of polycomb repressive complex 2 indirectly reactivates most-and only-hypomethylated developmental enhancers. Embryonic and fetal transcriptional programs re-emerge as a result, in reverse chronology to cis element inactivation during development. Thus, hypomethylated DNA in adult cells preserves a "fossil record" of tissue-specific developmental enhancers, stably marking decommissioned sites and enabling recovery of this epigenetic memory.

Anatomic Evaluation of the Quadriceps Tendon in Cadaveric Specimens: Application for Anterior Cruciate Ligament Reconstruction Graft Choice.

PURPOSE: The quadriceps tendon (QT) is an autograft option for primary and revision anterior cruciate ligament (ACL) reconstruction. Techniques for predicting the appropriate graft size are limited. The purpose of this study was to evaluate the morphologic features of the QT in cadaveric specimens and compare the findings to recent MRI studies. MATERIALS AND METHODS: Macroscopic dissections were performed on 10 cadaveric knees. Using the distal myotendinous junction of the rectus femoris and superior pole of the patella as anatomic landmarks, the QT was isolated. Tendon length, width, and thickness were recorded at 10 millimeter (mm) increments. A central 80 mm x 10 mm graft was harvested, after which the graft was measured in an identical fashion. Specimen anthropometric data was collected. Subgroup analysis and linear regression were then performed using Microsoft Excel 2011 Office Analysis ToolPak. RESULTS: The mean QT length was 83.3 +/- 14.4 mm, ranging from 63 to 108 mm. The mean percentage of remaining QT volume following graft harvesting was 63.3%. QT length showed significant correlation with patient height (correlation coefficient: 0.719, p = 0.027). QT thickness remained relatively constant, while the width is greatest at its patellar insertion and gradually decreases proximally towards the myotendinous junction. CONCLUSION: The QT has the anatomical features to produce a robust autograft for ACL reconstruction. During preoperative evaluation of graft size and quality, patient height should be considered as it is strongly correlated with the length of the potential graft. Our findings support the use of MRI as a way to preoperatively assess the QT as an autograft when performing an ACL reconstruction.

Radiation Dose Reduction of Computed Tomography in Complex Distal Femur Fractures: A Cadaver Study to Develop a Low Dose Scanning Protocol.

CONTEXT: Recent advances in diagnostic imaging have made computed tomography (CT) a widely used test in trauma patients. Consequently, the collective radiation burdened sustained by this patient population has increased substantially. The purpose of this cadaveric study was to determine if a significantly lowered CT radiation dose protocol would provide adequate imaging studies for the surgeon, using the distal femur as a model. METHODS: Ten adult cadaveric knee specimens were used to create Orthopaedic Trauma Association (AO/OTA) 33-C3 distal femur fractures with associated coronal plane Hoffa fractures and varying intra-articular displacements. Using a single CT scanner, each cadaver was scanned at 5 separate protocols defined by sequentially lowered radiation doses, the highest of which was one-third the value of our institution's current protocol. These images were then evaluated by fellowship-trained orthopedic surgeons, an orthopedic trauma fellow, and residents. Observer reliability and confidence levels were calculated for measuring fracture displacement, assessing the quality of 3D reconstructions, and developing treatment plans. RESULTS: Across all reviewers and specimens, there was an average difference of 0.66 millimeters (mm) between the measured fracture gap and true fracture gap. The highest intraclass correlation coefficient (ICC) calculated for the inter-rater reliability of gap measurements was 0.983 at 75 mAs (95% CI: 0.955-0.996), followed by 0.973 (95% CI: 0.930-0.993) and 0.958 (95% CI: 0.896-0.988) at 15 mAs and 60 mAs, respectively. All 3D reconstructions obtained at 75 mAs and 45 mAs values (N = 8) were of acceptable imaging quality to all reviewers, while only 3 of 4 3D reconstructions obtained at 15 mAs were considered acceptable. There was no difference in treatment plans across all reviewers, regardless of radiation dose. CONCLUSIONS: In summary, our results indicate that CT scans of complex distal femur fractures at one-third the amount of radiation exposure may provide adequate imaging necessary to develop an appropriate treatment plan. At significantly lowered doses, the reviewers were able to accurately measure the amount of fracture displacement and identify the presence of each Hoffa fracture. Future studies are necessary to compare this protocol's diagnostic capacity and limitations in evaluating complex fractures with that of our institution's standard protocol in a clinical setting.

Enhancer, transcriptional, and cell fate plasticity precedes intestinal determination during endoderm development.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

RORα-expressing T regulatory cells restrain allergic skin inflammation.

Atopic dermatitis is an allergic inflammatory skin disease characterized by the production of the type 2 cytokines in the skin by type 2 innate lymphoid cells (ILC2s) and T helper 2 (TH2) cells, and tissue eosinophilia. Using two distinct mouse models of atopic dermatitis, we show that expression of retinoid-related orphan receptor α (RORα) in skin-resident T regulatory cells (Tregs) is important for restraining allergic skin inflammation. In both models, targeted deletion of RORα in mouse Tregs led to exaggerated eosinophilia driven by interleukin-5 (IL-5) production by ILC2s and TH2 cells. Expression of RORα in skin-resident Tregs suppressed IL-4 expression and enhanced expression of death receptor 3 (DR3), which is the receptor for tumor necrosis factor (TNF) family cytokine, TNF ligand-related molecule 1 (TL1A), which promotes Treg functions. DR3 is expressed on both ILC2s and skin-resident Tregs Upon deletion of RORα in skin-resident Tregs, we found that Tregs were no longer able to sequester TL1A, resulting in enhanced ILC2 activation. We also documented higher expression of RORα in skin-resident Tregs than in peripheral blood circulating Tregs in humans, suggesting that RORα and the TL1A-DR3 circuit could be therapeutically targeted in atopic dermatitis.

Single incision pediatric flexible intramedullary tibial nailing.

BACKGROUND: There has been a trend towards flexible intramedullary nailing for unstable tibial shaft fractures in the pediatric population, traditionally, utilizing a 2-incision technique with passage of one nail medially and one nail laterally. Our study aims to compare a single incision approach for flexible nailing of unstable tibial shaft fractures in pediatric patients to the traditional 2-incision approach. METHODS: Patients were selected for operative fixation if they had a length unstable tibial shaft fracture confirmed by fluoroscopy. Exclusion criteria included length stable tibial fractures that could undergo nonoperative treatment. Single incision technique utilized the medial incision only. Patients were monitored in the hospital for one postoperative day and followed up at 4 week, 8 week, and 12 week marks. Radiographic analysis was performed to evaluate for malunion or nonunion. Operative times, infection rates and complications were recorded and analyzed. RESULTS: All patients achieved complete fracture healing at the 12-week follow up. There were no delayed unions, nonunions or malunions in either treatment group. CONCLUSIONS: Single medial incision for tibial flexible nails had equivalent outcomes with no difference in primary healing rate, malunion or nonunion rate when compared to the dual incision technique.

Dynamic Reorganization of Chromatin Accessibility Signatures during Dedifferentiation of Secretory Precursors into Lgr5+ Intestinal Stem Cells.

Replicating Lgr5+ stem cells and quiescent Bmi1+ cells behave as intestinal stem cells (ISCs) in vivo. Disrupting Lgr5+ ISCs triggers epithelial renewal from Bmi1+ cells, from secretory or absorptive progenitors, and from Paneth cell precursors, revealing a high degree of plasticity within intestinal crypts. Here, we show that GFP+ cells from Bmi1GFP mice are preterminal enteroendocrine cells and we identify CD69+CD274+ cells as related goblet cell precursors. Upon loss of native Lgr5+ ISCs, both populations revert toward an Lgr5+ cell identity. While active histone marks are distributed similarly between Lgr5+ ISCs and progenitors of both major lineages, thousands of cis elements that control expression of lineage-restricted genes are selectively open in secretory cells. This accessibility signature dynamically converts to that of Lgr5+ ISCs during crypt regeneration. Beyond establishing the nature of Bmi1GFP+ cells, these findings reveal how chromatin status underlies intestinal cell diversity and dedifferentiation to restore ISC function and intestinal homeostasis.

Transcription factor-dependent 'anti-repressive' mammalian enhancers exclude H3K27me3 from extended genomic domains.

Compacted chromatin and nucleosomes are known barriers to gene expression; the nature and relative importance of other transcriptional constraints remain unclear, especially at distant enhancers. Polycomb repressor complex 2 (PRC2) places the histone mark H3K27me3 predominantly at promoters, where its silencing activity is well documented. In adult tissues, enhancers lack H3K27me3, and it is unknown whether intergenic H3K27me3 deposits affect nearby genes. In primary intestinal villus cells, we identified hundreds of tissue-restricted enhancers that require the transcription factor (TF) CDX2 to prevent the incursion of H3K27me3 from adjoining areas of elevated basal marking into large well-demarcated genome domains. Similarly, GATA1-dependent enhancers exclude H3K27me3 from extended regions in erythroid blood cells. Excess intergenic H3K27me3 in both TF-deficient tissues is associated with extreme mRNA deficits, which are significantly rescued in intestinal cells lacking PRC2. Explaining these observations, enhancers show TF-dependent binding of the H3K27 demethylase KDM6A. Thus, in diverse cell types, certain genome regions far from promoters accumulate H3K27me3, and optimal gene expression depends on enhancers clearing this repressive mark. These findings reveal new "anti-repressive" function for hundreds of tissue-specific enhancers.

Acquired Tissue-Specific Promoter Bivalency Is a Basis for PRC2 Necessity in Adult Cells.

Bivalent promoters in embryonic stem cells (ESCs) carry methylation marks on two lysine residues, K4 and K27, in histone3 (H3). K4me2/3 is generally considered to promote transcription, and Polycomb Repressive Complex 2 (PRC2) places K27me3, which is erased at lineage-restricted genes when ESCs differentiate in culture. Molecular defects in various PRC2 null adult tissues lack a unifying explanation. We found that epigenomes in adult mouse intestine and other self-renewing tissues show fewer and distinct bivalent promoters compared to ESCs. Groups of tissue-specific genes that carry bivalent marks are repressed, despite the presence of promoter H3K4me2/3. These are the predominant genes de-repressed in PRC2-deficient adult cells, where aberrant expression is proportional to the H3K4me2/3 levels observed at their promoters in wild-type cells. Thus, in adult animals, PRC2 specifically represses genes with acquired, tissue-restricted promoter bivalency. These findings provide new insights into specificity in chromatin-based gene regulation.

Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

Multilayer films assembled from naturally-derived materials for controlled protein release.

Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(ß-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.

Detection of novel polyomaviruses, TSPyV, HPyV6, HPyV7, HPyV9 and MWPyV in feces, urine, blood, respiratory swabs and cerebrospinal fluid.

Eight novel human polyomaviruses have been discovered since 2007. Prevalence rates and tissue tropism for the most recent members HPyV 6, 7, 9, TSPyV and MWPyV are largely unknown. We used real-time PCR to determine the presence of HPyV 6, 7, 9, TSPyV and MWPyV in feces (n = 263), urine (n = 189), blood (n = 161), respiratory swabs (n = 1385) and cerebrospinal fluid (n = 171) from both healthy control children and children and adults undergoing diagnostic testing. Whole genome sequencing was able to be performed on 9 MWPyV positive specimens. Novel polyomaviruses were only detected in respiratory swabs and feces, with no detections of HPyV 9 in any sample type. MWPyV was found to be the most prevalent novel polyomavirus, being detected in 18 (1.5%) respiratory specimens from symptomatic patients, 16 (9.8%) respiratory sample from healthy control children, 11 (5.9%) fecal specimens from patient suffering gastrointestinal illness, and in 13 (15.3%) of feces from healthy control children. MWPyV was found only in respiratory and fecal specimens from children, the oldest being 9 years old. HPyV 6, 7, 9 and TSPyV were also detected in respiratory specimens and fecal specimens at low prevalence (<1.3%). The majority of these detections were found in immunocompromised patients. Our findings suggest that MWPyV can result in a subclinical infection, persistent or intermittent shedding, particularly in young children. The other novel polyomaviruses were also found in respiratory and fecal specimens, but at lower prevalence and most commonly in immunocompromised individuals.