Proteomic Barcoding Platform for Macromolecular Screening and Delivery.

Engineered macromolecules offer compelling means for the therapy of conventionally undruggable interactions in human disease. However, their efficacy is limited by barriers to tissue and intracellular delivery. Inspired by recent advances in molecular barcoding and evolution, we developed BarcodeBabel, a generalized method for the design of libraries of peptide barcodes suitable for high-throughput mass spectrometry proteomics. Combined with PeptideBabel, a Monte Carlo sampling algorithm for the design of peptides with evolvable physicochemical properties and sequence complexity, we developed a barcoded library of cell penetrating peptides (CPPs) with distinct physicochemical features. Using quantitative targeted mass spectrometry, we identified CPPS with improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell while maintaining minimal membrane disruption and negligible toxicity in vitro. These studies provide a proof of concept for peptide barcoding as a homogeneous high-throughput method for macromolecular screening and delivery. BarcodeBabel and PeptideBabel are available open-source from https://github.com/kentsisresearchgroup/.

Are DNA Repair Gene Alterations Associated With the Response to Platinum-Based Regimen and Immune Checkpoint Inhibitors in Patients With Solid Tumors?

We analyzed the antitumor activity of platinum-based chemotherapies and then immune checkpoint inhibitors (ICI) in all-comers patients with solid tumors having a somatic DNA damage repair gene alteration (DDR-GA) identified through a prospective precision medicine study (NCT02534649). Each DDR-GA was classified as pathogenic (Pa), probably pathogenic (PPa), and unknown pathogenicity (UPa) according to OncoKB and ClinVAR databases. Between January 2018 and May 2020, 662 patients were screened. One hundred ninety-nine tumors with DDR-GA were found in 121 (18.3%) patients. Ninety-six patients received platinum-based chemotherapy in the advanced setting. No difference in objective response rate (ORR) under platinum regimen was observed between the 3 DDR-GA groups. The only predictor of worse progression-free survival (PFS) in Cox regression was the existence of a Pa alteration compared to the UPa group: HR = 2.11 (95% CI = 1.2-3.7), P = .009. Forty-eight patients received ICI alone or in combination. We observed a significant trend in better ORR to ICI according to the DDR-GA status: 1/11 (9%) patients in UPa, 5/17 (29.4%) patients in PPa, and 9/20 (45%) patients in Pa (P = .003, Cochran-Armitage trend test), and an increased 6-month PFS probability of 11%, 44%, and 50% in the UPa, PPa, and Pa groups, respectively (P = .37, log-rank test). Overall, somatic pathogenic DDR-GAs were not associated with ORR or PFS to platinum-based chemotherapy in patients with unselected advanced solid tumors. However, DDR-GA seemed to impact ORR and PFS to ICI, paving the way for a therapeutic combination with ICI and molecules targeting the DDR mechanisms, which are currently evaluated in ongoing clinical trials.

Abbott realtime MTB assay for detecting Mycobacterium tuberculosis complex in respiratory specimens: a cost-benefit analysis.

PURPOSE: Molecular screening for Mycobacterium tuberculosis (MTB) can lead to rapid empirical treatment inception and reduce hospitalization time and complementary diagnostic tests. However, in low-prevalence settings, the cost-benefit balance remains controversial due to the high cost. METHODS: We used a Markov model to perform an economic analysis to evaluate the profit after implementing molecular MTB screening (Period B) compared with conventional culture testing (Period A) in respiratory samples from 7,452 consecutive subjects with presumed tuberculosis (TB). RESULTS: The proportion of positivity was comparable between both periods (P > 0.05), with a total of 2.16 and 1.78 samples/patient requested in periods A and B, respectively (P < 0.001). The mean length of hospital stay was 8.66 days (95%CI: 7.63-9.70) in Period B and 11.51 days (95%CI: 10.15-12.87) in Period A (P = 0.001). The healthcare costs associated with diagnosing patients with presumed TB were reduced by €717.95 per patient with PCR screening. The probability of remaining hospitalized and the need for a greater number of outpatient specialty care visits were the variables with the most weight in the model. CONCLUSION: Employing PCR as an MTB screening method in a low-prevalence setting may increase the profits to the system.

MKRN3 circulating levels in girls with central precocious puberty caused by MKRN3 gene mutations.

PURPOSE: MKNR3 is a paternally expressed gene whose mutations are the main cause of central precocious puberty (CPP). Protein circulating levels can be easily measured, as demonstrated in idiopathic CPP and healthy controls. No data are available for patients harboring an MKRN3 mutation. Our aim was to perform MKRN3 mutation screening and to investigate if circulating protein levels could be a screening tool to identify MKRN3 mutation in CPP patients. METHODS: We enrolled 140 CPP girls and performed MKRN3 mutation analysis. Patients were stratified into two groups: idiopathic CPP (iCPP) and MKRN3 mutation-related CPP (MKRN3-CPP). Clinical characteristics were collected. Serum MKRN3 values were measured by a commercially available ELISA assay kit in MKRN3-CPP and a subgroup of 15 iCPP patients. RESULTS: We identified 5 patients with MKRN3 mutations: one was a novel mutation (p.Gln352Arg) while the others were previously reported (p.Arg328Cys, p.Arg345Cys, p.Pro160Cysfs*14, p.Cys410Ter). There was a significant difference in circulating MKRN3 values in MKRN3-CPP compared to iCPP (p < 0.001). In MKRN3-CPP, the subject harboring Pro160Cysfs*14 presented undetectable levels. Subjects carrying the missense mutations p.Arg328Cys and p.Gln352Arg showed divergent circulating protein levels, respectively 40.56 pg/mL and undetectable. The patient with the non-sense mutation reported low but measurable MKRN3 levels (12.72 pg/mL). CONCLUSIONS: MKRN3 defect in patients with CPP cannot be predicted by MKRN3 circulating levels, although those patients presented lower protein levels than iCPP. Due to the great inter-individual variability of the assay and the lack of reference values, no precise cut-off can be identified to suspect MKRN3 defect.

A large-scale screening identified in USH2A gene the P3272L founder pathogenic variant explaining familial Usher syndrome in Sardinia, Italy.

BACKGROUND: Usher syndrome (USH) encompasses a group of disorders characterized by congenital sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP). We described the clinical findings, natural history, and molecular analyses of USH patients identified during a large-scale screening to identify quantitative traits related to ocular disorders in the SardiNIA project cohort. METHODS: We identified 3 USH-affected families out of a cohort of 6,148 healthy subjects. 9 subjects presented a pathological phenotype, with SNHL and RP. All patients and their family members underwent a complete ophthalmic examination including best-corrected visual acuity, slit-lamp biomicroscopy, fundoscopy, fundus autofluorescence, spectral-domain optical coherence tomography, and electrophysiological testing. Audiological evaluation was performed with a clinical audiometer. Genotyping was performed using several arrays integrated with whole genome sequence data providing approximately 22 million markers equally distributed for each subject analyzed. Molecular diagnostics focused on analysis of the following candidate genes: MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31, CLRN1, and PDZD7. RESULTS: A single missense causal variant in USH2A gene was identified in homozygous status in all patients and in heterozygous status in unaffected parents. The presence of multiple homozygous patients with the same phenotypic severity of the syndromic form suggests that the Sardinian USH phenotype is the result of a founder effect on a specific pathogenic variant related haplotype. The frequency of heterozygotes in general Sardinian population is 1.89. Additionally, to provide new insights into the structure of usherin and the pathological mechanisms caused by small pathogenic in-frame variants, like p.Pro3272Leu, molecular dynamics simulations of native and mutant protein-protein and protein-ligand complexes were performed that predicted a destabilization of the protein with a decrease in the free energy change. CONCLUSIONS: Our results suggest that our approach is effective for the genetic diagnosis of USH. Based on the heterozygous frequency, targeted screening of this variant in the general population and in families at risk or with familial USH can be suggested. This can lead to more accurate molecular diagnosis, better genetic counseling, and improved molecular epidemiology data that are critical for future intervention plans. TRIAL REGISTRATION: We did not perform any health-related interventions for the participants.

A drug screening to identify novel combinatorial strategies for boosting cancer immunotherapy efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cells and immune checkpoint blockades (ICBs) have made remarkable breakthroughs in cancer treatment, but the efficacy is still limited for solid tumors due to tumor antigen heterogeneity and the tumor immune microenvironment. The restrained treatment efficacy prompted us to seek new potential therapeutic methods. METHODS: In this study, we conducted a small molecule compound library screen in a human BC cell line to identify whether certain drugs contribute to CAR T cell killing. Signaling pathways of tumor cells and T cells affected by the screened drugs were predicted via RNA sequencing. Among them, the antitumor activities of JK184 in combination with CAR T cells or ICBs were evaluated in vitro and in vivo. RESULTS: We selected three small molecule drugs from a compound library, among which JK184 directly induces tumor cell apoptosis by inhibiting the Hedgehog signaling pathway, modulates B7-H3 CAR T cells to an effector memory phenotype, and promotes B7-H3 CAR T cells cytokine secretion in vitro. In addition, our data suggested that JK184 exerts antitumor activities and strongly synergizes with B7-H3 CAR T cells or ICBs in vivo. Mechanistically, JK184 enhances B7-H3 CAR T cells infiltrating in xenograft mouse models. Moreover, JK184 combined with ICB markedly reshaped the tumor immune microenvironment by increasing effector T cells infiltration and inflammation cytokine secretion, inhibiting the recruitment of MDSCs and the transition of M2-type macrophages in an immunocompetent mouse model. CONCLUSION: These data show that JK184 may be a potential adjutant in combination with CAR T cells or ICB therapy.

Detection of human papillomavirus in fresh and dried urine through an automated system for cervical cancer screening in low- and middle-income countries.

The majority of cervical cancer cases and associated deaths occur in low- and middle-income countries (LMICs), where sociocultural barriers, poor access to prevention and care, and technical and practical difficulties hinder screening coverage improvement. Using urine specimens for human papillomaviruses (HPV) molecular screening through automated testing platforms can help to overcome these problems. We evaluated the high-risk (HR) HPV detection performance of the Xpert® HPV test on GeneXpert® System (Cepheid), on fresh and dried urine (Dried Urine Spot [DUS]) samples as compared to an in-house polymerase chain reaction (PCR) genotyping assay. Forty-five concentrated urine samples collected from women with known cytological and HPV infection status, determined through in-house PCR and genotyping assays, were tested "as is" and as DUS with the Xpert® HPV test. This system detected HR-HPV in 86.4% of fresh and in 77.3% of dried urine samples collected from HPV+ women, correctly identifying HR-HPV infection in 100% of women with low- and high-grade lesions. High concordance (91.4%, k = 0.82) was found between PCR test and Xpert® HPV Test from urine. Urine-based Xpert® HPV test seems to be a suitable screening test for detection of HR-HPV infections associated with low- and high-grade lesions requiring follow-up monitoring or treatment. This methodology, relying on noninvasively collected samples and on available rapid testing platforms, could facilitate large, at-scale screening programs, particularly in LMICs and rural areas, thus reducing adverse outcomes of HPV infection and facilitating achievement of the WHO cervical cancer elimination goal.

A new DLK1 defect in a family with idiopathic central precocious puberty: elucidation of the male phenotype.

PURPOSE: We aimed to investigate a cohort of female and male patients with idiopathic central precocious puberty (CPP), negative for Makorin Ring Finger Protein 3 (MKRN3) defect, by molecular screening for Delta-like 1 homolog (DLK1) defects. DLK1 is an imprinted gene, whose mutations have been described as a rare cause of CPP in girls and adult women with precocious menarche, obesity and metabolic derangement. METHODS: We enrolled 14 girls with familial CPP and 13 boys with familial or sporadic CPP from multiple academic hospital centers. Gene sequencing of DLK1 gene was performed. Circulating levels of DLK1 were measured and clinical and biochemical characteristics were described in those with DLK1 defects. RESULTS: A novel heterozygous mutation in DLK1, c.288_289insC (p.Cys97Leufs*16), was identified in a male proband, his sister and their father. Age at onset of puberty was in line with previous reports in the girl and 8 years in the boy. The father with untreated CPP showed short stature. No metabolic derangement was present in the father except hypercholesterolemia. Undetectable Dlk1 serum levels indicated the complete lack of protein production in the three affected patients. CONCLUSION: A DLK1 defect has been identified for the first time in a boy, underscoring the importance of genetic testing in males with idiopathic or sporadic CPP. The short stature reported by his untreated father suggests the need for timely diagnosis and treatment of subjects with DLK1 defects.

An Overall View of the Functional and Structural Characterization of Glucose-6-Phosphate Dehydrogenase Variants in the Mexican Population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.

The Impact of Routine Molecular Screening for SARS-CoV-2 in Patients Receiving Anticancer Therapy: An Interim Analysis of the Observational COICA Study.

Introduction: Cancer aggravates COVID-19 prognosis. Nosocomial transmission of SARS-CoV-2 is particularly frequent in cancer patients, who need to attend hospitals regularly. Since March 2020, all cancer patients having access to the Oncology Unit at the "Andrea Tortora" Hospital (Pagani, Salerno - referred to as "the Hospital") as inpatients or outpatients receiving intravenous therapy have been screened for SARS-CoV-2 using RT-PCR nasal swab. The ongoing COICA (COVID-19 infection in cancer patients) study is an ambispective, multicenter, observational study designed to assess the prognosis of SARS-CoV-2 infection in cancer patients. The aim of the study presented here was to explore potential differences in COVID-19-related outcomes among screening-detected versus nonscreening-detected SARS-CoV-2-infected patients. Methods: The COICA study enrolled cancer patients who had received any anticancer systemic therapy within 3 months since the day they tested positive for SARS-CoV-2 on RT-PCR. The target accrual is 128 patients, and the study was approved by the competent Ethics Committee. Only the subgroup of patients enrolled at the Hospital was considered in this unplanned interim analysis. Logistic regression analysis was used to evaluate the association of screening-based versus nonscreening-based diagnosis. Results: Since March 15, 2020, until August 15, 2021, a total of 931 outpatients and 230 inpatients were repeatedly screened for SARS-CoV-2 using RT-PCR nasal swab at the Hospital. Among these, 71 asymptomatic patients were positive on routine screening and 5 patients were positive for SARS-CoV-2 outside the institutional screening. Seven patients died because of COVID-19. At univariate analysis, nonscreening- versus screening-detected SARS-CoV-2 infection was associated with significantly higher odds of O2 therapy (OR = 16.2; 95% CI = 2.2-117.1; p = 0.006), hospital admission (OR = 31.5; 95% CI = 3.1-317.8; p = 0.003), admission to ICU (OR = 23.0; 95% CI = 2.4-223.8; p = 0.007), and death (OR = 8.8; 95% CI = 1.2-65.5; p = 0.034). Conclusion: Routine screening with RT-PCR may represent a feasible and effective strategy in reducing viral circulation and possibly COVID-19 mortality in patients with active cancer having repeated access to hospital facilities.

The Effect of Vaccination against COVID-19 in Cancer Patients: Final Results of the COICA Trial.

Background: The COICA study is an ambispective, observational trial that was conceived to assess the clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cancer patients. A recently published, population-based, case-control study reported a reduced vaccine efficacy at 3-6 months in cancer patients compared to individuals without cancer. Objectives: The aim of the study was to describe coronavirus disease 19 (COVID-19) outcomes in cancer patients and analyze differences in SARS-CoV-2 outcomes between vaccinated and unvaccinated patients. Methods: Descriptive statistics and frequency counts were used to summarize characteristics of the study population. χ2 test and the log-rank test were used to compare outcomes between vaccinated and unvaccinated patients. Results: A total of 141 cancer patients (80 males, 61 females) were recruited at two participating Institutions from March 2020 until April 2022 and observed from the time of positive SARS-CoV-2 test to the time of negativization or death. Approximately 35% of patients had been vaccinated at the time of infection with 2 (16 patients) or 3 (33 patients) vaccine doses. Vaccinated patients consistently and significantly showed improved COVID-19 outcomes compared to unvaccinated patients, with CT-diagnosed pneumonia, hospitalization, O2 therapy, and death reported in 0% versus 48.6%, 2.0% versus 15.2%, 0% versus 14.1%, and 0% versus 7.6%, respectively, of assessable patients (p < 0.05). Vaccinated versus unvaccinated patients showed a significantly shorter time to negativization, with a median (95% confidence interval) time of 12 (10-14) versus 20 (17-23) days, respectively (p < 0.001). Conclusions: Vaccination consistently improved all COVID-19 outcomes. No death was recorded among vaccinated patients. Additional research is especially warranted to establish optimal timing and patient selection for administration of the fourth vaccination dose.

Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity.

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies.

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at-risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review-curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high-throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease-causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease-causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high-throughput sequencing data. Due to the availability of a comprehensive phenotype-genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD-DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.

Computational screening, ensemble docking and pharmacophore analysis of potential gefitinib analogues against epidermal growth factor receptor.

The observable mutated isoforms of epidermal growth factor receptor (EGFR) are important considerable therapeutic benchmarks in moderating the non-small cell lung cancer (NSCLC). Recently, quinazoline-based ATP competitive inhibitors have been developed against the EGFR; however, these imply the mutation probabilities, which contribute to the discovery of high probable novel inhibitors for EGFR mutants. Therefore, SAR-based bioactivity analysis, molecular docking and computational toxicogenomics approaches were performed to identify and evaluate new analogs of gefitinib against the ligand-binding domain of the EGFR double-mutated model. From the diverse groups of molecular clustering and molecular screening strategies, top high-binding gefitinib-analogues were identified and studied against EGFR core cavity through three-phase ensemble docking approach. Resulted high possible leads showed good binding orientations than gefitinib (positive control) thus they were subjected to pharmacophore analysis that possesses possible molecular assets to tight binding with EGFR domain. Residues Ser720, Arg841 and Trp880 were observed as novel hot spots and involved in H-bonds, pi-stacking and π-cation interactions that contribute additional electrostatic potency to sustain stability and complexity of protein-ligand complexes, thus they have ability to profoundly adopted by pharmacophoric features. Furthermore, lead molecules have an inhibition percent probability, anticancer potency, toxic impacts, flexible pharmacokinetics, potential gene-chemical interactions towards EGFR were revealed by computational systems biology tools. Our multiple screening strategies confirmed that the druggable sub-pocket was crucial to strong EGFR-ligand binding. The essential pharmacophoric features of ligands provided viewpoints for new inhibitors envisaging, and predicted scaffolds could used as anticancer agents against selected EGFR mutated isoforms.

Clinical Application of Targeted Deep Sequencing in Solid-Cancer Patients and Utility for Biomarker-Selected Clinical Trials.

Molecular profiling of actionable mutations in refractory cancer patients has the potential to enable "precision medicine," wherein individualized therapies are guided based on genomic profiling. The molecular-screening program was intended to route participants to different candidate drugs in trials based on clinical-sequencing reports. In this screening program, we used a custom target-enrichment panel consisting of cancer-related genes to interrogate single-nucleotide variants, insertions and deletions, copy number variants, and a subset of gene fusions. From August 2014 through April 2015, 654 patients consented to participate in the program at Samsung Medical Center. Of these patients, 588 passed the quality control process for the 381-gene cancer-panel test, and 418 patients were included in the final analysis as being eligible for any anticancer treatment (127 gastric cancer, 122 colorectal cancer, 62 pancreatic/biliary tract cancer, 67 sarcoma/other cancer, and 40 genitourinary cancer patients). Of the 418 patients, 55 (12%) harbored a biomarker that guided them to a biomarker-selected clinical trial, and 184 (44%) patients harbored at least one genomic alteration that was potentially targetable. This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase. IMPLICATIONS FOR PRACTICE: This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase.

Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women.

OBJECTIVES: DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples. METHODS: We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined. RESULTS: Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4-80.6) at a specificity of 67.8% (95%CI: 62.7-73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8-80.1) at a 76.4% (95%CI: 70.2-82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4-95.6) and 46.0% (95%CI: 40.4-51.5) for lavage self-samples, and 84.7% (95%CI: 76.4-93.0) and 54.9% (95%CI: 47.7-62.2) for brush self-samples. CONCLUSIONS: FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity.

Common asymptomatic and submicroscopic malaria infections in Western Thailand revealed in longitudinal molecular and serological studies: a challenge to malaria elimination.

BACKGROUND: Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals. RESULTS: Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers. CONCLUSIONS: Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.

Small-molecule inhibitors of JC polyomavirus infection.

The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals, the infection remains dormant and asymptomatic, but in immuno-suppressed patients, it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection nor for the treatment of PML. Here, we report the development of small-molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively, these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics.