Open-Source Data Analysis Tool for Spectral Small-Angle X-ray Scattering Using Spectroscopic Photon-Counting Detector.

Spectral small-angle X-ray scattering (sSAXS) is a powerful technique for material characterization from thicker samples by capturing elastic X-ray scattering data in angle- and energy-dispersive modes at small angles. This approach is enabled by the use of a 2D spectroscopic photon-counting detector that provides energy and position information of scattered photons when a sample is irradiated by a polychromatic X-ray beam. Here, we describe an open-source tool with a graphical interface for analyzing sSAXS data obtained from a 2D spectroscopic photon-counting detector with a large number of energy bins. The tool takes system geometry parameters and raw detector data to output 1D scattering patterns and a 2D spatially-resolved scattering map in the energy range of interest. We validated these features using data from samples of caffeine powder with well-known scattering peaks. This open-source tool will facilitate sSAXS data analysis for various material characterization applications.

Patient Access to Health Data: A Review of Philosophic and Healthcare Issues.

In 2012 Australia created a national Personal Controlled Electronic Health Record (PCEHR) known as "My Health Record" (MHR). However, MHR has seen low patient utilization. Debate regarding MHR has centered on utility and moral issues (e.g. data privacy). We conducted a narrative review to assess patient perception and clinical utility of PCEHRs worldwide. Results show patient and clinician support for PCEHRs but little evidence of improved outcomes and patient concerns regarding data providence.

Excited State Properties of Point Defects in Semiconductors and Insulators Investigated with Time-Dependent Density Functional Theory.

We present a formulation of spin-conserving and spin-flip hybrid time-dependent density functional theory (TDDFT), including the calculation of analytical forces, which allows for efficient calculations of excited state properties of solid-state systems with hundreds to thousands of atoms. We discuss an implementation on both GPU- and CPU-based architectures along with several acceleration techniques. We then apply our formulation to the study of several point defects in semiconductors and insulators, specifically the negatively charged nitrogen-vacancy and neutral silicon-vacancy centers in diamond, the neutral divacancy center in 4H silicon carbide, and the neutral oxygen-vacancy center in magnesium oxide. Our results highlight the importance of taking into account structural relaxations in excited states in order to interpret and predict optical absorption and emission mechanisms in spin defects.

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVß5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/ß5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVß5 complex docking model. Irisin binds very tightly to an alternative interface on αVß5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

The application of artificial intelligence to accelerate G protein-coupled receptor drug discovery.

The application of artificial intelligence (AI) approaches to drug discovery for G protein-coupled receptors (GPCRs) is a rapidly expanding area. Artificial intelligence can be used at multiple stages during the drug discovery process, from aiding our understanding of the fundamental actions of GPCRs to the discovery of new ligand-GPCR interactions or the prediction of clinical responses. Here, we provide an overview of the concepts behind artificial intelligence, including the subfields of machine learning and deep learning. We summarise the published applications of artificial intelligence to different stages of the GPCR drug discovery process. Finally, we reflect on the benefits and limitations of artificial intelligence and share our vision for the exciting potential for further development of applications to aid GPCR drug discovery. In addition to making the drug discovery process "faster, smarter and cheaper," we anticipate that the application of artificial intelligence will create exciting new opportunities for GPCR drug discovery.

Lumbar Disc Herniation: Diagnosis and Management.

Lumbar disc herniations are common causes of lower back pain, neurological dysfunction, and buttock/leg pain. Herniation refers to the displacement of the nucleus pulposus of the intervertebral disc through the annulus fibrosus, thereby causing pressure on the neural elements. The sequalae of lumbar disc herniations range in severity from mild low back and buttock pain to severe cases of inability to ambulate and cauda equina syndrome. Diagnosis is achieved with a thorough history and physical examination along with advanced imaging. Treatment plans are dictated by corresponding patient symptoms and examination findings with their imaging. Most patients can experience relief with nonsurgical measures. However, if symptoms persist or worsen, surgery may be appropriate.

Lactate regulates cell cycle by remodelling the anaphase promoting complex.

Lactate is abundant in rapidly dividing cells owing to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here we use a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we identify a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodelling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We find that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. This mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient-replete growth phase to stimulate timed opening of APC/C, cell division and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodelling and can overcome anti-mitotic pharmacology via mitotic slippage. In sum, we define a biochemical mechanism through which lactate directly regulates protein function to control the cell cycle and proliferation.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Systematic Assessment of Tumor Purity and Its Clinical Implications.

PURPOSE: The tumor microenvironment is complex, comprising heterogeneous cellular populations. As molecular profiles are frequently generated using bulk tissue sections, they represent an admixture of multiple cell types (including immune, stromal, and cancer cells) interacting with each other. Therefore, these molecular profiles are confounded by signals emanating from many cell types. Accurate assessment of residual cancer cell fraction is crucial for parameterization and interpretation of genomic analyses, as well as for accurately interpreting the clinical properties of the tumor. MATERIALS AND METHODS: To benchmark cancer cell fraction estimation methods, 10 estimators were applied to a clinical cohort of 333 patients with prostate cancer. These methods include gold-standard multiobserver pathology estimates, as well as estimates inferred from genome, epigenome, and transcriptome data. In addition, two methods based on genomic and transcriptomic profiles were used to quantify tumor purity in 4,497 tumors across 12 cancer types. Bulk mRNA and microRNA profiles were subject to in silico deconvolution to estimate cancer cell-specific mRNA and microRNA profiles. RESULTS: We present a systematic comparison of 10 tumor purity estimation methods on a cohort of 333 prostate tumors. We quantify variation among purity estimation methods and demonstrate how this influences interpretation of clinico-genomic analyses. Our data show poor concordance between pathologic and molecular purity estimates, necessitating caution when interpreting molecular results. Limited concordance between DNA- and mRNA-derived purity estimates remained a general pan-cancer phenomenon when tested in an additional 4,497 tumors spanning 12 cancer types. CONCLUSION: The choice of tumor purity estimation method may have a profound impact on the interpretation of genomic assays. Taken together, these data highlight the need for improved assessment of tumor purity and quantitation of its influences on the molecular hallmarks of cancers.

Bottom-Up Construction of Electrochemically Active Living Filters: From Graphene Oxide Mediated Formation of Bacterial Cables to 3D Assembly of Hierarchical Architectures.

Living composites comprising of both biotic and abiotic modules are shifting the paradigm of materials science, yet challenges remain in effectively converging their distinctive structural and functional attributes. Here we present a bottom-up hybridization strategy to construct functionally coherent, electrochemically active biohybrids with optimal mass/charge transport, mechanical integrity, and biocatalytic performance. This biohybrid can overcome several key limitations of traditional biocarrier designs and demonstrate superior efficiency in metabolizing low-concentration toxic ions with minimal environmental impact. Overall, this work exemplifies a biointegration strategy that complements existing synthetic biology toolsets to further expand the range of material attributes and functionalities.

Resolving the full spectrum of human genome variation using Linked-Reads.

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2 Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer.

Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture.

Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of ΔF508-cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR).

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.

Characterization of HPV and host genome interactions in primary head and neck cancers.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome.

Aneuploidy has been recognized as a hallmark of cancer for more than 100 years, yet no general theory to explain the recurring patterns of aneuploidy in cancer has emerged. Here, we develop Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method that analyzes the patterns of mutational signatures in tumors and predicts the likelihood that any individual gene functions as a tumor suppressor (TSG) or oncogene (OG). By analyzing >8,200 tumor-normal pairs, we provide statistical evidence suggesting that many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. Integrating our driver predictions with information on somatic copy number alterations, we find that the distribution and potency of TSGs (STOP genes), OGs, and essential genes (GO genes) on chromosomes can predict the complex patterns of aneuploidy and copy number variation characteristic of cancer genomes. We propose that the cancer genome is shaped through a process of cumulative haploinsufficiency and triplosensitivity.

Method for non-optical quantification of in situ local soft tissue biomechanics.

Soft tissues exhibit significant biomechanical changes as they grow, adapt, and remodel under a variety of normal and pathogenic stimuli. Biomechanical measurement of intact soft tissues is challenging because of its large strain and nonlinear behavior. Tissue distention through applied vacuum pressure is an attractive method for acquiring local biomechanical information minimally invasive and non-destructive, but the current requirement for optical strain measurement limits its use. In this study, we implemented a novel flexible micro-electrode array placed within a cylindrical probe tip. We hypothesized that upon tissue distention, contact with each electrode would result in a precipitous voltage drop (from the resistive connection formed between input and output electrodes) across the array. Hence, tissue distention (strain) can be derived directly from the electrode array geometry. In pilot studies, we compared the electrode array measurements directly against optical deformation measurements in-situ of agar tissue phantoms and freshly isolated porcine tissue. Our results demonstrate that the probe derived stress-strain profiles and modulus measurements were statistically indistinguishable from optical measurement. We further show that electrode geometry can be scaled down to 50µm in size (length and width) and spaced 50µm apart without impairing measurement accuracy. These results establish a promising new method for minimally invasive local soft tissue biomechanical measurement, which may be useful for applications such as disease diagnosis and health monitoring.