GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

Low-Cost Online Monitoring System for the Etching Process in Fiber Optic Sensors by Computer Vision.

The present research exposes a novel methodology to manufacture fiber optic sensors following the etching process by Hydrofluoric Acid deposition through a real-time monitoring diameter measurement by computer vision. This is based on virtual instrumentation developed with the National Instruments® technology and a conventional digital microscope. Here, the system has been tested proving its feasibility by the SMS structure diameter reduction from its original diameter of 125 µ until approximately 42.5 µm. The results obtained have allowed us to demonstrate a stable state behavior of the developed system during the etching process through diameter measurement at three different structure sections. Therefore, this proposal will contribute to the etched fiber optic sensor development that requires reaching an enhanced sensitivity. Finally, to demonstrate the previously mentioned SMS without chemical corrosion, and the etched manufactured SMS, both have been applied as glucose concentration sensors.

Design and On-Field Validation of an Embedded System for Monitoring Second-Life Electric Vehicle Lithium-Ion Batteries.

In the last few years, the growing demand for electric vehicles (EVs) in the transportation sector has contributed to the increased use of electric rechargeable batteries. At present, lithium-ion (Li-ion) batteries are the most commonly used in electric vehicles. Although once their storage capacity has dropped to below 80-70% it is no longer possible to use these batteries in EVs, it is feasible to use them in second-life applications as stationary energy storage systems. The purpose of this study is to present an embedded system that allows a Nissan® LEAF Li-ion battery to communicate with an Ingecon® Sun Storage 1Play inverter, for control and monitoring purposes. The prototype was developed using an Arduino® microcontroller and a graphical user interface (GUI) on LabVIEW®. The experimental tests have allowed us to determine the feasibility of using Li-ion battery packs (BPs) coming from the automotive sector with an inverter with no need for a prior disassembly and rebuilding process. Furthermore, this research presents a programming and hardware methodology for the development of the embedded systems focused on second-life electric vehicle Li-ion batteries. One second-life battery pack coming from a Nissan® Leaf and aged under real driving conditions was integrated into a residential microgrid serving as an energy storage system (ESS).

An Indoor Navigation Methodology for Mobile Devices by Integrating Augmented Reality and Semantic Web.

Indoor navigation systems incorporating augmented reality allow users to locate places within buildings and acquire more knowledge about their environment. However, although diverse works have been introduced with varied technologies, infrastructure, and functionalities, a standardization of the procedures for elaborating these systems has not been reached. Moreover, while systems usually handle contextual information of places in proprietary formats, a platform-independent model is desirable, which would encourage its access, updating, and management. This paper proposes a methodology for developing indoor navigation systems based on the integration of Augmented Reality and Semantic Web technologies to present navigation instructions and contextual information about the environment. It comprises four modules to define a spatial model, data management (supported by an ontology), positioning and navigation, and content visualization. A mobile application system was developed for testing the proposal in academic environments, modeling the structure, routes, and places of two buildings from independent institutions. The experiments cover distinct navigation tasks by participants in both scenarios, recording data such as navigation time, position tracking, system functionality, feedback (answering a survey), and a navigation comparison when the system is not used. The results demonstrate the system's feasibility, where the participants show a positive interest in its functionalities.

DNAJB1-PRKACA fusion kinase interacts with ß-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma.

A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1-PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of ß-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1-PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.

The evolutionarily conserved transcription factor Sp1 controls appendage growth through Notch signaling.

The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms by which they participate in control limb growth are mostly unknown. We analyzed here the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. We show that Sp1 plays a more prominent role controlling leg growth than does btd We identified a regulatory function of Sp1 in Notch signaling, and performed a genome-wide transcriptome analysis to identify other potential Sp1 target genes contributing to leg growth. Our data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling.

Development of a lateral flow test for the rapid detection of Avibacterium paragallinarum in chickens suspected of having infectious coryza.

BACKGROUND: Infectious coryza (IC) is an acute respiratory disease of growing chickens and layers caused by Avibacterium paragallinarum. The development of tools that allow rapid pathogen detection is necessary in order to avoid disease dissemination and economic losses in poultry. An Av. paragallinarum-specific Ma-4 epitope of the TonB-dependent transporter (TBDT) was selected using bioinformatic tools in order to immunize a BalbC mouse and to produce monoclonal antibodies to be used in a lateral flow test (LFT) developed for Av. paragallinarum detection in chicken nasal mucus samples. RESULTS: The 1G7G8 monoclonal antibody was able to detect TBDT in Av. paragallinarum cultures (serogroups: A, B and C) by Western blot and indirect ELISA assay. Consequently, we developed a self-pairing prototype LFT. The limit of detection of the prototype LFT using Av. paragallinarum cultures was 1 × 104 colony-forming units (CFU)/mL. Thirty-five nasal mucus samples from chickens suspected of having infectious coryza were evaluated for the LFT detection capacity and compared with bacterial isolation (B.I) and polymerase chain reaction (PCR). Comparative indicators such as sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive values (NPV) and the kappa index (K) were obtained. The values were 100.0% Se, 50% Sp, 65.4% PPV, 100% NPV, and 0.49 K and 83.9% Se, 100% Sp, 100% PPV, 44.4% NPV, and 0.54 K for the comparison of the LFT with B.I and PCR, respectively. Additionally, the LFT allowed the detection of Av. paragallinarum from coinfection cases of Av. paragallinarum with Gallibacterium anatis. CONCLUSIONS: The results indicate that the self-pairing prototype LFT is suitable for the detection of TBDT in Av. paragallinarum cultures as well as in field samples such as nasal mucus from Av. paragallinarum-infected chickens. Therefore, this prototype LFT could be considered a rapid and promising tool to be used in farm conditions for Av. paragallinarum diagnosis.

Establishment of a Developmental Compartment Requires Interactions between Three Synergistic Cis-regulatory Modules.

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMs.

A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance.

BACKGROUND: Tuberculosis (TB) is a major global health problem and drug resistance compromises the efforts to control this disease. Pyrazinamide (PZA) is an important drug used in both first and second line treatment regimes. However, its complete mechanism of action and resistance remains unclear. RESULTS: We genotyped and sequenced the complete genomes of 68 M. tuberculosis strains isolated from unrelated TB patients in Peru. No clustering pattern of the strains was verified based on spoligotyping. We analyzed the association between PZA resistance with non-synonymous mutations and specific genes. We found mutations in pncA and novel genes significantly associated with PZA resistance in strains without pncA mutations. These included genes related to transportation of metal ions, pH regulation and immune system evasion. CONCLUSIONS: These results suggest potential alternate mechanisms of PZA resistance that have not been found in other populations, supporting that the antibacterial activity of PZA may hit multiple targets.

De novo assembly and functional annotation of Myrciaria dubia fruit transcriptome reveals multiple metabolic pathways for L-ascorbic acid biosynthesis.

BACKGROUND: Myrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis. RESULTS: In total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs. CONCLUSIONS: This study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with the empirically observed capability of M. dubia to synthesize and accumulate AsA and other important molecules, and adds to our current knowledge of the molecular biology and biochemistry of their production in plants. By providing insights into the mechanisms underpinning these metabolic processes, these results can be used to direct efforts to genetically manipulate this organism in order to enhance the production of these bioactive phytochemicals. The accumulation of AsA precursor and discovery of genes associated with their biosynthesis and metabolism in M. dubia is intriguing and worthy of further investigation. The sequences and pathways produced here present the genetic framework required for further studies. Quantitative transcriptomics in concert with studies of the genome, proteome, and metabolome under conditions that stimulate production and accumulation of AsA and their precursors are needed to provide a more comprehensive view of how these pathways for AsA metabolism are regulated and linked in this species.

Studying the dynamics of the drug processing of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a key drug in the treatment of Mycobacterium tuberculosis. Although not completely understood yet, the bactericidal mechanism of PZA starts with its diffusion into the cell and subsequent conversion into pyrazinoic acid (POA) after the hydrolysis of ammonia group. This leads to the acidification cycle, which involves: (1) POA extrusion into the extracellular environment, (2) reentry of protonated POA, and (3) release of a proton into the cytoplasm, resulting in acidification of the cytoplasm and accumulation of intracellular POA. To better understand this process, we developed a system of coupled non-linear differential equations, which successfully recapitulates the kinetics of PZA/POA observed in M. tuberculosis. The parametric space was explored, assessing the impact of different PZA and pH concentrations and variations in the kinetic parameters, finding scenarios of PZA susceptibility and resistance. Furthermore, our predictions show that the acidification cycle alone is not enough to result in significant intracellular accumulation of POA in experimental time scales when compared to other neutral pH scenarios. Thus, revealing the need of novel hypotheses and experimental evidence to determine the missing mechanisms that may explain the pH-dependent intracellular accumulation of POA and their subsequent effects.

Increased Protein Kinase A Activity Induces Fibrolamellar Hepatocellular Carcinoma Features Independent of DNAJB1.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.

Assembly and phylogeographical analysis of novel Taenia solium mitochondrial genomes suggest stratification within the African-American genotype.

BACKGROUND: Taenia solium is a parasite of public health concern, causing human taeniasis and cysticercosis. Two main genotypes have been identified: Asian and African-American. Although characterizing T. solium genotypes is crucial to understanding the genetic epidemiology of its diseases, not much is known about the differences between T. solium mitochondrial genomes from different genotypes. Also, little is known about whether genotypes are further subdivided. Therefore, this study aimed to identify a set of point mutations distributed throughout the T. solium mitochondrial genome that differentiate the African-American from the Asian genotype. Another objective was to identify whether T. solium main genotypes are further stratified. METHODS: One Mexican and two Peruvian T. solium mitochondrial genomes were assembled using reads available in the NCBI Sequence Read Archive and the reference genome from China as a template. Mutations with respect to the Chinese reference were identified by multiple genome alignment. Jensen-Shannon and Grantham scores were computed for mutations in protein-coding genes to evaluate whether they affected protein function. Phylogenies by Bayesian inference and haplotype networks were constructed using cytochrome c oxidase subunit 1 and cytochrome b from these genomes and other isolates to infer phylogeographical relationships. RESULTS: A set of 31 novel non-synonymous point mutations present in all genomes of the African-American genotype were identified. These mutations were distributed across the mitochondrial genome, differentiating the African-American from the Asian genotype. All occurred in non-conserved protein positions. Furthermore, the analysis suggested a stratification of the African-American genotypes into an East African and a West African sublineage. CONCLUSIONS: A novel set of 31 non-synonymous mutations differentiating the main T. solium genotypes was identified. None of these seem to be causing differences in mitochondrial protein function between parasites of the two genotypes. Furthermore, two sublineages within the African-American genotype are proposed for the first time. The presence of the East African sublineage in the Americas suggests an underestimated connection between East African and Latin American countries that might have arisen in the major slave trade between Portuguese Mozambique and the Americas. The results obtained here help to complete the molecular epidemiology of the parasite.

Disruption of proteome by an oncogenic fusion kinase alters metabolism in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.

Epitope-Evaluator: An interactive web application to study predicted T-cell epitopes.

Multiple immunoinformatic tools have been developed to predict T-cell epitopes from protein amino acid sequences for different major histocompatibility complex (MHC) alleles. These prediction tools output hundreds of potential peptide candidates which require further processing; however, these tools are either not graphical or not friendly for non-programming users. We present Epitope-Evaluator, a web tool developed in the Shiny/R framework to interactively analyze predicted T-cell epitopes. Epitope-Evaluator contains six tools providing the distribution of epitopes across a selected set of MHC alleles, the promiscuity and conservation of epitopes, and their density and location within antigens. Epitope-Evaluator requires as input the fasta file of protein sequences and the output prediction file coming out from any predictor. By choosing different cutoffs and parameters, users can produce several interactive plots and tables that can be downloaded as JPG and text files, respectively. Using Epitope-Evaluator, we found the HLA-B*40, HLA-B*27:05 and HLA-B*07:02 recognized fewer epitopes from the SARS-CoV-2 proteome than other MHC Class I alleles. We also identified shared epitopes between Delta, Omicron, and Wuhan Spike variants as well as variant-specific epitopes. In summary, Epitope-Evaluator removes the programming barrier and provides intuitive tools, allowing a straightforward interpretation and graphical representations that facilitate the selection of candidate epitopes for experimental evaluation. The web server Epitope-Evaluator is available at https://fuxmanlab.shinyapps.io/Epitope-Evaluator/.

Immunoinformatic analysis of the whole proteome for vaccine design: An application to Clostridium perfringens.

Clostridium perfringens is a dangerous bacterium and known biological warfare weapon associated with several diseases, whose lethal toxins can produce necrosis in humans. However, there is no safe and fully effective vaccine against C. perfringens for humans yet. To address this problem, we computationally screened its whole proteome, identifying highly immunogenic proteins, domains, and epitopes. First, we identified that the proteins with the highest epitope density are Collagenase A, Exo-alpha-sialidase, alpha n-acetylglucosaminidase and hyaluronoglucosaminidase, representing potential recombinant vaccine candidates. Second, we further explored the toxins, finding that the non-toxic domain of Perfringolysin O is enriched in CTL and HTL epitopes. This domain could be used as a potential sub-unit vaccine to combat gas gangrene. And third, we designed a multi-epitope protein containing 24 HTL-epitopes and 34 CTL-epitopes from extracellular regions of transmembrane proteins. Also, we analyzed the structural properties of this novel protein using molecular dynamics. Altogether, we are presenting a thorough immunoinformatic exploration of the whole proteome of C. perfringens, as well as promising whole-protein, domain-based and multi-epitope vaccine candidates. These can be evaluated in preclinical trials to assess their immunogenicity and protection against C. perfringens infection.

Human liver organoids for disease modeling of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Enteropathogen Changes After Rotavirus Vaccine Scale-up.

OBJECTIVES: To inform next steps in pediatric diarrhea burden reduction by understanding the shifting enteropathogen landscape after rotavirus vaccine implementation. METHODS: We conducted a case-control study of 1788 medically attended children younger than 5 years, with and without gastroenteritis, after universal rotavirus vaccine implementation in Peru. We tested case and control stools for 5 viruses, 19 bacteria, and parasites; calculated coinfection-adjusted attributable fractions (AFs) to determine pathogen-specific burdens; and evaluated pathogen-specific gastroenteritis severity using Clark and Vesikari scales. RESULTS: Six pathogens were independently positively associated with gastroenteritis: norovirus genogroup II (GII) (AF 29.1, 95% confidence interval [CI]: 28.0-32.3), rotavirus (AF 8.9, 95% CI: 6.8-9.7), sapovirus (AF 6.3, 95% CI: 4.3-7.4), astrovirus (AF 2.8, 95% CI: 0.0-4.0); enterotoxigenic Escherichia coli heat stable and/or heat labile and heat stable (AF 2.4, 95% CI: 0.6-3.1), and Shigella spp. (AF 2.0, 95% CI: 0.4-2.2). Among typeable rotavirus cases, we most frequently identified partially heterotypic strain G12P[8] (54 of 81, 67%). Mean severity was significantly higher for norovirus GII-positive cases relative to norovirus GII-negative cases (Vesikari [12.7 vs 11.8; P < .001] and Clark [11.7 vs 11.4; P = .016]), and cases in the 6- to 12-month age range relative to cases in other age groups (Vesikari [12.7 vs 12.0; P = .0002] and Clark [12.0 vs 11.4; P = .0016]). CONCLUSIONS: Norovirus is well recognized as the leading cause of pediatric gastroenteritis in settings with universal rotavirus vaccination. However, sapovirus is often overlooked. Both norovirus and sapovirus contribute significantly to the severe pediatric disease burden in this setting. Decision-makers should consider multivalent vaccine acquisition strategies to target multiple caliciviruses in similar countries after successful rotavirus vaccine implementation.

Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

Identification of Novel Candidate Epitopes on SARS-CoV-2 Proteins for South America: A Review of HLA Frequencies by Country.

Coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, is already responsible for more than 4.3 million confirmed cases and 295,000 deaths worldwide as of May 15, 2020. Ongoing efforts to control the pandemic include the development of peptide-based vaccines and diagnostic tests. In these approaches, HLA allelic diversity plays a crucial role. Despite its importance, current knowledge of HLA allele frequencies in South America is very limited. In this study, we have performed a literature review of datasets reporting HLA frequencies of South American populations, available in scientific literature and/or in the Allele Frequency Net Database. This allowed us to enrich the current scenario with more than 12.8 million data points. As a result, we are presenting updated HLA allelic frequencies based on country, including 91 alleles that were previously thought to have frequencies either under 5% or of an unknown value. Using alleles with an updated frequency of at least ≥5% in any South American country, we predicted epitopes in SARS-CoV-2 proteins using NetMHCpan (I and II) and MHC flurry. Then, the best predicted epitopes (class-I and -II) were selected based on their binding to South American alleles (Coverage Score). Class II predicted epitopes were also filtered based on their three-dimensional exposure. We obtained 14 class-I and four class-II candidate epitopes with experimental evidence (reported in the Immune Epitope Database and Analysis Resource), having good coverage scores for South America. Additionally, we are presenting 13 HLA-I and 30 HLA-II novel candidate epitopes without experimental evidence, including 16 class-II candidates in highly exposed conserved areas of the NTD and RBD regions of the Spike protein. These novel candidates have even better coverage scores for South America than those with experimental evidence. Finally, we show that recent similar studies presenting candidate epitopes also predicted some of our candidates but discarded them in the selection process, resulting in candidates with suboptimal coverage for South America. In conclusion, the candidate epitopes presented provide valuable information for the development of epitope-based strategies against SARS-CoV-2, such as peptide vaccines and diagnostic tests. Additionally, the updated HLA allelic frequencies provide a better representation of South America and may impact different immunogenetic studies.