It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study.

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.

Enhanced etching resolution of self-assembled PS-b-PMMA block copolymer films by ionic liquid additives.

Polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) is one of the most widely studied block copolymers for direct self-assembly because of its excellent compatibility with traditional processes. However, pattern transfer of PS-b-PMMA block copolymers (BCPs) remains a great challenge for its applications due to the insufficient etching resolution. In this study, the effect of ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (HMHF) additives on the line edge roughness (LER) performances of PS-b-PMMA self-assembled patterns was studied. Trace addition of HMHF kept the photolithography compatibility of PS-b-PMMA block copolymer films, but obviously increased their Flory-Huggins interaction parameter (χ) and enabled phase separation of disordered low molecular weight BCPs. LER value was effectively decreased by blending HMHF directly with PS-b-PMMA or from a supplying top layer of polyvinylpyrrolidone containing HMHF additives. This study shows an excellent strategy to improve the deficiencies of existing block copolymers.

Abnormalities of Rest-Activity and Light Exposure Rhythms Associated with Cognitive Function in Patients with Mild Cognitive Impairment (MCI).

We aimed to examine the difference in rest-activity rhythm (RAR) and light exposure rhythm (LER) between patients with mild cognitive impairment (MCI) and normal controls (NC), and to verify their relationships with cognitive functions. The neuropsychological battery was administered to participants above 50 years old. The MCI diagnosis was made according to Petersen's criteria. Ten patients with MCI (77.90 ± 6.95 years) and eight NC (74.75 ± 5.06 years) were studied. Actigraphy (Actiwatch 2; Philips Respironics) was recorded at home for 5 days. RAR and LER variables, including interdaily stability (IS), intradaily variability (IV) and relative amplitude, were calculated using nonparametric analyses. The associations between cognitive performance and RAR and LER variables were explored using generalized linear models. There were no significant differences in RAR or LER variables between MCI and NC. There was a significant main effect of RAR-IS on the Stroop Color and Word Test (SCWT), indicating a positive relationship between RAR stability and SCWT performance. There was a significant group by RAR-IS interaction on Trail Making Test-A, indicating a negative relationship in MCI compared to NC. There was a significant group by LER-IV interaction on the Boston Naming Test, indicating a positive relationship in MCI compared to NC. There was no disruption in RAR and LER in patients with MCI. Our study showed that circadian rhythm abnormality was associated with a decline in executive function. However, circadian rhythm abnormality was not associated with declines in processing speed and language function in patients with MCI, implying an altered pathophysiology compared to NC.

Serum high mobility group box-1 levels associated with cardiovascular events after lower extremity revascularization: a prospective study of a diabetic population.

BACKGROUND: Peripheral arterial disease (PAD) is one of the most disabling cardiovascular complications of type 2 diabetes mellitus and is indeed associated with a high risk of cardiovascular and limb adverse events. High mobility group box-1 (HMGB-1) is a nuclear protein involved in the inflammatory response that acts as a pro-inflammatory cytokine when released into the extracellular space. HMBG-1 is associated with PAD in diabetic patients. The aim of this study was to evaluate the association between serum HMGB-1 levels and major adverse cardiovascular events (MACE) and major adverse limb events (MALE) after lower-extremity endovascular revascularization (LER) in a group of diabetic patients with chronic limb-threatening ischemia (CLTI). METHODS: We conducted a prospective observational study of 201 diabetic patients with PAD and CLTI requiring LER. Baseline serum HMGB-1 levels were determined before endovascular procedure. Data on cardiovascular and limb outcomes were collected in a 12-month follow-up. RESULTS: During the follow-up period, 81 cases of MACE and 93 cases of MALE occurred. Patients who subsequently developed MACE and MALE had higher serum HMGB-1 levels. Specifically, 7.5 ng/mL vs 4.9 ng/mL (p < 0.01) for MACE and 7.2 ng/mL vs 4.8 ng/mL (p < 0.01) for MALE. After adjusting for traditional cardiovascular risk factors, the association between serum HMGB-1 levels and cardiovascular outcomes remained significant in multivariable analysis. In our receiver operating characteristic (ROC) curve analysis, serum HMGB-1 levels were a good predictor of MACE incidence (area under the curve [AUC] = 0.78) and MALE incidence (AUC = 0.75). CONCLUSIONS: This study demonstrates that serum HMGB-1 levels are associated with the incidence of MACE and MALE after LER in diabetic populations with PAD and CLTI.

Genome-wide characterization of the PDI gene family in Medicago truncatula and their roles in response to endoplasmic reticulum stress.

Protein disulfide isomerases (PDIs) are pivotal protein folding catalysts in the endoplasmic reticulum (ER) through formation of disulfide bond, isomerization, and inhibition of misfolded protein aggregation. When protein folding capacity is overwhelmed by the demands during transitions between growth phases or under environmental changes, the accumulation of unfolded or misfolded proteins in the ER triggers ER stress. However, little is known about the PDI gene family in the model legume Medicago truncatula, especially the responses to ER stress. Therefore, we identified 17 putative PDI genes from the genome of M. truncatula and present their gene and protein structures, phylogenetic relationships, chromosomal distributions, and synteny analysis with the orthologs in four other eudicot species, including Arabidopsis thaliana, Glycine max, Brassica rapa, and Vitis vinifera. Moreover, expression profiles derived from transcriptome data showed distinct expression patterns of MtPDI genes among plant organs, while real-time quantitative PCR analysis and data from the proteome revealed the potential roles of MtPDI genes in response to ER stress. Our study provides a foundation for further investigations of the biological roles of PDI genes in Medicago, especially their roles in response to ER stress.

Reduction of EUV resist damage by neutral beam etching.

Even though EUV lithography has the advantage of implenting a finer pattern compared to ArF immersion lithography due to the use of 13.5 nm instead of 193 nm as the wavelength of the light source, due to the low energy of EUV light source, EUV resist has a thinner thickness than conventional ArF resist. EUV resist having such a thin thickness is more vulnerable to radiation damage received during the etching because of its low etch resistance and also tends to have a problem of low etch selectivity. In this study, the radiation damage to EUV resist during etching of hardmask materials such as Si3N4, SiO2, etc using CF4gas was compared between neutral beam etching (NBE) and ion beam etching (IBE). When NBE was used, after the etching of 20 nm thick EUV resist, the line edge roughness increase and the critical dimension change of EUV resist were reduced by ∼1/3 and ∼1/2, respectively, compared to those by IBE. Also, at that EUV etch depth, the root mean square surface roughness value of EUV resist etched by NBE was ∼2/3 compared to that by IBE on the average. It was also confirmed that the etching selectivity between SiO2, Si3N4, etc and EUV resist was higher for NBE compared to IBE. The less damage to the EUV resist and the higher etch selectivity of materials such as Si3N4and SiO2over EUV resist for NBE compared to IBE are believed to be related to the no potential energy released by the neutralization of the ions during the etching by NBE.

The Arabidopsis Accessions Selection Is Crucial: Insight from Photosynthetic Studies.

Natural genetic variation in photosynthesis is strictly associated with the remarkable adaptive plasticity observed amongst Arabidopsis thaliana accessions derived from environmentally distinct regions. Exploration of the characteristic features of the photosynthetic machinery could reveal the regulatory mechanisms underlying those traits. In this study, we performed a detailed characterisation and comparison of photosynthesis performance and spectral properties of the photosynthetic apparatus in the following selected Arabidopsis thaliana accessions commonly used in laboratories as background lines: Col-0, Col-1, Col-2, Col-8, Ler-0, and Ws-2. The main focus was to distinguish the characteristic disparities for every accession in photosynthetic efficiency that could be accountable for their remarkable plasticity to adapt. The biophysical and biochemical analysis of the thylakoid membranes in control conditions revealed differences in lipid-to-protein contribution, Chlorophyll-to-Carotenoid ratio (Chl/Car), and xanthophyll cycle pigment distribution among accessions. We presented that such changes led to disparities in the arrangement of the Chlorophyll-Protein complexes, the PSI/PSII ratio, and the lateral mobility of the thylakoid membrane, with the most significant aberrations detected in the Ler-0 and Ws-2 accessions. We concluded that selecting an accession suitable for specific research on the photosynthetic process is essential for optimising the experiment.

1.6 Million transactions replicate distributed PV market slowdown by COVID-19 lockdown.

Solar PV has seen a spectacular market development in recent years and has become a cost competitive source of electricity in many parts of the world. Yet, prospective observations show that the coronavirus pandemic could impact renewable energy projects, especially in the distributed market. Tracking and attributing the economic footprint of COVID-19 lockdowns in the photovoltaic sector poses a significant research challenge. Based on millions of financial transaction records and 44 thousand photovoltaic installation records, we tracked the spatio-temporal sale network of the distributed photovoltaic market and explored the extent of market slowdown. We found that a two-month lockdown duration can be assessed as a high-risk threshold value. When the lockdown duration exceeds the threshold value, the monthly value-added loss reaches 67.7%, and emission reduction capacity is cut by 64.2% over the whole year. We show that risks of a slowdown in PV deployment due to COVID-19 lockdowns can be mitigated by comprehensive incentive strategies for the distributed PV market amid market uncertainties.

Transcriptional and posttranscriptional regulation of the locus of enterocyte effacement in Escherichia albertii.

The diarrheic bacterium Escherichia albertii is a recent addition to the attaching and effacing (A/E) morphotype of pathogens. A/E pathogens cause disease by tightly attaching to intestinal cells, destroying their actin-rich microvilli, and triggering re-localization and repolymerization of actin at the bacterial-host interface to form actin-filled membranous protrusions, termed A/E lesions, beneath the adherent bacterium. The locus of enterocyte effacement (LEE) is required for the biogenesis of these lesions. Whereas regulation of the LEE has been intensively investigated in EPEC and EHEC, it remains cryptic in E. albertii. In this study we characterized the very first transcriptional and posttranscriptional regulators of the LEE in this emerging pathogen. Our results suggest that Ler and GrlA globally activate transcription from the LEE, whereas GrlR negatively regulates the LEE. Additionally, we demonstrate that the RNA chaperone Hfq posttranscriptionally represses the LEE by specifically targeting the 5' UTR of grlR. In summary, our findings provide the very first glimpse of the regulatory landscape of the LEE in E. albertii - a bacterium that has been implicated in multiple diarrheal outbreaks worldwide.

Dietary taste patterns by sex and weight status in the Netherlands.

Taste is a key driver of food choice and intake. Taste preferences are widely studied, unlike the diet's taste profile. This study assessed dietary taste patterns in the Netherlands by sex, BMI, age and education. A taste database, containing 476 foods' taste values, was combined with 2-d 24-h recalls in two study populations. The percentage of energy intake from six taste clusters was assessed in the Dutch National Food Consumption Survey (DNFCS 2007-2010; n 1351) and in an independent observational study: the Nutrition Questionnaires plus (NQplus) study (2011-2013; n 944). Dietary taste patterns were similar across study populations. Men consumed relatively more energy from 'salt, umami and fat' (DNFCS; 24 % energy, NQplus study; 23 %)- and 'bitter' (7 %)-tasting foods compared with women (21 %, P<0·001, 22 %, P=0·005; 3 %, P<0·001, 4 %, P<0·001, respectively). Women consumed more % energy from 'sweet and fat' (15 %)- and 'sweet and sour' (13 %, 12 %, respectively)-tasting foods compared with men (12 %, P<0·001, 13 %, P=0·001; 10 %, P<0·001). Obese individuals consumed more % energy from 'salt, umami and fat'- and less from 'sweet and fat'-tasting foods than normal-weight individuals ('salt, umami and fat', men; obese both studies 26 %, normal-weight DNFCS 23 %, P=0·037, NQplus 22 %, P=0·001, women; obese 23 %, 24 %, normal weight 20 %, P=0·004, P=0·011, respectively, 'sweet and fat', men; obese 11 %, 10 %, normal weight 13 %, P<0·05, 14 %, P<0·01, women; obese 14 %, 15 %, normal weight 16 %, P=0·12, P=0·99). In conclusion, our taste database can be used to deepen our understanding of the role of taste in dietary intake in the Netherlands by sex, BMI, age and education.

Investigation of the kinetics and mechanism of low endotoxin recovery in a matrix for biopharmaceutical drug products.

The inability to detect endotoxin added to undiluted drug samples has been called: Low Endotoxin Recovery (LER). The phenomenon has caused concerns amongst drug manufacturing quality control scientists in that manufactured solutions contaminated with endotoxin could show false-negative results via routine Limulus-based tests. The time-dependent appearance of LER has been analyzed in detail to provide a better understanding of the mechanism. The assumption has been that the root-cause of LER involves the interplay of endotoxin with surfactants and results in aggregate structures that are complexed with surfactants. The endotoxin molecules when complexed with surfactants are not accessible for Limulus-based detection. The results demonstrate a predominant role of complex-forming agents. It was shown that although the presence of surfactants is a strong prerequisite for masking, it does not determine the kinetics of endotoxin masking. Interestingly, the endotoxin concentration itself had no substantial impact on LER kinetics. By adjusting the ratios of complex-forming constituents, including surfactant, chelator and endotoxin, and by testing the order in which the constituents are added, a new model for simulating masking kinetics has been determined. Our work provides for the first time a model to simulate masking kinetics of endotoxin which lends a better understanding of LER.

The mechanism underlying Ler-mediated alleviation of gene repression by H-NS.

Secretion of effector proteins in Enteropathogeneic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5 promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on LEE5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of H-NS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on LEE5 promoter resulting in a derepression.

Results of a harmonized endotoxin recovery study protocol evaluation by 14 BioPhorum Operations Group (BPOG) member companies.

Significant regulatory and biopharmaceutical manufacturing attention has been recently generated regarding the inability to recover purified lipopolysaccharide in certain biopharmaceutical products, specifically those containing a chelator and surfactant. Disparate data has been generated and submitted during regulatory review. Study design is thought to significantly impact, and cause the confounding data. The BioPhorum Operations Group (BPOG) is an industry consortium of biopharmaceutical manufacturers. A BPOG team developed a collaborative experiment to determine if multiple laboratories could execute a harmonized protocol and reach equivalent conclusions. Twenty-one laboratories across fourteen different companies participated, yielding a robust study and large dataset. The experiment was successful as nearly all of the laboratories reached the same conclusions despite allowable variation in methods, suppliers, reagents, locations and analysts. The results of the study indicate a naturally occurring endotoxin analyte activity was recovered in a chelating buffer containing surfactant (citrate and polysorbate), and a buffer without surfactant (phosphate), while the purified lipopolysaccharide analyte activity was not recovered in the chelating buffer containing surfactant, but was recovered in the buffer without surfactant. Because the harmonized protocol yields consistent results across many different laboratories, the BPOG LER team recommends adoption of this protocol for use in endotoxin recovery studies.

Energy Efficient Real-Time Scheduling Using DPM on Mobile Sensors with a Uniform Multi-Cores.

In wireless sensor networks (WSNs), sensor nodes are deployed for collecting and analyzing data. These nodes use limited energy batteries for easy deployment and low cost. The use of limited energy batteries is closely related to the lifetime of the sensor nodes when using wireless sensor networks. Efficient-energy management is important to extending the lifetime of the sensor nodes. Most effort for improving power efficiency in tiny sensor nodes has focused mainly on reducing the power consumed during data transmission. However, recent emergence of sensor nodes equipped with multi-cores strongly requires attention to be given to the problem of reducing power consumption in multi-cores. In this paper, we propose an energy efficient scheduling method for sensor nodes supporting a uniform multi-cores. We extend the proposed T-Ler plane based scheduling for global optimal scheduling of a uniform multi-cores and multi-processors to enable power management using dynamic power management. In the proposed approach, processor selection for a scheduling and mapping method between the tasks and processors is proposed to efficiently utilize dynamic power management. Experiments show the effectiveness of the proposed approach compared to other existing methods.

Maize-grain legume intercropping for enhanced resource use efficiency and crop productivity in the Guinea savanna of northern Ghana.

Smallholder farmers in the Guinea savanna practise cereal-legume intercropping to mitigate risks of crop failure in mono-cropping. The productivity of cereal-legume intercrops could be influenced by the spatial arrangement of the intercrops and the soil fertility status. Knowledge on the effect of soil fertility status on intercrop productivity is generally lacking in the Guinea savanna despite the wide variability in soil fertility status in farmers' fields, and the productivity of within-row spatial arrangement of intercrops relative to the distinct-row systems under on-farm conditions has not been studied in the region. We studied effects of maize-legume spatial intercropping patterns and soil fertility status on resource use efficiency, crop productivity and economic profitability under on-farm conditions in the Guinea savanna. Treatments consisted of maize-legume intercropped within-row, 1 row of maize alternated with one row of legume, 2 rows of maize alternated with 2 rows of legume, a sole maize crop and a sole legume crop. These were assessed in the southern Guinea savanna (SGS) and the northern Guinea savanna (NGS) of northern Ghana for two seasons using three fields differing in soil fertility in each agro-ecological zone. Each treatment received 25 kg P and 30 kg K ha-1 at sowing, while maize received 25 kg (intercrop) or 50 kg (sole) N ha-1 at 3 and 6 weeks after sowing. The experiment was conducted in a randomised complete block design with each block of treatments replicated four times per fertility level at each site. Better soil conditions and rainfall in the SGS resulted in 48, 38 and 9% more maize, soybean and groundnut grain yield, respectively produced than in the NGS, while 11% more cowpea grain yield was produced in the NGS. Sole crops of maize and legumes produced significantly more grain yield per unit area than the respective intercrops of maize and legumes. Land equivalent ratios (LERs) of all intercrop patterns were greater than unity indicating more efficient and productive use of environmental resources by intercrops. Sole legumes intercepted more radiation than sole maize, while the interception by intercrops was in between that of sole legumes and sole maize. The intercrop however converted the intercepted radiation more efficiently into grain yield than the sole crops. Economic returns were greater for intercrops than for either sole crop. The within-row intercrop pattern was the most productive and lucrative system. Larger grain yields in the SGS and in fertile fields led to greater economic returns. However, intercropping systems in poorly fertile fields and in the NGS recorded greater LERs (1.16-1.81) compared with fertile fields (1.07-1.54) and with the SGS. This suggests that intercropping is more beneficial in less fertile fields and in more marginal environments such as the NGS. Cowpea and groundnut performed better than soybean when intercropped with maize, though the larger absolute grain yields of soybean resulted in larger net benefits.

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon.

Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called "Low Endotoxin Recovery (LER)". LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration's (FDA's) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.

Masking of endotoxin in surfactant samples: Effects on Limulus-based detection systems.

Over the last few decades Limulus Amebocyte Lysate (LAL) has been the most sensitive method for the detection of endotoxins (Lipopolysaccharides) and is well accepted in a broad field of applications. Recently, Low Endotoxin Recovery (LER) in biopharmaceutical drug products has been noticed, whereby the detection of potential endotoxin contaminations is not ensured. Notably, most of these drug products contain surfactants, which can have crucial effects on the detectability of endotoxin. In order to analyze the driving forces of LER, endotoxin detection in samples containing nonionic surfactants in various buffer systems was investigated. The results show that the process of LER is kinetically controlled and temperature-dependent. Furthermore, only the simultaneous presence of nonionic surfactants and components capable of forming metal complexes resulted in LER. In addition, capacity experiments show that even hazardous amounts of endotoxin can remain undetectable within such formulation compositions. In conclusion, the LER phenomenon is caused by endotoxin masking and not by test interference. In this process, the supramolecular structure of endotoxin is altered and exhibits only a limited susceptibility in binding to the Factor C of Limulus-based detection systems. We propose a two-step mechanism of endotoxin masking by complex forming agents and nonionic surfactants.

Locus of enterocyte effacement-encoded regulator (Ler) of pathogenic Escherichia coli competes off histone-like nucleoid-structuring protein (H-NS) through noncooperative DNA binding.

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.

DELLA activity is required for successful pollen development in the Columbia ecotype of Arabidopsis.

Excessive gibberellin (GA) signalling, mediated through the DELLA proteins, has a negative impact on plant fertility. Loss of DELLA activity in the monocot rice (Oryza sativa) causes complete male sterility, but not in the dicot model Arabidopsis (Arabidopsis thaliana) ecotype Landsberg erecta (Ler), in which DELLA function has been studied most extensively, leading to the assumption that DELLA activity is not essential for Arabidopsis pollen development. A novel DELLA fertility phenotype was identified in the Columbia (Col-0) ecotype that necessitates re-evaluation of the general conclusions drawn from Ler. Fertility phenotypes were compared between the Col-0 and Ler ecotypes under conditions of chemical and genetic GA overdose, including mutants in both ecotypes lacking the DELLA paralogues REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI). Ler displays a less severe fertility phenotype than Col-0 under GA treatment. Col-0 rga gai mutants, in contrast with the equivalent Ler phenotype, were entirely male sterile, caused by post-meiotic defects in pollen development, which were rescued by the reintroduction of DELLA into either the tapetum or developing pollen. We conclude that DELLA activity is essential for Arabidopsis pollen development. Differences between the fertility responses of Col-0 and Ler might be caused by differences in downstream signalling pathways or altered DELLA expression.

Phenotypic diversity of type III secretion system activity in enteropathogenic Escherichia coli clinical isolates.

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins.Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation.Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression.Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity.Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression.Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.