Identifying lupus Patient Subsets Through Immune Cell Deconvolution of Gene Expression Data in Two Atacicept Phase II Studies.

OBJECTIVE: To use cell-based gene signatures to identify patients with systemic lupus erythematous (SLE) in the phase II/III APRIL-SLE and phase IIb ADDRESS II trials most likely to respond to atacicept. METHODS: A published immune cell deconvolution algorithm based on Affymetrix gene array data was applied to whole blood gene expression from patients entering APRIL-SLE. Five distinct patient clusters were identified. Patient characteristics, biomarkers, and clinical response to atacicept were assessed per cluster. A modified immune cell deconvolution algorithm was developed based on RNA sequencing data and applied to ADDRESS II data to identify similar patient clusters and their responses. RESULTS: Patients in APRIL-SLE (N = 105) were segregated into the following five clusters (P1-5) characterized by dominant cell subset signatures: high neutrophils, T helper cells and natural killer (NK) cells (P1), high plasma cells and activated NK cells (P2), high B cells and neutrophils (P3), high B cells and low neutrophils (P4), or high activated dendritic cells, activated NK cells, and neutrophils (P5). Placebo- and atacicept-treated patients in clusters P2,4,5 had markedly higher British Isles Lupus Assessment Group (BILAG) A/B flare rates than those in clusters P1,3, with a greater treatment effect of atacicept on lowering flares in clusters P2,4,5. In ADDRESS II, placebo-treated patients from P2,4,5 were less likely to be SLE Responder Index (SRI)-4, SRI-6, and BILAG-Based Combined Lupus Assessment responders than those in P1,3; the response proportions again suggested lower placebo effect and a greater treatment differential for atacicept in P2,4,5. CONCLUSION: This exploratory analysis indicates larger differences between placebo- and atacicept-treated patients with SLE in a molecularly defined patient subset.

Revealing the immune cell subtype reconstitution profile in patients from the CLARITY study using deconvolution algorithms after cladribine tablets treatment.

Immune Cell Deconvolution methods utilizing gene expression profiling to quantify immune cells in tissues and blood are an appealing alternative to flow cytometry. Our objective was to investigate the applicability of deconvolution approaches in clinical trial settings to better investigate the mode of action of drugs for autoimmune diseases. Popular deconvolution methods CIBERSORT and xCell were validated using gene expression from the publicly available GSE93777 dataset that has comprehensive matching flow cytometry. As shown in the online tool, ~ 50% of signatures show strong correlation (r > 0.5) with the remainder showing moderate correlation, or in a few cases, no correlation. Deconvolution methods were then applied to gene expression data from the phase III CLARITY study (NCT00213135) to evaluate the immune cell profile of relapsing multiple sclerosis patients treated with cladribine tablets. At 96 weeks after treatment, deconvolution scores showed the following changes vs placebo: naïve, mature, memory CD4+ and CD8+ T cells, non-class switched, and class switched memory B cells and plasmablasts were significantly reduced, naïve B cells and M2 macrophages were more abundant. Results confirm previously described changes in immune cell composition following cladribine tablets treatment and reveal immune homeostasis of pro- vs anti-inflammatory immune cell subtypes, potentially supporting long-term efficacy.

Identification of HMGA2 as a predictive biomarker of response to bintrafusp alfa in a phase 1 trial in patients with advanced triple-negative breast cancer.

Background: We report the clinical activity, safety, and identification of a predictive biomarker for bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGFßRII (a TGF-ß "trap") fused to a human IgG1 mAb blocking PD-L1, in patients with advanced triple-negative breast cancer (TNBC). Methods: In this expansion cohort of a global phase 1 study, patients with pretreated, advanced TNBC received bintrafusp alfa 1200 mg every 2 weeks intravenously until disease progression, unacceptable toxicity, or withdrawal. The primary objective was confirmed best overall response by RECIST 1.1 assessed per independent review committee (IRC). Results: As of May 15, 2020, a total of 33 patients had received bintrafusp alfa, for a median of 6.0 (range, 2.0-48.1) weeks. The objective response rate was 9.1% (95% CI, 1.9%-24.3%) by IRC and investigator assessment. The median progression-free survival per IRC was 1.3 (95% CI, 1.2-1.4) months, and median overall survival was 7.7 (95% CI, 2.1-10.9) months. Twenty-five patients (75.8%) experienced treatment-related adverse events (TRAEs). Grade 3 TRAEs occurred in 5 patients (15.2%); no patients had a grade 4 TRAE. There was 1 treatment-related death (dyspnea, hemolysis, and thrombocytopenia in a patient with extensive disease at trial entry). Responses occurred independently of PD-L1 expression, and tumor RNAseq data identified HMGA2 as a potential biomarker of response. Conclusions: Bintrafusp alfa showed clinical activity and manageable safety in patients with heavily pretreated advanced TNBC. HMGA2 was identified as a potential predictive biomarker of response. ClinicalTrialsgov identifier: NCT02517398.

Risk assessment of drug-drug interaction potential for bintrafusp alfa with cytochrome P4503A4 substrates: A totality of evidence approach.

Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-ßRII (a TGF-ß "trap") fused to a human IgG1 mAb blocking PD-L1, is being evaluated for efficacy and safety in solid tumor indications as monotherapy and in combination with small-molecule drugs. We evaluated the perpetrator drug-drug interaction (DDI) potential of bintrafusp alfa via cytochrome P4503A4 (CYP3A4) enzyme modulation, which is responsible for the metabolism of a majority of drugs. The holistic approach included (1) evaluation of longitudinal profiles of cytokines implicated in CYP3A4 modulation and serum 4ß-hydroxycholesterol, an endogenous marker of CYP3A4 activity, in a phase I clinical study, and (2) transcriptomics analysis of the CYP3A4 mRNA levels vs the TGFB gene expression signature in normal hepatic tissues. Bintrafusp alfa was confirmed not to cause relevant proinflammatory cytokine modulation or alterations in 4ß-hydroxycholesterol serum concentrations in phase I studies. Transcriptomics analyses revealed no meaningful correlations between TGFB gene expression and CYP3A4 mRNA expression, supporting the conclusion that the risk of CYP3A4 enzyme modulation due to TGF-ß neutralization by bintrafusp alfa is low. Thus, bintrafusp alfa is not expected to have DDI potential as a perpetrator with co-administered drugs metabolized by CYP3A4; this information is relevant to clinical evaluations of bintrafusp alfa in combination settings.

Comparison of Two Illumina Whole Transcriptome RNA Sequencing Library Preparation Methods Using Human Cancer FFPE Specimens.

Objective: RNA extraction and library preparation from formalin-fixed, paraffin-embedded (FFPE) samples are crucial pre-analytical steps towards achieving optimal downstream RNA sequencing (RNASeq) results. In this study, we assessed 2 Illumina library preparation methods for RNA-Seq analysis using archived FFPE samples from human cancer indications at 2 independent vendors. Methods: Twenty-five FFPE samples from 5 indications (non-small cell lung cancer, colorectal cancer, renal carcinoma, breast cancer, and hepatocellular carcinoma) were included, covering a wide range of sample storage durations (3-25 years-old), sample qualities, and specimen types (resection vs core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20 pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry in paired-end mode with a read length of 2 × 50 bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions for each gene in each sample to compare 2 library kits at 2 different vendors. Results: Our data showed that TruSeq RNA Access libraries yield over 80% exonic reads across different quality samples, indicating higher selectivity of the exome pull down by the capture approach compared to the random priming of the TruSeq Stranded Total kit. The overall QC data for FFPE RNA extraction, library preparation, and sequencing generated by the 2 vendors are comparable, and downstream gene expression quantification results show high concordance as well. With the TruSeq Stranded Total kit, the mean Spearman correlation between vendors was 0.87 and the mean Pearson correlation was 0.76. With the TruSeq RNA Access kit, the mean Spearman correlation between vendors was 0.89 and the mean Pearson correlation was 0.73. Interestingly, examination of the cross-vendor correlations compared to various common QC statistics suggested that library concentration is better correlated with consistency between vendors than is the RNA quantity. Conclusions: Our analyses provide evidence to guide selection of sequencing methods for FFPE samples in which the sample quality may be severely compromised.

Bintrafusp alfa, a bifunctional fusion protein targeting TGF-ß and PD-L1, in patients with human papillomavirus-associated malignancies.

BACKGROUND: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-ßRII (a TGF-ß 'trap') fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa. METHODS: In these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety. RESULTS: As of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred. CONCLUSION: Bintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers.

Identification of tumor-associated macrophage subsets that are associated with breast cancer prognosis.

BACKGROUND: Breast cancer is the leading cause of cancer-related deaths in women, demanding new treatment options. With the advent of immune checkpoint blockade, immunotherapy emerged as a treatment option. In addition to lymphocytes, tumor-associated macrophages exert a significant, albeit controversial, impact on tumor development. Pro-inflammatory macrophages are thought to hinder, whereas anti-inflammatory macrophages promote tumor growth. However, molecular markers to identify prognostic macrophage populations remain elusive. METHODS: We isolated two macrophage subsets, from 48 primary human breast tumors, distinguished by the expression of CD206. Their transcriptomes were analyzed via RNA-Seq, and potential prognostic macrophage markers were validated by PhenOptics in tissue microarrays of patients with invasive breast cancer. RESULTS: Normal human breast tissue contained mainly CD206+ macrophages, while increased relative amounts of CD206- macrophages were observed in tumors. The presence of CD206+ macrophages correlated with a pronounced lymphocyte infiltrate and subsets of CD206+ macrophages, expressing SERPINH1 and collagen 1, or MORC4, were unexpectedly associated with improved survival of breast cancer patients. In contrast, MHCIIhi CD206- macrophages were linked with a poor survival prognosis. CONCLUSION: Our data highlight the heterogeneity of tumor-infiltrating macrophages and suggest the use of multiple phenotypic markers to predict the impact of macrophage subpopulations on cancer prognosis. We identified novel macrophage markers that correlate with the survival of patients with invasive mammary carcinoma.

Bintrafusp alfa, a bifunctional fusion protein targeting TGF-ß and PD-L1, in advanced squamous cell carcinoma of the head and neck: results from a phase I cohort.

BACKGROUND: We report the clinical activity and safety of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor ß (TGF-ß)RII receptor (a TGF-ß 'trap') fused to a human IgG1 monoclonal antibody blocking programmed death-ligand 1 (PD-L1), in patients with heavily pretreated squamous cell carcinoma of the head and neck (SCCHN). METHODS: In this phase I dose-expansion cohort, patients with advanced SCCHN not amenable to curative therapy that progressed/recurred after platinum therapy in the recurrent/metastatic setting, or <6 months after platinum therapy in the locally advanced setting, received bintrafusp alfa 1200 mg intravenously every 2 weeks. The primary endpoint was confirmed best overall response (BOR; Response Evaluation Criteria for Solid Tumors (RECIST) 1.1) per independent review committee (IRC); other endpoints included BOR per investigator and safety. RESULTS: As of August 24, 2018, 32 patients had received bintrafusp alfa (median follow-up 86.4 weeks; range 2-97). Per IRC, the confirmed objective response rate (ORR) was 13% (95% CI 4% to 29%; 4 partial responses (PR)); 4 patients had stable disease (SD) (disease control rate 34%; 95% CI 12% to 43%). Per investigator, there were 5 PRs (ORR, 16%), including 2 patients who developed delayed PRs after initial disease increase (total clinical response rate 22%). Responses (ORRs) were observed in patients with PD-L1-positive (12%), PD-L1-negative (17%; 73-10 antibody for immunohistochemistry), human papillomavirus (HPV)-positive (33%) and HPV-negative tumors (5%). Grade 3 treatment-related adverse events (TRAEs) were reported in 11 patients (34%), with no grade 4 TRAEs or treatment-related deaths. CONCLUSIONS: Bintrafusp alfa showed clinical activity across subgroups of PD-L1 expression and in HPV-positive tumors and had a manageable safety profile in patients with heavily pretreated advanced SCCHN. Activity in HPV-positive tumors is favorable compared with historical data from PD-L1 inhibitors and is being further investigated in an ongoing study of HPV-associated tumors. TRIAL REGISTRATION NUMBER: NCT02517398.

Observational Study of PD-L1, TGF-ß, and Immune Cell Infiltrates in Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) typically develops in cirrhotic livers, with increased programed death ligand 1 (PD-L1) and transforming growth factor beta (TGF-ß) activity implicated in immunosuppression. Methods: In an observational study of HCC liver samples, we determined the incidence of PD-L1 and immune cell (IC) infiltrates, and signs of TGF-ß activity. HCCs were characterized by the incidence and distribution of PD-L1+ cells, and CD8+, CD68+, and FoxP3+ infiltrating ICs in HCC and surrounding liver. Gene expression signatures (GESs) associated with TGF-ß activity and ICs were evaluated by RNAseq. Results: In non-neoplastic cirrhotic and non-cirrhotic liver, PD-L1 occurred on sinusoidal lining cells (mostly Kupffer cells), endothelial cells and ICs. In HCC, PD-L1+ tumor cells were rare. Most PD-L1+ cells were identified as ICs. CD8+, CD68+, and FoxP3+ ICs were associated with HCC, particularly in the invasive margin. CD8+ cell incidence correlated with PD-L1+ cells, consistent with PD-L1 being upregulated in response to pre-existing cytotoxic T-lymphocyte activity. TGFB1 mRNA levels and TGF-ß activation GES correlated with the strength of the tumor-associated macrophage GES. Conclusion: Inhibition of PD-L1+ ICs and TGF-ß activity and their respective immunomodulatory pathways may contribute to antitumor effects in HCC.

Comparative assessment of faecal diagnostics for detection and predictive modelling of endemic Fasciola hepatica infection in sheep and cattle on Australian farms.

The diagnosis, monitoring and flukicide efficacy testing of fasciolosis on-farm is reliant on non-terminal methods. The coproantigen ELISA (cELISA) has been recommended for diagnosis of fasciolosis and associated flukicide efficacy testing as an alternative to fluke egg counts for monitoring parasitism. Recently experimental multi-age infections have suggested that the reliability of efficacy results can be improved by a second cELISA testing at 6 weeks post-treatment (wpt) in addition to the generally accepted 1 wpt. A field study was conducted to determine the suitability of faecal fluke egg counts (FFEC) and cELISA as diagnostic, drug efficacy testing and epidemiological tools on Australian sheep and cattle farms. Faecal samples from sheep and/or cattle on three endemic farms were taken at monthly intervals for 12 months and examined by both methods. Normal farm management was maintained during the study period and opportunistic efficacy testing, in line with each farm's normal flukicide management was undertaken. Additionally, the suitability of the Ollerenshaw Index as a predictive model for fasciolosis under Australian conditions was examined. While both diagnostics demonstrated their value in the farm environment, the current data demonstrate a distinct and significant increase in diagnostic sensitivity for epidemiological studies by using the two tests in parallel. The agreement between the two diagnostics was found to be higher in cattle, despite the poor sensitivity of FFEC in this species. Similar levels of agreement between the two tests were demonstrated at both sheep properties, regardless of the marked difference in the intensity of F. hepatica challenge. Linear regression models demonstrated the results of the two diagnostics utilized in parallel were explained substantially (R2 = 0.91) as were series data (R2 = 0.88) when the respective models were fitted. In contrast, the fitted models for FFEC (R2 = 0.54) and cELISA (R2 = 0.58) were poor explanations for test outcomes. The outcomes of these models support previous findings that suggest that the two diagnostic tests are best utilized together, particularly in parallel. The application of the Ollerenshaw Index to Australian conditions requires further investigation.

Comparative assessment of faecal diagnostics for detection and predictive modelling of endemic Fasciola hepatica infection in sheep and cattle on Australian farms.

The diagnosis, monitoring and flukicide efficacy testing of fasciolosis on-farm is reliant on non-terminal methods. The coproantigen ELISA (cELISA) has been recommended for diagnosis of fasciolosis and associated flukicide efficacy testing as an alternative to fluke egg counts for monitoring parasitism. Recently experimental multi-age infections have suggested that the reliability of efficacy results can be improved by a second cELISA testing at 6 weeks post-treatment (wpt) in addition to the generally accepted 1 wpt. A field study was conducted to determine the suitability of faecal fluke egg counts (FFEC) and cELISA as diagnostic, drug efficacy testing and epidemiological tools on Australian sheep and cattle farms. Faecal samples from sheep and/or cattle on three endemic farms were taken at monthly intervals for 12 months and examined by both methods. Normal farm management was maintained during the study period and opportunistic efficacy testing, in line with each farm's normal flukicide management was undertaken. Additionally, the suitability of the Ollerenshaw Index as a predictive model for fasciolosis under Australian conditions was examined. While both diagnostics demonstrated their value in the farm environment, the current data demonstrate a distinct and significant increase in diagnostic sensitivity for epidemiological studies by using the two tests in parallel. The agreement between the two diagnostics was found to be higher in cattle, despite the poor sensitivity of FFEC in this species. Similar levels of agreement between the two tests were demonstrated at both sheep properties, regardless of the marked difference in the intensity of F. hepatica challenge. Linear regression models demonstrated the results of the two diagnostics utilized in parallel were explained substantially (R2 = 0.91) as were series data (R2 = 0.88) when the respective models were fitted. In contrast, the fitted models for FFEC (R2 = 0.54) and cELISA (R2 = 0.58) were poor explanations for test outcomes. The outcomes of these models support previous findings that suggest that the two diagnostic tests are best utilized together, particularly in parallel. The application of the Ollerenshaw Index to Australian conditions requires further investigation.

Characterization of multiple life stages of two Australian Fasciola hepatica isolates in sheep.

Information on the susceptibility status of Fasciola hepatica isolates is lacking in the literature, even for those isolates considered to be laboratory reference strains. Four controlled efficacy studies were conducted on two Fasciola hepatica isolates from Australia, viz. 'Oberon' and 'Sunny Corner' with treatment at either 2, 6 or 10 weeks post-infection (wpi) as defined in each study. Fluke burdens and examination of livers occurred at necropsy in weeks 12 (Study 1) or 13 (Studies 2, 3 and 4) post-infection. The triclabendazole (TCBZ) resistance status of the Oberon isolate was confirmed in 6 and 10-week old F. hepatica, utilizing the drug alone (Fasinex; 71.5% and 31.1%, respectively) and in combination with oxfendazole (Flukazole C; 79.9% and 0%, respectively). The susceptibility of this isolate to albendazole, as well as salicylanilide and sulphonamide drugs was confirmed. The Sunny Corner isolate was confirmed as susceptible to TCBZ (>99% all stages) and closantel (>90% at ≥6 wpi).

Application of a coproantigen ELISA as an indicator of efficacy against multiple life stages of Fasciola hepatica infections in sheep.

At present diagnosis of true resistance and determination of drug efficacy in Fasciola hepatica infection rely solely on terminal experiments. The coproantigen ELISA (cELISA) has been reported previously as a sensitive and specific tool appropriate to detect treatment failure, and potentially drug resistance. Two studies were conducted to determine whether the cELISA was appropriate for on-farm efficacy and resistance testing in Australian Merino sheep. In Study 1 sheep were infected orally with 50 F. hepatica metacercariae on three occasions, twelve, six and two weeks prior to a single flukicide treatment with triclabendazole, closantel or albendazole. Sheep were sampled weekly for a further seven weeks prior to necropsy. Following effective treatment, no faecal antigen was detected from 1 week. When immature stages (≤6 weeks) survived treatment, coproantigen reappeared from 6 weeks post-treatment. Therefore, cELISA conducted 1-4 weeks after treatment will demonstrate obvious treatment failure against adult F. hepatica, but is not sufficiently sensitive to detect survival of immature fluke until these reach maturity. In study 2, fluke burdens of sheep necropsied 13 weeks post single infection were compared to fecal worm egg counts (FWEC) and cELISA at necropsy. Regression analysis demonstrated that cELISA correlated strongly with fluke burden, whilst FWEC correlated weakly with cELISA. The correlation between FWEC and fluke burden was also weak, although stronger than that of FWEC with cELISA. The cELISA is an appropriate tool for monitoring effectiveness of treatments against Fasciola hepatica if an adult infection is present, however when immature stages of the parasite are present it is not as reliable. Where immature parasites are present it is recommended that initial cELISA be followed with a secondary cELISA at least 6 weeks after treatment to ensure resistance to immature stages is detected. Further testing is justified for monitoring the effectiveness of control programs by detecting adult populations that have survived a treatment regime.

Combination Therapy with NHS-muIL12 and Avelumab (anti-PD-L1) Enhances Antitumor Efficacy in Preclinical Cancer Models.

Purpose: To determine whether combination therapy with NHS-muIL12 and the anti-programmed death ligand 1 (PD-L1) antibody avelumab can enhance antitumor efficacy in preclinical models relative to monotherapies.Experimental Design: BALB/c mice bearing orthotopic EMT-6 mammary tumors and µMt- mice bearing subcutaneous MC38 tumors were treated with NHS-muIL12, avelumab, or combination therapy; tumor growth and survival were assessed. Tumor recurrence following remission and rechallenge was evaluated in EMT-6 tumor-bearing mice. Immune cell populations within spleen and tumors were evaluated by FACS and IHC. Immune gene expression in tumor tissue was profiled by NanoString® assay and plasma cytokine levels were determined by multiplex cytokine assay. The frequency of tumor antigen-reactive IFNγ-producing CD8+ T cells was evaluated by ELISpot assay.Results: NHS-muIL12 and avelumab combination therapy enhanced antitumor efficacy relative to either monotherapy in both tumor models. Most EMT-6 tumor-bearing mice treated with combination therapy had complete tumor regression. Combination therapy also induced the generation of tumor-specific immune memory, as demonstrated by protection against tumor rechallenge and induction of effector and memory T cells. Combination therapy enhanced cytotoxic NK and CD8+ T-cell proliferation and T-bet expression, whereas NHS-muIL12 monotherapy induced CD8+ T-cell infiltration into the tumor. Combination therapy also enhanced plasma cytokine levels and stimulated expression of a greater number of innate and adaptive immune genes compared with either monotherapy.Conclusions: These data indicate that combination therapy with NHS-muIL12 and avelumab increased antitumor efficacy in preclinical models, and suggest that combining NHS-IL12 and avelumab may be a promising approach to treating patients with solid tumors. Clin Cancer Res; 23(19); 5869-80. ©2017 AACR.

Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by next-generation sequencing.

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.

Effective control of a suspected cyromazine-resistant strain of Lucilia cuprina using commercial spray-on formulations of cyromazine or dicyclanil.

OBJECTIVE: To demonstrate the protection of Merino sheep from flystrike by Lucilia cuprina with cyromazine or dicyclanil in an implant study and in the field. METHODS: In the implant study, sheep were treated with cyromazine or dicyclanil and implanted with 1st-stage larvae from a newly isolated field strain of L. cuprina (CYR-LS) or a reference strain (DZR50), then assessed over 3 days and compared with the implants on untreated control sheep. In the field study, weaner lambs were treated with cyromazine or dicyclanil and monitored weekly for flystrike over 18 weeks of grazing on the same farm from which the L. cuprina were isolated. RESULTS: Implant study: cyromazine (6%) provided effective protection against CYR-LS and DZR50 L. cuprina for a minimum of 13 and 10 weeks, respectively. Dicyclanil (5%) provided at least 18 weeks' protection against both strains. Field study: only 1 of 386 lambs in the cyromazine-treated group was struck in the first 14 weeks of the trial. No strikes occurred in the 198 sheep treated with dicyclanil (5%). Rainfall, temperature and flytrap data indicated consistent fly pressure during the study. CONCLUSIONS: Based on the results of these studies, there was no evidence of reduced susceptibility to cyromazine or dicyclanil and the periods of protection of sheep against L. cuprina were unaffected and consistent with the registered label claims.

Rapid resistome fingerprinting and clonal lineage profiling of carbapenem-resistant Klebsiella pneumoniae isolates by targeted next-generation sequencing.

Thirty-two carbapenem-resistant Klebsiella pneumoniae isolates, representative of different resistance mechanisms and clonal lineages, were analyzed with the Pathogenica HAI BioDetection system, based on targeted next-generation sequencing (NGS) technology. With most strains, the system simultaneously yielded comprehensive information on relevant ß-lactam resistance determinants and accurate discrimination of clonal lineages, in a shorter time frame and in a less labor-intensive manner than currently available methods for molecular epidemiology analysis. Results supported the usefulness of targeted NGS-based technologies for similar applications.

A multi-parametric flow cytometric assay to analyze DNA-protein interactions.

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.

Ruler arrays reveal haploid genomic structural variation.

Despite the known relevance of genomic structural variants to pathogen behavior, cancer, development, and evolution, certain repeat based structural variants may evade detection by existing high-throughput techniques. Here, we present ruler arrays, a technique to detect genomic structural variants including insertions and deletions (indels), duplications, and translocations. A ruler array exploits DNA polymerase's processivity to detect physical distances between defined genomic sequences regardless of the intervening sequence. The method combines a sample preparation protocol, tiling genomic microarrays, and a new computational analysis. The analysis of ruler array data from two genomic samples enables the identification of structural variation between the samples. In an empirical test between two closely related haploid strains of yeast ruler arrays detected 78% of the structural variants larger than 100 bp.

RLS adaptive filtering for physiological interference reduction in NIRS brain activity measurement: a Monte Carlo study.

The non-invasive measurement of cerebral functional haemodynamics using near-infrared spectroscopy (NIRS) instruments is often affected by physiological interference. The suppression of this interference is crucial for reliable recovery of brain activity measurements because it can significantly affect the signal quality. In this study, we present a recursive least-squares (RLS) algorithm for adaptive filtering to reduce the magnitude of the physiological interference component. To evaluate it, we implemented Monte Carlo simulations based on a five-layer slab model of a human adult head with a multidistance source-detector arrangement, of a short pair and a long pair, for NIRS measurement. We derived measurements by adopting different interoptode distances, which is relevant to the process of optimizing the NIRS probe configuration. Both RLS and least mean squares (LMS) algorithms were used to attempt the removal of physiological interference. The results suggest that the RLS algorithm is more capable of minimizing the effect of physiological interference due to its advantages of faster convergence and smaller mean squared error (MSE). The influence of superficial layer thickness on the performance of the RLS algorithm was also investigated. We found that the near-detector position is an important variable in minimizing the MSE and a short source-detector separation less than 9 mm is robust to superficial layer thickness variation.