Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen.

The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.

Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines.

Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.

The PRRA insert at the S1/S2 site modulates cellular tropism of SARS-CoV-2 and ACE2 usage by the closely related Bat RaTG13.

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

Optimization of O-GIG for O-Glycopeptide Characterization with Sialic Acid Linkage Determination.

O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O-glycans. Moreover, chemical releasing methods, such as ß-elimination/Michael addition, are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived from intestinal bacteria and expressed in Escherichia coli, has become commercially available. The digestion of O-glycoprotein yields O-glycopeptides cleaved at the N-terminal end of serine and threonine, with O-glycan remaining intact. The enzyme has a broad substrate specificity and includes mammalian cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins, and it has been suggested that this acidic residue be removed prior to digestion, thus sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, varying pH, sialic acid modification, and digestion temperature, in order to optimize the enzymatic activity, with a special emphasis on sialylated glycosites. Conditions derived in this work facilitate the OpeRATOR digestion of fully sialylated O-glycopeptides that are mass tagged to identify the sialyl linkage, thus facilitating the analysis of these charged O-glycopeptides, which are often important in biological processes.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Novel reference genes in colorectal cancer identify a distinct subset of high stage tumors and their associated histologically normal colonic tissues.

BACKGROUND: Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls METHODS: Because Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 "classical" reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs. RESULTS: We affirmed that 17 out of 21 classical reference genes had upregulated expression in tumors compared to normal colonic epithelial tissue and dramatically so in some cases. Indeed, tumors were distinguished from normal controls in both unsupervised hierarchical clustering analyses (HCA) and principal component analyses (PCA). We then identified 42 novel potential reference genes with minimal coefficients of variation (CV) across 79 CRC tumor pairs. Though largely consistently expressed across tumors and normal control tissues, a subset of high stage tumors (HSTs) as well as some normal tissue samples (HSNs) located adjacent to these HSTs demonstrated dysregulated expression, thus identifying a subset of tumors with a potentially distinct and aggressive biological profile. CONCLUSION: While classical CRC reference genes were found to be differentially expressed between tumors and normal controls, novel reference genes, identified via NGS, were more consistently expressed across malignant and normal colonic tissues. Nonetheless, a subset of HST had profound dysregulation of such genes as did many of the histologically normal tissues adjacent to such HSTs, indicating that the HSTs so distinguished may have unique biological properties and that their histologically normal tissues likely harbor a small population of microscopically undetected but metabolically active tumors.

Altered learning, memory, and social behavior in type 1 taste receptor subunit 3 knock-out mice are associated with neuronal dysfunction.

The type 1 taste receptor member 3 (T1R3) is a G protein-coupled receptor involved in sweet-taste perception. Besides the tongue, the T1R3 receptor is highly expressed in brain areas implicated in cognition, including the hippocampus and cortex. As cognitive decline is often preceded by significant metabolic or endocrinological dysfunctions regulated by the sweet-taste perception system, we hypothesized that a disruption of the sweet-taste perception in the brain could have a key role in the development of cognitive dysfunction. To assess the importance of the sweet-taste receptors in the brain, we conducted transcriptomic and proteomic analyses of cortical and hippocampal tissues isolated from T1R3 knock-out (T1R3KO) mice. The effect of an impaired sweet-taste perception system on cognition functions were examined by analyzing synaptic integrity and performing animal behavior on T1R3KO mice. Although T1R3KO mice did not present a metabolically disrupted phenotype, bioinformatic interpretation of the high-dimensionality data indicated a strong neurodegenerative signature associated with significant alterations in pathways involved in neuritogenesis, dendritic growth, and synaptogenesis. Furthermore, a significantly reduced dendritic spine density was observed in T1R3KO mice together with alterations in learning and memory functions as well as sociability deficits. Taken together our data suggest that the sweet-taste receptor system plays an important neurotrophic role in the extralingual central nervous tissue that underpins synaptic function, memory acquisition, and social behavior.

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

BACKGROUND: Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. RESULTS: Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. CONCLUSIONS: A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

Deciphering Protein O-Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment.

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on O-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of O-glycans. Although O-glycosidase can catalyze the removal of core 1 and core 3 O-linked disaccharides from glycoproteins, analysis of other types of O-glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available O-protease and solid phase platform. This method enables the assignment of O-glycosylated peptides, N-glycan profile, sialyl O-glycopeptides linkage, and mapping of heterogeneous O-glycosylation. For the first time, we can analyze intact O-glycopeptides generated by O-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known O-glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant O-glycosites and setting a basis set of O-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein O-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where O-glycosylation is central.

3D brain Organoids derived from pluripotent stem cells: promising experimental models for brain development and neurodegenerative disorders.

Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), appear to recapitulate the brain's 3D cytoarchitectural arrangement and provide new opportunities to explore disease pathogenesis in the human brain. Human iPSC (hiPSC) reprogramming methods, combined with 3D brain organoid tools, may allow patient-derived organoids to serve as a preclinical platform to bridge the translational gap between animal models and human clinical trials. Studies using patient-derived brain organoids have already revealed novel insights into molecular and genetic mechanisms of certain complex human neurological disorders such as microcephaly, autism, and Alzheimer's disease. Furthermore, the combination of hiPSC technology and small-molecule high-throughput screening (HTS) facilitates the development of novel pharmacotherapeutic strategies, while transcriptome sequencing enables the transcriptional profiling of patient-derived brain organoids. Finally, the addition of CRISPR/Cas9 genome editing provides incredible potential for personalized cell replacement therapy with genetically corrected hiPSCs. This review describes the history and current state of 3D brain organoid differentiation strategies, a survey of applications of organoids towards studies of neurodevelopmental and neurodegenerative disorders, and the challenges associated with their use as in vitro models of neurological disorders.

Complex Nature of Protein Carbonylation Specificity After Metal-Catalyzed Oxidation.

PURPOSE: Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood. METHODS: In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry. RESULTS: Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45. CONCLUSIONS: Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.

Cost and cost-effectiveness of tuberculosis treatment shortening: a model-based analysis.

BACKGROUND: Despite improvements in treatment success rates for tuberculosis (TB), current six-month regimen duration remains a challenge for many National TB Programmes, health systems, and patients. There is increasing investment in the development of shortened regimens with a number of candidates in phase 3 trials. METHODS: We developed an individual-based decision analytic model to assess the cost-effectiveness of a hypothetical four-month regimen for first-line treatment of TB, assuming non-inferiority to current regimens of six-month duration. The model was populated using extensive, empirically-collected data to estimate the economic impact on both health systems and patients of regimen shortening for first-line TB treatment in South Africa, Brazil, Bangladesh, and Tanzania. We explicitly considered 'real world' constraints such as sub-optimal guideline adherence. RESULTS: From a societal perspective, a shortened regimen, priced at USD1 per day, could be a cost-saving option in South Africa, Brazil, and Tanzania, but would not be cost-effective in Bangladesh when compared to one gross domestic product (GDP) per capita. Incorporating 'real world' constraints reduces cost-effectiveness. Patient-incurred costs could be reduced in all settings. From a health service perspective, increased drug costs need to be balanced against decreased delivery costs. The new regimen would remain a cost-effective option, when compared to each countries' GDP per capita, even if new drugs cost up to USD7.5 and USD53.8 per day in South Africa and Brazil; this threshold was above USD1 in Tanzania and under USD1 in Bangladesh. CONCLUSION: Reducing the duration of first-line TB treatment has the potential for substantial economic gains from a patient perspective. The potential economic gains for health services may also be important, but will be context-specific and dependent on the appropriate pricing of any new regimen.

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

BACKGROUND: Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. METHODS: Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. RESULTS: Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. CONCLUSIONS: The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer.

Personalized therapy for breast cancer.

Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies.

The intersection of TB and health financing: defining needs and opportunities.

TB is an airborne public health threat, so the reponse to TB has been defined mainly through the lens of vertical, public-sector national TB programs (NTPs). However, TB exists within a broader health systems and health financing context. Here, we examine the intersection between the particular needs of TB programs and the broader health financing landscape. This includes the strategies needed to finance both the clinical and public health aspects of the TB response. In high-burden countries, the resource mobilization and strategic purchasing approaches described here will be critical if we are to maximize the reach and impact of the TB response.

La TB est une menace de santé publique transmise par l'air, de sorte que la réponse à la TB a été définie principalement à travers le prisme des programmes nationaux de lutte contre la tuberculose (NTP), verticaux et relevant du secteur public. Cependant, la TB s'inscrit dans un contexte plus large de systèmes de santé et de financement de la santé. Nous examinons ici l'intersection entre les besoins particuliers des NTP et le paysage plus large du financement de la santé. Il s'agit notamment des stratégies nécessaires pour financer les aspects cliniques et de santé publique de la lutte contre la TB. Dans les pays à forte charge de morbidité, les approches de mobilisation des ressources et d'achat stratégique décrites ici seront essentielles si nous voulons maximiser la portée et l'impact de la lutte contre la TB.

VESCL: an open source 2D vessel contouring library.

PURPOSE: VESCL (pronounced 'vessel') is a novel vessel contouring library for computer-assisted 2D vessel contouring and segmentation. VESCL facilitates manual vessel segmentation in 2D medical images to generate gold-standard datasets for training, testing, and validating automatic vessel segmentation. METHODS: VESCL is an open-source C++ library designed for easy integration into medical image processing systems. VESCL provides an intuitive interface for drawing variable-width parametric curves along vessels in 2D images. It includes highly optimized localized filtering to automatically fit drawn curves to the nearest vessel centerline and automatically determine the varying vessel width along each curve. To support a variety of segmentation paradigms, VESCL can export multiple segmentation representations including binary segmentations, occupancy maps, and distance fields. RESULTS: VESCL provides sub-pixel resolution for vessel centerlines and vessel widths. It is optimized to segment small vessels with single- or sub-pixel widths that are visible to the human eye but hard to segment automatically via conventional filters. When tested on neurovascular digital subtraction angiography (DSA), VESCL's intuitive hand-drawn input with automatic curve fitting increased the speed of fully manual segmentation by 22× over conventional methods and by 3× over the best publicly available computer-assisted manual segmentation method. Accuracy was shown to be within the range of inter-operator variability of gold standard manually segmented data from a publicly available dataset of neurovascular DSA images as measured using Dice scores. Preliminary tests showed similar improvements for segmenting DSA of coronary arteries and RGB images of retinal arteries. CONCLUSION: VESCL is an open-source C++ library for contouring vessels in 2D images which can be used to reduce the tedious, labor-intensive process of manually generating gold-standard segmentations for training, testing, and comparing automatic segmentation methods.

Impact of sunitinib resistance on clear cell renal cell carcinoma therapeutic sensitivity in vitro.

Sunitinib resistance creates a major clinical challenge for the treatment of advanced clear cell renal cell carcinoma (ccRCC) and functional and metabolic changes linked to sunitinib resistance are not fully understood. We sought to characterize the molecular and metabolic changes induced by the development of sunitinib resistance in ccRCC by developing and characterizing two human ccRCC cell lines resistant to sunitinib. Consistent with the literature, sunitinib-resistant ccRCC cell lines presented an aberrant overexpression of Axl and PD-L1, as well as a metabolic rewiring characterized by enhanced OXPHOS and glutamine metabolism. Therapeutic challenges of sunitinib-resistant ccRCC cell lines in vitro using small molecule inhibitors targeting Axl, AMPK and p38, as well as using PD-L1 blocking therapeutic antibodies, showed limited CTL-mediated cytotoxicity in a co-culture model. However, the AMPK activator metformin appears to sensitize the effect of PD-L1 blocking therapeutic antibodies and to enhance CTLs' cytotoxic effects on ccRCC cells. These effects were not broadly observed with the Axl and the p38 inhibitors. Taken together, these data suggest that targeting certain pathways aberrantly activated by sunitinib resistance such as the AMPK/PDL1 axis might sensitize ccRCC to immunotherapies as a second-line therapeutic approach.

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format.

Glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein drug-product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here, we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by proteinase K, selective N-glycan reduction, and O-glycan release by ß-elimination during permethylation of both N- and O-glycans. Glycan structural assignments and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is a reliable approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.