Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effect of Lignin on Bamboo Biomass Self-Bonding During Hot-Pressing: Lignin Structure and Characterization

To achieve high value-added utilization of lignin extracted from the biorefinery process in the wood composite industry, binderless particle boards were manufactured by bamboo materials combined with alkaline lignin (AL) in various proportions under various hot-pressing conditions. To elucidate the reactivity and chemical transformations of lignin macromolecules during the hot-pressing process, lignin samples were isolated from the corresponding boards and characterized by high-performance anion-exchange chromatography (HPAEC), gel permeation chromatography (GPC), quantitative 31P-NMR, and 2D-HSQC NMR. The best bonding strength (1.36 MPa) of the binderless particle boards was obtained under the conditions of 180 °C, pressure 5 MPa, and lignin/bamboo mass ratio 0.4. The molecular weight of the lignin samples decreased from 3260 to 1420 g/mol during hot-pressing. The NMR results showed that the contents of β-O-4' and β-β' linkages were reduced and β-5' linkages were increased as the hot-pressing temperature rose. Simultaneously, the percentage of G-type and H-type lignins as well as the content of phenolic OH increased

Chemical Changes of Raw Materials and Manufactured Binderless Boards during Hot Pressing: Lignin Isolation and Characterization

Thermomechanical pulp (TMP) is used for fiber production in binderless boards industries. Milled wood lignin (MWL) and enzymatic mild acidolysis lignin (EMAL) isolated from raw material and from binderless boards (BB) were comparatively analyzed to investigate the effects of chemical changes on the bonding performance in BB. The results showed that acid-insoluble lignin of the BB were increased during the sodium silicate solution pretreatment after hot-pressing. The lignin fractions obtained were characterized by gel permeation chromatography (GPC), Fourier transform infrared (FT-IR) spectroscopy, and 1H-13C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy. Results showed that 31.1% of EMAL (based on Klason lignin) with low molecular weight (Mw=1630 g/mol) was isolated from the BB. The increased total phenolic OH groups (3.97 mmol/g) of EMAL from sodium silicate solution pretreated BB indicated that there was degradation of lignin and cleavage of lignin-carbohydrate linkages during hot-pressing. In addition, the content of β-O-4' aryl ether linkages of EMAL from the BB increased to 69.2%, which was higher than that of the untreated sample (60.1%). It was found that S units (syringyl-like lignin structures) were preferentially condensed by hot pressing over G (guaiacyl-like lignin structures) units, and the S/G ratio increased after the hot-pressing process

Comparing biweekly single-dose actinomycin D with multiday methotrexate therapy for low-risk gestational trophoblastic neoplasia (FIGO Score 0–4): study protocol for a prospective, multicentre, randomized trial

Abstract Background Single-agent chemotherapy using methotrexate or actinomycin D is the first-line treatment for patients with low-risk gestational trophoblastic neoplasia. Various methotrexate-based and actinomycin D-based single-agent regimens can be used. However, there is insufficient evidence to determine the superior regimen. To guide doctors in selecting a single-agent chemotherapy regimen for patients with low-risk gestational trophoblastic neoplasia, we will compare two regimens. Methods We will conduct a multicentre, randomized, prospective clinical trial. Selected low-risk gestational trophoblastic neoplasia patients (FIGO score 0–4) will be randomized 1:1 to a biweekly single-dose actinomycin D group or a multiday methotrexate therapy group. The actinomycin D group will receive IV pulse actinomycin D (1.25 mg/m2) every 14 days, and the methotrexate group will receive methotrexate (50 mg) intramuscularly on days 1, 3, 5, and 7 (4 doses per cycle) and leucovorin (15 mg) intramuscularly on days 2, 4, 6, and 8. This process will be repeated every 14 days. The primary endpoints will include the complete remission rate by single-agent therapy and the overall complete remission rate. The secondary endpoints will include the duration needed to achieve complete remission after single-agent chemotherapy, number of courses needed to achieve complete remission after single-agent chemotherapy, incidence and severity of adverse effects, effects on menstrual conditions and ovarian function based on the anti-Mullerian hormone level, and patient-reported quality of life. Discussion Previous clinical trials comparing biweekly single-dose actinomycin D with multiday methotrexate therapy for treating low-risk gestational trophoblastic neoplasia patients failed to meet the expected case number. Through this multicentre study, the complete remission ratio and efficacy difference between biweekly single-dose actinomycin D and multiday methotrexate therapy will be obtained. This study will also provide the basis for formulating a preferred regimen for treating patients with low-risk gestational trophoblastic neoplasia. Trial registration ClinicalTrials.gov: NCT04562558, Registered on 13 September 2020 (Protocol version 2020–9-24, version 1.0)

Global fungal-host interactome mapping identifies host targets of candidalysin

Abstract Candidalysin, a cytolytic peptide toxin secreted by the human fungal pathogen Candida albicans, is critical for fungal pathogenesis. Yet, its intracellular targets have not been extensively mapped. Here, we performed a high-throughput enhanced yeast two-hybrid (HT-eY2H) screen to map the interactome of all eight Ece1 peptides with their direct human protein targets and identified a list of potential interacting proteins, some of which were shared between the peptides. CCNH, a regulatory subunit of the CDK-activating kinase (CAK) complex involved in DNA damage repair, was identified as one of the host targets of candidalysin. Mechanistic studies revealed that candidalysin triggers a significantly increased double-strand DNA breaks (DSBs), as evidenced by the formation of γ-H2AX foci and colocalization of CCNH and γ-H2AX. Importantly, candidalysin binds directly to CCNH to activate CAK to inhibit DNA damage repair pathway. Loss of CCNH alleviates DSBs formation under candidalysin treatment. Depletion of candidalysin-encoding gene fails to induce DSBs and stimulates CCNH upregulation in a murine model of oropharyngeal candidiasis. Collectively, our study reveals that a secreted fungal toxin acts to hijack the canonical DNA damage repair pathway by targeting CCNH and to promote fungal infection