Method for the selection of Lactococcus lactis mutants producing excess carbon dioxide

One of the characteristics of Roquefort cheese is the presence of irregularly shaped openings. Although many factors affect the development of opening in blue-veined cheeses (Martley & Crow, 1996), the limiting step in the case of Roquefort cheese is the production of CO2 by lactic acid bacteria (J.-P. Reverbel, pers. comm.). Most of the opening occurs after moulding; the process is difficult to control and many manufacturing runs result in cheeses with an insufficient opening. Concentrated suspensions of Leuconostoc strains are used to increase the production of CO2 (Devoyod & Muller, 1969), but it would clearly be useful to have microorganisms that produce larger quantities of the gas. McKay & Baldwin (1974) isolated a spontaneous mutant of Lactococcus lactis that produced more acetoin and CO2 than the parent strain. This mutant was lactate dehydrogenase (LDH)-deficient, which favoured the conversion of pyruvate into end products other than lactate. Following nitrosoguanidine mutagenesis, we isolated three Lc. lactis mutants whose LDH activities were reduced to varying extents and which produced varying amounts of CO2 (Boumerdassi et al. 1997). Subsequent work showed that these mutants were unstable on successive subculture in milk or synthetic broth (El Attar et al. 2000).The aim of our current work was to select a large number of Lc. lactis mutants producing excess CO2. This would increase the probability of selecting stable mutants and also provide a collection of strains with differing gas production activities. Currently available screening methods are, however, unsuitable for processing large numbers of mutants, which is why we have developed an improved screening method.

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Prevalence and Characterization of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolates from 14 Cities in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00206-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 3642-3649 ◽

Cited By ~ 43

Author(s):

Wenjia Sun ◽

Hongbin Chen ◽

Yudong Liu ◽

Chunjiang Zhao ◽

Wright W. Nichols ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Population Analysis ◽

Screening Method ◽

Estimation Method ◽

Area Under The Curve ◽

Brain Heart Infusion ◽

Screening Tests ◽

Screening Methods ◽

Large Numbers ◽

Screening Cascade

ABSTRACT The prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) among 1,012 vancomycin-susceptible methicillin (meticillin)-resistant S. aureus isolates collected from 14 cities in China from 2005 to 2007 was 13 to 16%, as determined by a combination of (i) measurement by the modified population analysis profile-area under the curve method (PAP-AUC) and (ii) estimation from the measured sensitivity and specificity of a screening method. Two hundred isolates from blood were chosen as a subset for measurement of the sensitivities and the specificities of several previously described screening methods by using the results of PAP-AUC as the reference. During this testing, one isolate was found to be a vancomycin-intermediate S. aureus (VISA) strain so was not used in the evaluation of the screening tests. Of the other 199 isolates, 26 (13.1%) were hVISA, as assessed by PAP-AUC. A screening cascade of culturing the isolates on brain heart infusion agar containing teicoplanin (5 mg/liter) and then subjecting the positive isolates to a macro-Etest method was applied to the 812 non-blood isolates, yielding 149 positive results. From these results and by adjusting for sensitivity (0.423) and specificity (0.861), the prevalence was estimated to be 15.7%. The precision of that estimate was assessed by reapplying the screening cascade to 120 randomly selected isolates from the 812 non-blood isolates and simultaneously determining their heterogeneous vancomycin-intermediate susceptibility status by PAP-AUC. Because PAP-AUC is impractical for use with large numbers of isolates, the screening-based estimation method is useful as a first approximation of the prevalence of hVISA. Of the 27 VISA or hVISA isolates from blood, 22.2% and 74.1% were staphylococcal chromosome cassette mec types II and III, respectively, while 77.8% and 22.2% were agr type 1 and agr type 2, respectively; the MIC ranges were 0.5 to 4 mg/liter for vancomycin and 0.25 to 1 mg/liter for daptomycin.

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Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow Assay

10.26434/chemrxiv.12709538 ◽

2021 ◽

Author(s):

David Cate ◽

Helen Hsieh ◽

Veronika Glukhova ◽

Joshua D Bishop ◽

H Gleda Hermansky ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Point Of Care ◽

Lateral Flow ◽

Screening Methods ◽

Antibody Screening ◽

Liquid Handling ◽

Large Numbers ◽

Accurate Performance ◽

Further Development ◽

Assay Conditions

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow immunoassay (LFA) tests in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.

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Impact of Nisin and Nisin-Producing Lactococcus lactis ssp. lactis on Clostridium tyrobutyricum and Bacterial Ecosystem of Cheese Matrices

Foods ◽

10.3390/foods10040898 ◽

2021 ◽

Vol 10 (4) ◽

pp. 898

Author(s):

Hebatoallah Hassan ◽

Daniel St-Gelais ◽

Ahmed Gomaa ◽

Ismail Fliss

Keyword(s):

Lactococcus Lactis ◽

Cheddar Cheese ◽

Economic Loss ◽

Gas Production ◽

Clostridium Tyrobutyricum ◽

Nisin Production ◽

Genus Clostridium ◽

Milk Pasteurization ◽

Cheese Slurry ◽

Significant Economic Loss

Clostridium tyrobutyricum spores survive milk pasteurization and cause late blowing of cheeses and significant economic loss. The effectiveness of nisin-producing Lactococcus lactis ssp. lactis 32 as a protective strain for control the C. tyrobutyricum growth in Cheddar cheese slurry was compared to that of encapsulated nisin-A. The encapsulated nisin was more effective, with 1.0 log10 reductions of viable spores after one week at 30 °C and 4 °C. Spores were not detected for three weeks at 4 °C in cheese slurry made with 1.3% salt, or during week 2 with 2% salt. Gas production was observed after one week at 30 °C only in the control slurry made with 1.3% salt. In slurry made with the protective strain, the reduction in C. tyrobutyricum count was 0.6 log10 in the second week at 4 °C with both salt concentration. At 4 °C, nisin production started in week 2 and reached 97 µg/g after four weeks. Metabarcoding analysis targeting the sequencing of 16S rRNA revealed that the genus Lactococcus dominated for four weeks at 4 °C. In cheese slurry made with 2% salt, the relative abundance of the genus Clostridium decreased significantly in the presence of nisin or the protective strain. The results indicated that both strategies are able to control the growth of Clostridium development in Cheddar cheese slurries.

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Efficacy of three greenhouse screening methods for the identification of physiological resistance to white mold in dry bean

Canadian Journal of Plant Science ◽

10.4141/cjps08230 ◽

2009 ◽

Vol 89 (4) ◽

pp. 755-762 ◽

Cited By ~ 11

Author(s):

H Terán ◽

S P Singh

Keyword(s):

Phaseolus Vulgaris ◽

Common Bean ◽

Sclerotinia Sclerotiorum ◽

Screening Method ◽

White Mold ◽

Screening Methods ◽

Post Inoculation ◽

Dry Bean ◽

Physiological Resistance ◽

Nueva Granada

White mold (WM) caused by Sclerotinia sclerotiorum (Lib.) de Bary is the most devastating disease of common bean (dry and snap or garden bean) (Phaseolus vulgaris L.) in North America. The use of a reliable screening method (SM) in common bean is crucial to improve physiological resistance to WM. The objective of this study was to compare the efficacy of three SM to identify physiological resistance in dry bean genotypes with different evolutionary origins and levels of resistance. Screening methods tested were: (i) the modified straw test or cut–stem (CSM); (ii) infected bean flower (IFL); and (iii) infected oat seed (IOS). A 195, ICA Bunsi, Othello, and VCW 54 dry bean were tested with the three SM. The experimental design was a split plot in randomized complete blocks with three replications in 2007 and 2008. Two independent inoculations 1 wk apart for each SM were made. The WM reaction was scored at 16, 23, and 33 d post-inoculation (DPI) using a 1 to 9 scale. There were highly significant differences between SM and its interaction with years. The CSM and IFL were the most consistent and highly correlated (r > 0.70, P < 0.01). Interspecific breeding line VCW 54 consistently had the highest WM resistance across years, SM, and evaluation dates, followed by A 195. White mold scores increased with delayed evaluations. Thus, CSM or IFL with disease assessed 33 DPI should be used for identifying common bean genotypes with high levels of physiological resistance to WM.Key words: Common bean, growth habit, race Mesoamerica, race Nueva Granada, Phaseolus vulgaris, Sclerotinia sclerotiorum

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Antimicrobial Activity of Lactococcus lactis subsp. lactis Isolated from a Stranded Cuvier’s Beaked Whale (Ziphius cavirostris) against Gram-Positive and -Negative Bacteria

Microorganisms ◽

10.3390/microorganisms9020243 ◽

2021 ◽

Vol 9 (2) ◽

pp. 243

Author(s):

Akihiko Suzuki ◽

Miwa Suzuki

Keyword(s):

Lactococcus Lactis ◽

Marine Organism ◽

Growth Conditions ◽

Gas Production ◽

Beaked Whale ◽

Carbohydrate Utilization ◽

Nisin Z ◽

Ziphius Cavirostris ◽

Cuvier’S Beaked Whale ◽

Subtilis Subsp

In the present study, we isolated and characterized Lactococcus lactis (L. lactis) subsp. lactis from a female Cuvier’s beaked whale (Ziphius cavirostris) stranded in Shizuoka, Japan. Only five isolates (CBW1-5), grown on Lactobacilli de Man Rogosa Sharpe (MRS) agar plates prepared using 50% artificial seawater, were positive in L. lactis species-specific primer PCR. Their 16S rRNA sequences were highly similar to those of L. lactis subsp. lactis JCM 5805T. The Gram reaction, motility, gas production from glucose, catalase production, and growth conditions were consistent with those of the type strain. Additionally, carbohydrate utilization of the strains was consistent with previously reported marine organism-derived strains. The pH-neutralized cell-free culture supernatant of strain CBW2 inhibited the growth of Bacillus subtilis subsp. subtilis ATCC 6051 and Vibrio alginolyticus ATCC 17749, whereas protease treatment eliminated or diminished its inhibitory activity. The strain possesses a precursor of the nisin structural gene (nisA), which showed 100% homology with nisin Z, and nisin biosynthesis-related genes (nisB, nisC, nisT, nisP, nisF, nisI, and nisRK), suggesting that the strain produces a nisin-like substance. This study provides fundamental information on whale-derived L. lactis subsp. lactis which may be useful for reducing the carriage of B. subtilis subsp. subtilis and V. alginolyticus.

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A New Screening Approach For Fast And Accurate Prediction Of Positive And Negative Urine Cultures By SediMAX Compared With The Standard Urine Culture

Technium BioChemMed ◽

10.47577/biochemmed.v1i1.2451 ◽

2021 ◽

Vol 1 (1) ◽

pp. 46-55

Author(s):

Massimo Pieri ◽

Flaminia Tomassetti ◽

Paola Cerini ◽

Roberta Felicetti ◽

Lucia Ceccaroni ◽

...

Keyword(s):

Blood Cell ◽

Bacterial Infections ◽

Urine Culture ◽

Screening Method ◽

Significant Proportion ◽

Bacterial Count ◽

Urine Samples ◽

Screening Methods ◽

Urine Sediment ◽

Tract Infections

Urinary tract infections (UTI) are the most frequent bacterial infections, and the detection of infection in urine samples is expensive and time-consuming. Also, in laboratories a significant proportion of samples processed yield negative results. For this, screening methods represent an important improvement towards the final UTI diagnosis. SediMAX is an automated microscopy, easier to use in laboratories due to its basic procedure and it is widely used for urine sediment analysis. In our study, we evaluated the performance of SediMAX, applying some screening parameters, compared with the gold standard methods, urine culture, to identify all the positive cases for UTI. We analysed 1185 urine samples from our daily laboratory routine. The basis of our screening model was to establish a cut-off for bacterial count (BACT), as 300 bacteria/µL in order to avoid missing positive cases. However, the sensitivity and the specificity achieved were not enough to identify all UTI infection in urine samples. So, in addition to BACT we have considered other parameters, such as White Blood Cell (WBC), Red Blood Cell (RBC), Yeasts (YEST), Age and Nitrates (NIT). The second screening method reached a sensitivity of 100%, that could be reliably employed in detect of UTIs.

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Therapeutic Potential of CUDC-907 (Fimepinostat) for Hepatocarcinoma Treatment Revealed by Tumor Spheroids-based Drug Screening

10.21203/rs.3.rs-125324/v1 ◽

2020 ◽

Author(s):

Wei Liao ◽

Wanren Yang ◽

Yue Zhang ◽

Fanhong Zeng ◽

Jiecheng Xu ◽

...

Keyword(s):

Cell Lines ◽

Drug Screening ◽

Screening Method ◽

Screening Methods ◽

Spheroid Culture ◽

3D Print ◽

Culture Plate ◽

Hcc Cells

Abstract Background: Cancer is the second leading cause of death globally. However, most of the new anti-cancer agents screened by traditional drug screening methods fail in the clinic because of lack of efficacy. One of the reasons for this dilemma is that the two-dimensional (2D) culture cancer cell lines could not represent the in vivo cancer cells well. Fortunately, the development of a three-dimensional (3D) culture technique helps in this problem. Methods: The high-throughput spheroid culture plate was fabricated by using 3D print technique and agarose. 4 hepatocarcinoma (HCC) cell lines were 3D cultured to screen 19 small molecular agents based on the spheroid culture plate. 3D cultured primary HCC cells and tumor-bearing mice model were established to verify the candidate anti-hepatocarcinoma agent. Cell function experiments and western blotting were conducted to explore the anti-hepatocarcinoma mechanism of the candidate agent. Results: Based on the previous study, we established an in vitro 3D drug screening method by using our invented spheroid culture device and found that CUDC-907 can serve as a potent anti-hepatocarcinoma agent. The study data show that CUDC-907 (fimepinostat), a novel dual acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC), has potent inhibitory effects on HCC cell lines and primary HCC cells in vitro, Animal studies have shown that CUDC-907 can also suppress HCC cells in vivo. Furthermore, we investigated the antitumor mechanism of CUDC-907 in HCC cells. We found that it inhibits the PI3K/AKT/mTOR pathway and downregulates the expression of c-Myc, leading to the suppression of HCC cells. Conclusion: Our results suggest that CUDC-907 can be a candidate anti-HCC drug, and the 3D in vitro drug screening method based on our novel spheroid culture device is promising for drug screening.

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Research on the Unstable Branch Screening Method for Power System With High-Proportion Wind Power

Frontiers in Energy Research ◽

10.3389/fenrg.2021.835440 ◽

2022 ◽

Vol 9 ◽

Author(s):

Fei Tang ◽

Xiaoqing Wei ◽

Yuhan Guo ◽

Junfeng Qi ◽

Jiarui Xie ◽

...

Keyword(s):

Power System ◽

Wind Power ◽

Wind Farm ◽

Simulation Analysis ◽

Screening Method ◽

Prediction Method ◽

Stability Margin ◽

Screening Methods ◽

Unstable Branch ◽

Oscillation Characteristics

The sooner the system instability is predicted and the unstable branches are screened, the timelier emergency control can be implemented for a wind power system. In this paper, aiming at the problem that the existing unstable branch screening methods are lack prejudgment, an unstable branch screening method for power system with high-proportion wind power is proposed. Firstly, the equivalent external characteristics model of the wind farm was deduced. And based on this, the out-of-step oscillation characteristics of the power system with high proportion wind power was analyzed. Secondly, based on the oscillation characteristics, line weak-connection index (LWcI) was proposed to quantify the stability margin of a branch. Then an instability prediction method and an unstable branch screening method were proposed based on LWcI and voltage phase angle difference. Finally, the rapidity and effectiveness of the proposed method are verified through the simulation analysis of IEEE-118 system.

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Generating Property-Matched Decoy Molecules Using Deep Learning

10.1101/2020.08.26.268193 ◽

2020 ◽

Author(s):

Fergus Imrie ◽

Anthony R. Bradley ◽

Charlotte M. Deane

Keyword(s):

Deep Learning ◽

Virtual Screening ◽

Method Development ◽

Screening Method ◽

Screening Methods ◽

Additional Risk ◽

Link Type ◽

Screening Performance ◽

And Training ◽

Virtual Screening Performance

An essential step in the development of virtual screening methods is the use of established sets of actives and decoys for benchmarking and training. However, the decoy molecules in commonly used sets are biased meaning that methods often exploit these biases to separate actives and decoys, rather than learning how to perform molecular recognition. This fundamental issue prevents generalisation and hinders virtual screening method development. We have developed a deep learning method (DeepCoy) that generates decoys to a user’s preferred specification in order to remove such biases or construct sets with a defined bias. We validated DeepCoy using two established benchmarks, DUD-E and DEKOIS 2.0. For all DUD-E targets and 80 of the 81 DEKOIS 2.0 targets, our generated decoy molecules more closely matched the active molecules’ physicochemical properties while introducing no discernible additional risk of false negatives. The DeepCoy decoys improved the Deviation from Optimal Embedding (DOE) score by an average of 81% and 66%, respectively, decreasing from 0.163 to 0.032 for DUD-E and from 0.109 to 0.038 for DEKOIS 2.0. Further, the generated decoys are harder to distinguish than the original decoy molecules via docking with Autodock Vina, with virtual screening performance falling from an AUC ROC of 0.71 to 0.63. The code is available at https://github.com/oxpig/DeepCoy. Generated molecules can be downloaded from http://opig.stats.ox.ac.uk/resources.

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