Reducing sources of variance in experimental procedures in in vitro research

Background: Lack of reproducibility in preclinical research poses ethical and economic challenges for biomedical science. Various institutional activities by society stakeholders of leading industrialised nations are currently underway with the aim of improving the situation. Such initiatives are usually concerned with high-level organisational issues and typically do not focus on improving experimental approaches per se. Addressing these is necessary in order to increase consistency and success rates of lab-to-lab repetitions. Methods: In this project, we statistically evaluated repetitive data of a very basic and widely applied lab procedure, namely quantifying the number of viable cells. The purpose of this was to assess the impact of different parameters and instrumentations which may constitute sources of variance in this procedure. Conclusion: By comparing the variability of data acquired under two different procedures, featuring improved stringency of protocol adherence, our project attempts to identify the sources and propose guidelines on how to reduce such fluctuations. We believe our work can contribute to tackling the repeatability crisis in biomedical research.

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Reducing sources of variance in experimental procedures in in vitro research

F1000Research ◽

10.12688/f1000research.73497.1 ◽

2021 ◽

Vol 10 ◽

pp. 1037

Author(s):

Igor Fischer ◽

Maria Victoria Martinez-Dominguez ◽

Daniel Hänggi ◽

Ulf Kahlert

Keyword(s):

Biomedical Science ◽

Success Rates ◽

Preclinical Research ◽

Viable Cells ◽

Leading Industry ◽

Experimental Approaches ◽

Per Se ◽

High Level ◽

The Impact

Background: Lack of reproducibility in preclinical research is a problem posing ethical and economic challenges for biomedical science. Various institutional activities from society stakeholders of leading industry nations are currently underway to improve the situation. Such initiatives usually attempt to tackle high-level organisational issues and do not typically focus on improving experimental approaches per se. Addressing these is necessary in order to increase consistency and success rates of lab-to-lab repetitions. Methods: In this project, we statistically evaluated repetitive data of a very basic and widely applied lab procedure, namely quantifying the number of viable cells. The purpose of this was to appreciate the impact of different parameters and instrumentations that may constitute sources of variance in this procedure. Conclusion: By comparing the variations of data acquired under two different procedures, featuring improved stringency of protocol adherence, our project attempts to propose guidelines on how to reduce such variations. We believe our work can contribute to tackling the repeatability crisis in biomedical research.

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Evaluation of amoxicillin-clavulanic acid resistance in the Bifidobacterium genus

Applied and Environmental Microbiology ◽

10.1128/aem.03137-20 ◽

2021 ◽

Author(s):

Leonardo Mancabelli ◽

Walter Mancino ◽

Gabriele Andrea Lugli ◽

Chiara Argentini ◽

Giulia Longhi ◽

...

Keyword(s):

Gut Microbiota ◽

Clavulanic Acid ◽

Acid Resistance ◽

Resistance Mechanisms ◽

Western World ◽

Beneficial Effects ◽

Amoxicillin Clavulanic Acid ◽

High Level ◽

The Impact

Amoxicillin-Clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding six months. Moreover, we evaluated AMC sensitivity by Minimal Inhibitory Concentration (MIC) test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains, which exhibit a high level of AMC insensitivity, and which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch-cultures. Importance Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, also affecting the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genome revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in-vitro experiments revealed that one strain, i.e. B. breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

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Clavulanate Induces Expression of the Pseudomonas aeruginosa AmpC Cephalosporinase at Physiologically Relevant Concentrations and Antagonizes the Antibacterial Activity of Ticarcillin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.4.882 ◽

1999 ◽

Vol 43 (4) ◽

pp. 882-889 ◽

Cited By ~ 39

Author(s):

Philip D. Lister ◽

Victoria M. Gardner ◽

Christine C. Sanders

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibacterial Activity ◽

Bactericidal Effect ◽

Pharmacodynamic Model ◽

Immunocompromised Patients ◽

Minimal Level ◽

Quantitative Studies ◽

Per Se ◽

The Impact

ABSTRACT Although previous studies have indicated that clavulanate may induce AmpC expression in isolates of Pseudomonas aeruginosa, the impact of this inducer activity on the antibacterial activity of ticarcillin at clinically relevant concentrations has not been investigated. Therefore, a study was designed to determine if the inducer activity of clavulanate was associated with in vitro antagonism of ticarcillin at pharmacokinetically relevant concentrations. By the disk approximation methodology, clavulanate induction of AmpC expression was observed with 8 of 10 clinical isolates of P. aeruginosa. Quantitative studies demonstrated a significant induction of AmpC when clavulanate-inducible strains were exposed to the peak concentrations of clavulanate achieved in human serum with the 3.2- and 3.1-g doses of ticarcillin-clavulanate. In studies with three clavulanate-inducible strains in an in vitro pharmacodynamic model, antagonism of the bactericidal effect of ticarcillin was observed in some tests with regimens simulating a 3.1-g dose of ticarcillin-clavulanate and in all tests with regimens simulating a 3.2-g dose of ticarcillin-clavulanate. No antagonism was observed in studies with two clavulanate-noninducible strains. In contrast to clavulanate, tazobactam failed to induce AmpC expression in any strains, and the pharmacodynamics of piperacillin-tazobactam were somewhat enhanced over those of piperacillin alone against all strains studied. Overall, the data collected from the pharmacodynamic model suggested that induction per se was not always associated with reduced killing but that a certain minimal level of induction by clavulanate was required before antagonism of the antibacterial activity of its companion drug occurred. Nevertheless, since clinically relevant concentrations of clavulanate can antagonize the bactericidal activity of ticarcillin, the combination of ticarcillin-clavulanate should be avoided when selecting an antipseudomonal β-lactam for the treatment of P. aeruginosa infections, particularly in immunocompromised patients. For piperacillin-tazobactam, induction is not an issue in the context of treating this pathogen.

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Tumor Cells Resistant to Alemtuzumab Complement Mediated Cytotoxicity in Patients with High Risk Previously Untreated Early Stage CLL: A Possible Mechanism of Treatment Failure.

Blood ◽

10.1182/blood.v106.11.2973.2973 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2973-2973

Author(s):

Clive S. Zent ◽

Nancy D. Bone ◽

Susan M. Geyer ◽

Neil E. Kay

Keyword(s):

Flow Cytometry ◽

B Cells ◽

High Risk ◽

B Cell ◽

Cell Clone ◽

Early Stage ◽

Cell Membrane Permeability ◽

Viable Cells ◽

The Impact

Abstract The monoclonal antibodies (MoAb) alemtuzumab and rituximab have proven efficacy in the treatment of CLL. In addition, alemtuzumab is effective in patients with defective p53 function responding poorly to purine analogue therapy. The action of both MoAb is not completely understood. Proposed mechanisms include complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and direct induction of apoptosis of CLL B cells. We have done correlative studies on CLL B cells from patients enrolled in a trial of alemtuzumab and rituximab in “high risk” early stage previously untreated CLL to determine: 1. Role of apoptosis induction and CDC in each MoAb and 2. If the addition of rituximab to alemtuzumab increases their in vitro cytotoxicity. Patients and Methods: Patients with early stage, previously untreated, high risk CLL are treated with subcutaneous alemtuzumab (dose escalation over 3 days then 30 mg Mon-Wed-Fri for 4 weeks) and rituximab (375 mg/m2/dose weekly from day 8 x 4 doses). High risk disease was defined as one or more of the following features of the CLL B cell clone: (1) 17p13−; (2) 11q22−; (3) unmutated IgVH (< 2%) and either CD38+ or ZAP-70+. Blood B lymphocytes collected prior to the start of therapy were tested for response to MoAb in vitro. Cells were cultured at 2 x 106/ml in AIM-V medium using standard conditions. Alemtuzumab and rituximab were used at 20 μg/ml and complement as 10% of 40 CH50 units/ml human serum. The impact of the MoAb was measured by counting viable cells (trypan blue negative) and measuring early apoptosis (annexin V) and cell death (cell membrane permeability to propidium iodide) using flow cytometry at 1 hour, and then daily for 3 days. Results: Treatment caused rapid resolution of lymphocytosis in all 7 patients and 3 patients were negative for circulating CLL cells using a highly sensitive 3 color flow cytometry (CD5+/CD19+/CD79b-) after therapy. All patients had a clinical response (2 CR, 5 PR). Alemtuzumab and complement were rapidly cytotoxic to most CLL cells. Mean cell viability was 39% (sd: 8%) after 1 hour of incubation. Cytotoxicity was similar in all samples irrespective of FISH defects, IgVH mutation status, and in vitro resistance to F-ara-A (n = 3). Alemtuzumab was inactive in the absence of complement for all samples. Rituximab alone and together with complement did not induce cytotoxicity or apoptosis. However, the addition of rituximab to alemtuzumab and complement did increase CDC where the number of viable cells was significantly lower at 1, 24, 48, and 72 hours incubation (p = 0.075, 0.047, 0.031, 0.027, respectively, for pairwise comparisons). CLL cells surviving alemtuzumab CDC subsequently had a lower level of apoptosis than control cells, implying a selection for resistant cells. Alemtuzumab CDC on this residual population was not increased at higher concentrations of alemtuzumab or complement. This mechanism of CDC resistance is currently under investigation. Conclusion: These data suggest that alemtuzumab CDC is an important mechanism of action in patients with CLL. However, alemtuzumab CDC kills only about 61% of CLL cells in vitro, and the surviving cells are more resistant to spontaneous apoptosis. This suggests that cells that survive alemtuzimab CDC contribute to disease progression or relapse. We intend to elucidate the mechanism of this resistance using our in vitro model with the hope that treatment strategies can be deployed to remove this residual CLL B cell clone.

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EGFRAmplification and Glioblastoma Stem-Like Cells

Stem Cells International ◽

10.1155/2015/427518 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 17

Author(s):

Katrin Liffers ◽

Katrin Lamszus ◽

Alexander Schulte

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Current Knowledge ◽

Growth Factor Receptor ◽

Model Systems ◽

Preclinical Research ◽

Epidermal Growth ◽

Methodological Approaches ◽

The Impact

Glioblastoma (GBM), the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells). GS-cells can be maintainedin vitrousing serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR) gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retainedEGFRamplification could overcome the limitations of currentin vitromodel systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with differentEGFRstatus in order to maintain EGFR-dependent intratumoral heterogeneityin vitro. Further, it will summarize the current knowledge about the impact ofEGFRamplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM.

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Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

10.1101/2021.06.07.447394 ◽

2021 ◽

Author(s):

Rui Yang ◽

Wenzhe Wang ◽

Meichen Dong ◽

Kristen Roso ◽

Paula Greer ◽

...

Keyword(s):

Cancer Cells ◽

Tumor Cells ◽

Target Genes ◽

De Novo ◽

Inhibitory Effect ◽

Nucleotide Synthesis ◽

High Level ◽

The Impact

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

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Evaluating the Rapid Emergence of Daptomycin Resistance in Corynebacterium: A Multi-Center Study

Journal of Clinical Microbiology ◽

10.1128/jcm.02052-20 ◽

2021 ◽

Author(s):

Kaitlin F. Mitchell ◽

Erin McElvania ◽

Meghan A. Wallace ◽

Lauren E. Droske ◽

Amy E. Robertson ◽

...

Keyword(s):

Antimicrobial Agents ◽

Exposure Period ◽

Corynebacterium Striatum ◽

Before And After ◽

Multi Center Study ◽

High Level ◽

The Impact ◽

Repeated Subculture ◽

Center Study

Background: Members of the genus Corynebacterium are increasingly recognized as pathobionts and can be very resistant to antimicrobial agents. Previous studies have demonstrated that Corynebacterium striatum can rapidly develop high-level daptomycin resistance (HLDR) (minimum inhibitory concentration [MIC] ≥256 μg/mL). Here we conducted a multi-center study to assay for this in vitro phenotype in diverse Corynebacterium species. Methods: Corynebacterium clinical isolates (n=157) from four medical centers were evaluated. MIC values to daptomycin, vancomycin, and telavancin were determined before and after overnight exposure to daptomycin to identify isolates able to rapidly develop daptomycin non-susceptibility. To investigate assay reproducibility, 18 isolates were evaluated at three study sites. In addition, stability of daptomycin non-susceptibility was tested using repeated subculture without selective pressure. The impact of different media brands was also investigated. Results: Daptomycin non-susceptibility emerged in 12 of 23 species evaluated in this study (C. afermentans, amycolatum, aurimucosum, bovis, jeikeium, macginleyi, pseudodiphtheriticum, resistens, simulans, striatum, tuberculostearicum, and ulcerans) and was detected in 50 of 157 (31.8%) isolates tested. All isolates displayed low (susceptible) MIC values to vancomycin and telavancin before and after daptomycin exposure. Repeated subculture demonstrated 2 of 9 isolates (22.2%) exhibiting HLDR reverted to a susceptible phenotype. Of 30 isolates tested on three media brands, 13 (43.3%) had differences in daptomycin MIC values between brands. Conclusions: Multiple Corynebacterium species can rapidly develop daptomycin non-susceptibility, including HLDR, after a short daptomycin exposure period.

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Modeling Prostate Cancer Treatment Responses in the Organoid Era: 3D Environment Impacts Drug Testing

Biomolecules ◽

10.3390/biom11111572 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1572

Author(s):

Annelies Van Hemelryk ◽

Lisanne Mout ◽

Sigrun Erkens-Schulze ◽

Pim J. French ◽

Wytske M. van Weerden ◽

...

Keyword(s):

Prostate Cancer ◽

Treatment Effect ◽

Drug Testing ◽

Nuclear Translocation ◽

Single Cells ◽

Great Promise ◽

Preclinical Research ◽

3D Scaffolds ◽

The Impact

Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, experimental variability in organoid drug testing complicates reproducibility. For example, we observed PCa organoids to be less affected by cabazitaxel, abiraterone and enzalutamide as compared to corresponding single cells prior to organoid assembly. We hypothesized that three-dimensional (3D) organoid organization and the use of various 3D scaffolds impact treatment efficacy. Live-cell imaging of androgen-induced androgen receptor (AR) nuclear translocation and taxane-induced tubulin stabilization was used to investigate the impact of 3D scaffolds, spatial organoid distribution and organoid size on treatment effect. Scaffolds delayed AR translocation and tubulin stabilization, with Matrigel causing a more pronounced delay than synthetic hydrogel as well as incomplete tubulin stabilization. Drug effect was further attenuated the more centrally organoids were located in the scaffold dome. Moreover, cells in the organoid core revealed a delayed treatment effect compared to cells in the organoid periphery, underscoring the impact of organoid size. These findings indicate that analysis of organoid drug responses needs careful interpretation and requires dedicated read-outs with consideration of underlying technical aspects.

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Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1853 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1853-1857 ◽

Cited By ~ 138

Author(s):

D. L. Williams ◽

L. Spring ◽

L. Collins ◽

L. P. Miller ◽

L. B. Heifets ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cross Resistance ◽

In Vitro Mutagenesis ◽

Missense Mutations ◽

Resistance Mutations ◽

Development Of Resistance ◽

High Level ◽

The Impact ◽

Level Resistance

ABSTRACT The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutantrpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoBmutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specificrpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

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Situating Second-Year Success: Understanding Second-Year STEM Experiences at a Liberal Arts College

CBE—Life Sciences Education ◽

10.1187/cbe.16-01-0044 ◽

2016 ◽

Vol 15 (3) ◽

pp. ar43 ◽

Cited By ~ 5

Author(s):

Leslie Gregg-Jolly ◽

Jim Swartz ◽

Ellen Iverson ◽

Joyce Stern ◽

Narren Brown ◽

...

Keyword(s):

Liberal Arts ◽

Student Attitudes ◽

Future Research ◽

Success Rates ◽

Science Courses ◽

Perceptions And Attitudes ◽

Second Year ◽

And Mathematics ◽

High Level ◽

The Impact

Challenges particular to second-year students have been identified that can impact persistence in science, technology, engineering, and mathematics (STEM) fields. We implemented a program to improve student success in intermediate-level science courses by helping students to feel they belonged and could succeed in STEM. We used survey measures of perceptions and attitudes and then qualitative measures to characterize the impact of support strategies, including peer mentoring, a second-year science student retreat, learning and advising support resources, and department-specific activities. Analysis of registration and transcript information revealed underperformance by students of color (SOC) and first-generation (FG) students in 200-level science courses. Comparison of these data before and during programming revealed significant improvement in success rates of these students in 200-level biology and chemistry courses, but success rates of SOC and FG students remain lower than the overall rate for 200-level science courses. Contemporaneous with the program, qualitative and quantitative measures of student attitudes revealed a high level of belongingness and support. The results suggest that a focus on students’ metacognition about their own abilities and strategic knowledge of how to succeed may be a fruitful direction for future research.

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