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The current four symptom screen recommended by the WHO is widely used as screen to initiate diagnostic testing for active pulmonary tuberculosis (TB), yet the performance is poor especially when TB prevalence is low. In contrast, more sensitive molecular tests are less suitable for the placement at primary care level in low resource settings. In order to meet the WHO End TB targets new diagnostic approaches are urgently needed to find the missing undiagnosed cases. Proteomics-derived blood host biomarkers have been explored because protein detection technologies are suitable for the point-of-care setting and could meet cost targets.This study aims to find a biomarker signature that fulfills WHO’s target product profile (TPP) for a TB screening. 12 blood-based protein biomarkers from three sample populations (Vietnam, Peru, South Africa) are analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin shows the most promising results for TB prediction throughout the datasets meeting the TPP for a triage test in adults from two countries (Peru and South Africa). The top performing individual markers identified at the global level (I-309 and SYWC) were also among the best performing markers at country level in South Africa and Vietnam.This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions based on one or two biomarkers with a performance that meets minimal WHO TPP criteria.Abstract ImportanceTuberculosis (TB) remains a serious worldwide health problem and diagnosis is hampered by the complexity of tests at primary care level in low resource settings or the low accuracy for screening settings. In order to meet the WHO End TB targets new diagnostic approaches are urgently needed to find the missing undiagnosed cases.This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions. We were able to construct an algorithm through statistical methods and machine learning algorithms whose performance meets the minimum of the WHO target product profile criteria. Thus, further work should be addressed at demonstrating that such as assay can be translated into a practical point-of-care test, and to better understand how to address regional differences in biomarker levels and responses.

In recent dog and cat experiments, a novel milk oligosaccharide biosimilar (GNU100) positively modulated fecal microbiota and metabolite profiles, suggesting benefits to gastrointestinal health. The objective of this study was to investigate the effects of GNU100 on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Twelve healthy adult female dogs (mean age: 3.74 ± 2.4 yr) were used in an 8-wk crossover design study (dogs underwent both treatments). All dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were orally administered metronidazole (20 mg/kg BW) twice daily. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, and 8 for measurement of pH, dry matter, microbiota populations, and BA, immunoglobulin A, and calprotectin concentrations. On weeks 0, 4, and 8, blood samples were collected for serum chemistry and hematology analysis. All data were analyzed as repeated measures using the Mixed Models procedure of SAS version 9.4, with significance considered P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools) and modified (P < 0.05) fecal microbiota and BA profiles. Using qPCR, metronidazole reduced fecal Blautia, Fusobacterium, Turicibacter, Clostridium hiranonis, and Faecalibacterium abundances, and increased fecal Streptococcus and Escherichia coli abundances. DNA sequencing analysis demonstrated that metronidazole reduced microbial alpha diversity and influenced the relative abundance of 20 bacterial genera and families. Metronidazole also increased primary BA and reduced secondary BA concentrations. Most antibiotic-induced changes returned to baseline by week 8. Fecal scores were more stable (P = 0.01) in GNU100-fed dogs than controls after antibiotic administration. GNU100 also influenced fecal microbiota and BA profiles, reducing (P < 0.05) the influence of metronidazole on microbial alpha diversity and returning some fecal microbiota and secondary BA to baseline levels at a quicker (P < 0.05) rate than controls. In conclusion, our results suggest that GNU100 supplementation provides benefits to dogs treated with antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and secondary BA profiles which play an essential role in gut health.

Only a fraction of patients benefits from immune checkpoint inhibitors (ICI). Tumor cells exhibit a distinct glycosylation pattern and supplementation of glycans has the potential to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a subcutaneous tumor mouse model, we investigated whether two glycopeptide-based test items reduce tumor burden when orally administered alone or in combination with ICI. C57/BL6 mice were supplemented with 3% GNU-201 (n=10) or 3% GNU-101 (n=10) in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice (n=10) received normal drinking water. On day 0, all mice were injected subcutaneously with YUMM1.7 melanoma cells in each flank. On days 6, 9 and 12, mice in each group received anti-PD1 antibody or an isotype control. Tumor size was measured every 3rd day. On day 15, the mice were sacrificed to analyze tumor weight and tumor infiltrating immune cells. 16S rDNA sequencing of fecal samples was performed on days -14, 0 and 14. GNU-201 and GNU-101 supplementation was well tolerated, no difference in water consumption and no abnormal behavior were detected that would indicate adverse effects of the products. While anti-PD1 on its own had no effect on tumor growth, we observed reduced tumor growth in the GNU-201+anti-PD1, GNU-101 and GNU-101+anti-PD1-treated groups, resulting in a significantly reduced tumor volume in these three groups on day 15. Administration of the two test items had a clear effect on anti-tumor T cell responses. In combination with anti-PD1, GNU-201 promoted the overall abundance of T cells, and among those the proportion of CD8+ T cells and IFNγ producing CD4+ T cells. Even in absence of anti-PD1, GNU-201 promoted the number of IFNγ+ CD8+ T cells, indicating that GNU-201 did not only promote tumor infiltration but also activation of CD8+ T cells, which are known to directly kill tumor cells. Finally, GNU-201+anti-PD1 resulted in significantly increased levels of Perforin and TNFα producing CD8+ T cells. With GNU-101 there was a trend towards elevated numbers of T cells and TNFα+ CD4 T cells. In mice that received GNU-101 with anti-PD1, we observed significantly elevated levels of TNFα+ CD8+ T cells and a trend towards higher numbers of IFNγ+ CD8+ T cells. GNU-201 and GNU-101 induced a significant modulation of the gut microbiota. In particular, the products promoted the growth of Akkermansia spp., a genus previously associated with anti-tumor immunity. In conclusion, GNU-201 and GNU-101 are both able to boost an anti-tumor immune response upon PD1 inhibition and GNU-101 shows moderate anti-tumor effects on its own. The response is linked with tumor-infiltration and activation of T cells and seems to be associated with changes in the gut microbiome. This study highlights a potential therapeutic role for glycopeptides in boosting anti-tumor immunity in response to ICI therapy. Citation Format: Marianne R. Spalinger, Romain Wyss, Sara Vidal, Yong Miao, Michael Scharl, Yemi Adesokan. Glycopeptides promote anti-cancer immune response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5600.

2565 Background: Only a minority of colorectal cancer (CRC) patients benefit from immune checkpoint inhibitors (ICI), with less than half of eligible patients responding to the treatment. Abnormal glycosylation of tumor cells is a signature of cancer development. Here, we described novel immunomodulatory molecules, named GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). We hypothesized that GLAAD, by sharing similarities with aberrant glycans expressed in many cancers, may be a safe and effective treatment strategy to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a CRC mouse model, we tested whether two GLAAD candidates reduce tumor burden when orally administered alone or in combination with ICI. Methods: C57/BL6J mice were supplemented with 3% GNU-201 or 3% GNU-101 in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice received normal drinking water. On day 0, all mice were injected subcutaneously with MC38 CRC cells in each flank. On days 6, 9, 12, and 15, mice in each group received anti-PD1 antibody (aPD1) or an isotype control. Tumor size was measured every 3rd day. On day 18, the mice were sacrificed. Results: The aPD1 therapy alone showed a limited anti-tumor efficacy overall. Groups treated with GNU-201, GNU-101, GNU-201+aPD1 or GNU-101+aPD1 exhibited reduced growth, resulting in a significantly reduced tumor volume vs control on day 18. In contrast, GLAAD candidates alone significantly reduced tumor volume compared with control and led to a numerically larger decrease than aPD1 treatment alone, suggesting a higher efficacy. When analyzing tumor infiltrating myeloid cells on day 18, no differences were seen for neutrophils or dendritic cells but the proportion of macrophages was decreased in mice receiving GNU-201 or GNU-101. GNU-101 resulted in reduced serum levels of IL-10, a molecule participating in the suppression of antitumor immune responses. The tested GLAAD candidates stimulated robust anti-tumor T cell responses. GNU-201 and GNU-101 supplementation alone or in combination with aPD1 increased the proportion of T cells and among those CD8+ T cells, IFNγ producing CD4+ T cells, IFNγ+ and perforin+ CD8+ T cells. GNU-101 and GNU-201 elicited memory T cell responses after priming of the mice, measured as the ex vivo recall response (CD69 expression) of CD8+ T cells isolated from the tumor. Conclusions: GNU-201 and GNU-101 as standalone treatment induce a strong anti-tumor response that is as good as or even better than aPD1 alone. The therapeutic efficacy is associated with increased tumor infiltration and activation of T cells, in particular CD8+ T cells, highlighting the role of these GLAAD candidates as potent adjuvants in supporting specific tumor-associated antigen recognition by the immune system.

A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone., A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone. We depict results based on performance targets set by the WHO in comparison with a laboratory-based C-reactive protein (CRP) assay. Of 201 patients included, 67 were culture positive for Mycobacterium tuberculosis. The areas under the concentration-time curve (AUCs) for Xpert-MTB-HR-Prototype were 0.89 (confidence interval [CI], 0.83 to 0.94) against the CMRS and 0.94 (CI, 0.89 to 0.98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at the nearest upper value of sensitivity to 90%), specificities were 55.8% (CI, 47.2 to 64.1%) compared to the CMRS and 85.9% (CI, 79.3 to 90.7%) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65.7% (CI, 53.7 to 75.9%), while the CRP assay achieved a sensitivity of only 13.6% (CI, 7.3 to 23.4%). In this first accuracy study of a prototype blood-based host marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.

Milk oligosaccharides (MO) are bioactive compounds in mammalian milk that provide health benefits to neonates beyond essential nutrients. GNU100, a novel animal MO biosimilar, was recently tested in vitro, with results showing beneficial shifts in microbiota and increased short-chain fatty acid (SCFA) production, but other effects of GNU100 were unknown. Three studies were conducted to evaluate the safety, palatability, and gastrointestinal (GI) tolerance of GNU100. In study 1, the mutagenic potential of GNU100 was tested using a bacterial reverse mutation assay and a mammalian cell micronucleus test. In study 2, palatability was assessed by comparing diets containing 0% vs. 1% GNU100 in 20 adult dogs. In study 3, 32 adult dogs were used in a completely randomized design to assess the safety and GI tolerance of GNU100 and explore utility. Following a 2-wk baseline, dogs were assigned to one of four treatments and fed for 26 wk: 0%, 0.5%, 1%, and 1.5% GNU100. On weeks 2, 4, and 26, fresh fecal samples were collected to measure stool quality, immunoglobulin A, and calprotectin, and blood samples were collected to measure serum chemistry, inflammatory markers, and hematology. On weeks 2 and 4, fresh fecal samples were collected to measure metabolites and microbiota. On week 4, total feces were collected to assess apparent total tract macronutrient digestibility. Although revertant numbers were greater compared with the solvent control in tester strain WP2uvrA(pKM101) in the presence of metabolic activation (S9) in the initial experiment, they remained below the threshold for a positive mutagenic response in follow-up confirmatory tests, supporting that GNU100 is not mutagenic. Similarly, no cytotoxicity or chromosome damage was observed in the cell micronucleus test. The palatability test showed that 1% GNU100 was strongly preferred (P < 0.05; 3.6:1 consumption ratio) over the control. In study 3, all dogs were healthy and had no signs of GI intolerance or illness. All diets were well accepted, and food intake, fecal characteristics, metabolite concentrations, and macronutrient digestibilities were not altered. GNU100 modulated fecal microbiota, increasing evenness and Catenibacterium, Megamonas, and Prevotella (SCFA producers) and reducing Collinsella. Overall, the results suggest that GNU100 is palatable and well-tolerated, causes no genotoxicity or adverse effects on health, and beneficially shifts the fecal microbiota, supporting the safety of GNU100 for the inclusion in canine diets.

GNU100 is a novel animal milk oligosaccharide (AMO) biosimilar. In a recent in vitro fermentation study, GNU100 was shown to be fermentable by feline gastrointestinal microbiota and lead to increased short-chain fatty acid production. Our objectives herein were to evaluate the palatability, safety, and gastrointestinal tolerance of GNU100 in healthy adult cats. Exploratory end-points were measured to assess utility. In study 1, 20 adult cats were used to test the palatability of diets containing 0% or 1% GNU100. In study 2, 32 (mean age = 1.9 yr; mean body weight = 4.6 kg) male (n = 12) and female (n = 20) adult cats were used in a completely randomized design. After a 2-wk baseline, cats were assigned to one of the following treatment groups and fed for 26 wk: control (CT, no GNU100), low dose (LD, 0.5% GNU100), medium dose (MD, 1.0% GNU100), and high dose (HD, 1.5% GNU100). On weeks 2, 4, and 26, fresh fecal samples were collected for the measurement of stool quality and immune and inflammatory markers and on weeks 2 and 4 for microbiota and metabolites. On week 4, total feces were collected to measure apparent total tract macronutrient digestibility. On weeks 2, 4, and 26, blood samples were collected for serum chemistry, hematology, and inflammatory marker measurement. The palatability test showed that 1% GNU100 was strongly preferred (P < 0.05), with GNU100 having a 17.6:1 consumption ratio compared with control. In the long-term study, all cats remained healthy, without any signs of gastrointestinal intolerance or illness. All diets were well accepted, resulting in similar (P > 0.05) food intake, fecal characteristics, immunoglobulin A, and calprotectin, and dry matter, organic matter, fat, and crude protein digestibilities. Fecal butyrate was greater (P = 0.02) in cats fed HD than cats fed LD or MD. Fecal indole was lower (P = 0.02) in cats fed HD than cats fed LD. Cats fed CT had a higher (P = 0.003) relative abundance of Actinobacteria than cats fed LD. The relative abundance of Peptococcus was impacted by diet and time. At 4 wk, Campylobacter was lower in fecal samples of cats fed HD. Overall, the data suggest that dietary GNU100 supplementation was highly palatable, well tolerated, did not cause detrimental effects on fecal quality or nutrient digestibility, increased fecal butyrate concentrations, and reduced fecal indole concentrations, supporting the safety of GNU100 for inclusion in feline diets and suggesting potential benefits on gastrointestinal health of cats.

Milk oligosaccharides (MO) confer multiple potential physiological benefits, such as the selective growth promotion of beneficial microbiota, inhibition of enteric pathogen growth and adhesion to enterocytes, maturation of the gut mucosal barrier, and modulation of the gastrointestinal immune system. This study was conducted to determine the fermentation potential of GNU100, an animal MO biosimilar, in an in vitro system using healthy canine and feline fecal inocula. Single feline and single canine fecal samples were used to inoculate a batch fermentation system. Tubes containing a blank control (BNC), GNU100 at 0.5% (5 g/L; GNU1), or GNU100 at 1.0% (10 g/L; GNU2) were incubated for 48 h. Gas pressure, pH, lactate, short-chain fatty acids (SCFA; acetate, propionate, and butyrate), and branched-chain fatty acids (BCFA; isobutyrate, isovalerate, and valerate) were measured after 6, 24, and 48 h. Ammonium and microbiota (total bacteria by flow cytometry and Pet-16Seq; Lactobacillus and Bifidobacterium by quantitative polymerase chain reaction ) were measured after 24 and 48 h. Data were analyzed using the Mixed Models procedure of SAS. Substrates were considered to be a fixed effect and replicates considered to be a random effect. Tukey’s multiple comparison analysis was used to compare least squares means, with differences considered significant with P < 0.05. In feline and canine incubations, SCFA increases were greater (P < 0.0001) in GNU100 compared with BNC, with acetate making up the largest SCFA proportion (P < 0.0001). GNU100 cultures led to greater increases (P < 0.0001) in lactate and ammonium than BNC in the feline incubations. GNU100 cultures led to greater increases (P < 0.0001) in ammonium than BNC in canine incubations and greater increases (P < 0.0001) in BCFA than BNC in feline incubations. Pet-16Seq microbial profiles from the feline and canine fecal incubations exhibited a modulation after GNU100 fermentation, with a reduction of the genera Escherichia/Shigella and Salmonella. In feline incubations, Bifidobacterium populations had greater increases (P < 0.0001) in GNU100 than BNC. In feline incubations, Lactobacillus populations had greater increases (P = 0.01) in GNU100 than BNC, with GNU1 leading to greater increases (P = 0.02) in Lactobacillus than BNC tubes in canine incubations. Overall, this study demonstrated that GNU100 was fermented in an in vitro fermentation system inoculated with canine and feline microbiota, resulting in the growth of beneficial bacteria and the production of SCFA, BCFA, and ammonium.

Introduction A highly sensitive triage test that captures most symptomatic patients at increased likelihood of having pulmonary tuberculosis (PTB) would ‘rule-out’ lower-risk patients from expensive confirmatory testing. Although studies have assessed the diagnostic accuracy of a C-reactive protein (CRP) triage test for PTB in HIV+ patients, limited data are available from HIV- cohorts. Materials and methods In this retrospective case-control study, 765 serum samples were selected from FIND’s biobank. Each sample had been collected from an adult presenting with respiratory symptomatology to district hospitals in South Africa and referral hospitals in Cambodia, Peru, Georgia and Vietnam between 2007–2017. Serum CRP measurements were obtained using a laboratory-based assay. CRP cutoff-points of ≥8mg/L and ≥10mg/L were predefined as a positive triage test result. The PTB reference standard was two contemporaneously collected sputum liquid culture results. Results CRP demonstrated an overall sensitivity for PTB of 79.8% (95%CI 75.5–83.5) and 77.7% (95%CI 73.4–81.6) for cutoff-points of 8mg/L and 10mg/L respectively. Specificity was 62.8% (95%CI 57.8–67.6%) and 66.6% (95%CI 61.1–70.7) respectively. Area-under-the-curve using Receiver Operating Characteristic analysis was 0.77 (95%CI 0.74–0.81). Threshold analysis showed optimal CRP cutoff-points were higher in HIV+ than HIV- participants. An algorithm in which CRP triage was followed by confirmatory Xpert MTB/Rif testing achieved a sensitivity of 75.1% (95%CI 69.0–80.4%) whilst decreasing Xpert usage by 40.6%. Discussion CRP may not meet the challenge of a catch-all TB triage test. However, it shows promise in HIV+ individuals. Further research is required in a prospective study using point-of-care platforms to further evaluate its capabilities.

A non-sputum triage test to rule out TB disease is a WHO high-priority diagnostic and a combinatory score based on a 3-gene host-signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge-assay (“Xpert MTB Host Response”, or Xpert-MTB-HR-Prototype) of this 3-gene signature on bio-banked blood-samples from PLHIV against a comprehensive microbiological reference standard (CMRS) and against Xpert® MTB/RIF on first sputum alone. We depict results based on performance targets set by WHO in comparison with a laboratory-based CRP assay. Of 201 patients included, 67 were culture-positive for Mycobacterium tuberculosis. AUC for the Xpert-MTB-HR-Prototype was 0·89 (CI 0·83-0·94) against the CMRS and 0·94 (CI 0·89-0·98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at nearest upper value of sensitivity to 90%), specificity was 55·8% (CI 47·2-64·1) compared to the CMRS and 85·9% (CI 79·3-90·7) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65·7% (CI 53·7-75·9) while CRP achieved a sensitivity of only 13·6% (CI 7·3-23·4). In this first accuracy study of a prototype blood-based host-marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.One Sentence SummaryThis is an accuracy study of a blood-based host-marker assay demonstrating its potential in triage and diagnosis of active tuberculosis (TB) in people living with HIV (PLHIV).

The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a noninvasive diagnostic and monitoring tool is especially promising for tuberculosis (TB), as it enables the detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting Mycobacterium tuberculosis DNA in the blood and urine of TB patients with nongenitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for the diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29% to 79% and 67% to 100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker.

The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease., The detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a noninvasive diagnostic and monitoring tool is especially promising for tuberculosis (TB), as it enables the detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting Mycobacterium tuberculosis DNA in the blood and urine of TB patients with nongenitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for the diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29% to 79% and 67% to 100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker.

International audience; There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

HsSAS-6 homodimers are present in the cytoplasm and assemble into ninefold symmetrical arrays at centrosomes, thus initiating procentriole formation., SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules., This study of fission yeast reveals that the active and inactive forms of the small GTPase Cdc42 have different rates of lateral diffusion in the membrane, providing insights into how it becomes spontaneously polarized, thereby determining the polarity of the cell., Author Summary Cell polarization is a critical feature of most cells that underlies their functional organization. A central polarity factor called Cdc42, a small GTPase targeted to the plasma membrane by prenylation, promotes cell polarization in its active GTP-bound form. Cdc42 is a key polarity factor because it accumulates at presumptive sites of polarity, which previous work suggested involves Cdc42 recycling on and off the plasma membrane. In addition, its activity can spontaneously polarize cells in a single location by self-enhancing positive feedback mechanisms, even in the absence of any pre-localized landmarks. In this study, we constructed the first functional fluorescently tagged allele of Cdc42 that replaces the endogenous genomic copy in Schizosaccharomyces pombe. This allowed measurements of Cdc42 dynamics at the plasma membrane by live microscopy. Unexpectedly, this approach revealed that Cdc42 primarily moves through lateral diffusion, rather than on and off the plasma membrane. Engineered Cdc42 alleles with alternative membrane-targeting mechanisms demonstrated that Cdc42 activity, indeed, polarizes in the absence of known pathways that recycle Cdc42 on and off the membrane. We further show that the active form, Cdc42-GTP, is less mobile than Cdc42-GDP. We thus propose that Cdc42 polarization occurs as a consequence of its local activation—either through self-enhanced feedback or in response to upstream cues—by a reduction in the active Cdc42 diffusion rate.

We report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.

Reference EPFL-CONF-201047View record in Web of Science Record created on 2014-08-29, modified on 2016-08-09

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.

Nanopores are under development for the detection of a variety of analytes and the investigation of chemical reactions at the single molecule level. In particular, the analysis of nucleic acid molecules is under intense investigation, including the development of systems for rapid, low-cost DNA sequencing. Here, we show that DNA can be translocated through an engineered alpha HL protein pore at pH 11.7, a value at which dsDNA is denatured. Therefore, the aHL pore is sufficiently stable to entertain the possibility of direct nanopore sequencing of genomic dsDNA samples, which are more readily obtained and handled than ssDNA. ispartof: Nano Letters vol:9 issue:11 pages:3831-3836 ispartof: location:United States status: published

Cellular signaling reactions are classically investigated by measuring optical or electrical properties of individual living cells or suspensions of cells. Here we show how the binding of ligands to cell surface receptors and the subsequent activation of signaling reactions can be monitored in single, sub-micrometer sized native vesicles with single molecule sensitivity. The native vesicles are derived from live mammalian cells either by incubation with chemicals (e.g. cytochalasin) known to destabilize the interaction of the cytoskeleton with the plasma membrane, or by micromanipulation using optical tweezers. Such native vesicles comprise parts of the plasma membrane and the cytosol of the mother cell. They represent the smallest autonomous containers capable of performing cellular signaling reactions thus functioning like minimal cells. To demonstrate this, we measured in individual vesicles derived from individual cells with single molecule resolution the different steps of G protein-coupled receptor mediated signalling like ligand binding to receptors, subsequent G protein activation and finally receptor deactivation by interaction with arrestin. The observation of cellular signalling reactions in individual (sub)micrometer sized vesicles opens the door to downscale the analysis of cellular functions to the atto- and femtoliter range for multiplexing single cell analysis or investigating receptor mediated signalling in multiarray format.

We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8711-5) contains supplementary material, which is available to authorized users.

A central goal in bioanalytics is to determine the concentration of, and interactions between biomolecules. Nanotechnology offers the possibility to perform such analyses in a highly parallel, low cost and miniaturized fashion. Here we report on label-free counting, mobility and volume analysis of single molecules and nanoparticles during their diffusion through a sub-attoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Non-fluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration and mobility of the non-fluorescent analytes through fluctuation analysis in both time and amplitude. In order to fully benefit from the array of holes and its high capacity of parallelization, the device allows wide-field illumination and CCD detection. This arrangement simplifies instrumentation compared to confocal microscopy and opens new possibilities to detect macromolecules and particles of pharmaceutical importance such as viruses.

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellulard communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.

Reference EPFL-CONF-186437View record in Web of Science Record created on 2013-05-13, modified on 2017-12-03

International audience; Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.