The evening complex integrates photoperiod signals to control flowering in rice.

Plants use photoperiodism to activate flowering in response to a particular daylength. In rice, flowering is accelerated in short-day conditions, and even a brief exposure to light during the dark period (night-break) is sufficient to delay flowering. Although many of the genes involved in controlling flowering in rice have been uncovered, how the long- and short-day flowering pathways are integrated, and the mechanism of photoperiod perception is not understood. While many of the signaling components controlling photoperiod-activated flowering are conserved between Arabidopsis and rice, flowering in these two systems is activated by opposite photoperiods. Here we establish that photoperiodism in rice is controlled by the evening complex (EC). We show that mutants in the EC genes LUX ARRYTHMO (LUX) and EARLY FLOWERING3 (ELF3) paralogs abolish rice flowering. We also show that the EC directly binds and suppresses the expression of flowering repressors, including PRR37 and Ghd7. We further demonstrate that light acts via phyB to cause a rapid and sustained posttranslational modification of ELF3-1. Our results suggest a mechanism by which the EC is able to control both long- and short-day flowering pathways.

Quorum sensing in bacteria: in silico protein analysis, ecophysiology, and reconstruction of their evolutionary history.

BACKGROUND: Quorum sensing (QS) is a sophisticated cell-to-cell signalling mechanism that allows the coordination of important processes in microbial populations. The AI-1 and AI-2 autoinducer systems are among the best characterized bacterial QS systems at the genetic level. RESULTS: In this study, we present data derived from in silico screening of QS proteins from bacterial genomes available in public databases. Sequence analyses allowed identifying candidate sequences of known QS systems that were used to build phylogenetic trees. Eight categories were established according to the number of genes from the two major QS systems present in each genome, revealing a correlation with specific taxa, lifestyles or metabolic traits. Many species had incomplete QS systems, encoding the receptor protein but not the biosynthesis of the quorum sensing molecule (QSMs). Reconstruction of the evolutionary history of the LuxR family and prediction of the 3D structure of the ancestral protein suggested their monomeric configuration in the absence of the signal molecule and the presence of a cavity for its binding. CONCLUSIONS: Here we correlate the taxonomic affiliation and lifestyle of bacteria from different genera with the QS systems encoded in their genomes. Moreover, we present the first ancestral reconstruction of the LuxR QS receptors, providing further insight in their evolutionary history.

Transcatheter tricuspid valve replacement with LuX-valve device: Overview of key echocardiographic considerations.

Tricuspid regurgitation is a common valve disease with high incidence and poor prognosis. For elderly patients and those with a history of open heart surgery, second thoracotomy and valve replacement carry a high risk. Transcatheter tricuspid valve replacement (TTVR) has become an alternative treatment for patients with high surgical risk. LuX-Valve is a novel self-expandable valve that does not rely on radial force to anchor the valve annulus. The preliminary results have been satisfactory, and this technology is gradually being adopted in China and around the world. Successful implementation of this technique depends on echocardiographic preoperative screening, intraoperative guidance, and postoperative follow-up. The purpose of this article is to provide a state-of-the-art review of the key points and technical considerations for preoperative screening, intraoperative guidance, and postoperative follow-up for TTVR.

Transcatheter tricuspid valve replacement for functional tricuspid regurgitation after left-sided valve surgery: A single-center experience.

BACKGROUND: Functional tricuspid regurgitation (FTR) following left-sided valve surgery (LSVS) is of clinical significance due to its high recurrence and mortality rates. Transcatheter therapy presents a potential solution to address this issue. AIMS: The study aimed to assess the safety and efficacy of transcatheter tricuspid valve replacement using the Lux-Valve system in a single center for patients with FTR after LSVS. METHODS: From June 2020 to April 2023, 20 patients with symptomatic severe FTR after LSVS were referred to our center. A multidisciplinary cardiac team evaluated these patients for suitability for transcatheter tricuspid valve replacement with Lux-Valve systems. Primary efficacy and safety endpoints were immediate postoperative tricuspid regurgitation severity ≤ moderate and major adverse events during follow-up. RESULTS: Twenty patients (average age 65.7 ± 7.4 years; 65.0% women) successfully underwent Lux-Valve system implantation after LSVS. All patients achieved ≤ moderate tricuspid regurgitation immediately after the procedure. Only one patient (5.0%) experienced a procedure-related major adverse event, leading to in-hospital mortality due to pulmonary infection. At the 6-month follow-up, 17 patients (89.5%) improved to New York Heart Association functional class I to II (p < 0.001). The overall Kansas City Cardiomyopathy Questionnaire score significantly improved (35.9 ± 6.7 points to 58.9 ± 5.8 points, p < 0.001). CONCLUSION: The Lux-Valve system was found to be safe and effective for treating FTR after LSVS. It resulted in positive early outcomes, including a significant reduction in FTR, improved functional status, and enhanced quality of life, especially in high-risk patients.

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation.

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

Resolving phenylephrine HCl and guaifenesin enantiomers on cellulose-based chiral stationary phases: Separation of four enantiomers on 50-mm column.

Chiral high performance liquid chromatographic technique usually employs polysaccharide-based stationary phases in a normal phase mode. This frequently generates large waste of organic solvents. Using shorter columns of 50 mm length as well as a mobile phase with a high water percentage are common approaches for greening this analytical technique. In this context, a new chiral chromatographic technique was developed for simultaneous enantio-separation of phenylephrine HCl and guaifenesin racemates. Four 50 mm cellulose-based columns were experimented to separate the four enantiomers in a reversed phase mode. A face centered design was then employed to optimize the mobile phase acetonitrile% and flow rate on Lux Cellulose-1 (50 × 4.6 mm, 5 µm). The simultaneous resolution of the cited drugs enantiomers was achieved using acetonitrile-water (30:70, by volume), with a flow rate of 0.5 ml min-1 . These optimized chromatographic conditions separate the enantiomers in 7 min running time, generating about 1.0 ml acetonitrile per run. The proposed method was favorably compared with other reported chiral ones in terms of waste volume generated and analysis time required.

Development of Fluorescent Bacteria with Lux and Riboflavin Genes.

Lumazine protein from marine luminescent bacteria of Photobacterium species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light emission of bacterial luminescent systems is used as a sensitive, rapid, and safe assay for an ever-increasing number of biological systems. Plasmid pRFN4, containing the genes encoding riboflavin from the rib operon of Bacillus subtilis, was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-lumP and pRFN4-Pp luxLP N-lumP) were constructed by amplifying the DNA encoding the N-lumP gene (luxL) from P. phosphoreum and the promoter region (luxLP) present upstream of the lux operon of the gene by PCR and ligating into the pRFN4-Pp N-lumP plasmid. A new recombinant plasmid, pRFN4-Pp luxLP-N-lumP, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into Escherichia coli. When this plasmid was transformed into E. coli 43R, the fluorescence intensity of transformants was 500 times greater than that of E. coli alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the lux promoter exhibited expression that was so high as to show fluorescence in single E. coli cells. The fluorescent bacterial systems developed in the present study using lux and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times.

OsLUX Confers Rice Cold Tolerance as a Positive Regulatory Factor.

During the early seedling stage, rice (Oryza sativa L.) must overcome low-temperature stress. While a few cold-tolerance genes have been characterized, further excavation of cold-resistance genes is still needed. In this study, we identified a cold-induced transcription factor-LUX ARRHYTHMO (LUX)-in rice. OsLUX was found to be specifically expressed in leaf blades and upregulated by both cold stress and circadian rhythm. The full-length OsLUX showed autoactivation activity, and the OsLUX protein localized throughout the entire onion cell. Overexpressing OsLUX resulted in increased cold tolerance and reduced ion leakage under cold-stress conditions during the seedling stage. In contrast, the knockout of OsLUX decreased seedling cold tolerance and showed higher ion leakage compared to the wild type. Furthermore, overexpressing OsLUX upregulated the expression levels of oxidative stress-responsive genes, which improved reactive oxygen species (ROS) scavenging ability and enhanced tolerance to chilling stress. Promoter analysis showed that the OsLUX promoter contains two dehydration-responsive element binding (DREB) motifs at positions -510/-505 (GTCGGa) and -162/-170 (cCACCGccc), which indicated that OsDREB1s and OsDREB2s probably regulate OsLUX expression by binding to the motif to respond to cold stress. Thus, OsLUX may act as a downstream gene of the DREB pathway. These results demonstrate that OsLUX serves as a positive regulatory factor of cold stress and that overexpressing OsLUX could be used in rice breeding programs to enhance abiotic stress tolerance.

100,000 lumens to treat seasonal affective disorder: A proof of concept RCT of Bright, whole-ROom, All-Day (BROAD) light therapy.

BACKGROUND: Seasonal affective disorder (SAD) is common and debilitating. The standard of care includes light therapy provided by a light box; however, this treatment is restrictive and only moderately effective. Advances in LED technology enable lighting solutions that emit vastly more light than traditional light boxes. Here, we assess the feasibility of BROAD (Bright, whole-ROom, All-Day) light therapy and get a first estimate for its potential effectiveness. METHODS: Patients were randomly assigned to a treatment for 4 weeks; either a very brightly illuminated room in their home for at least 6 h per day (BROAD light therapy) or 30 min in front of a standard 10,000 lux SAD light box. Feasibility was assessed by monitoring recruitment, adherence, and side effects. SAD symptoms were measured at baseline and after 2 and 4 weeks, with the Hamilton Depression Rating Scale-Seasonal Affective Disorders 29-items, self-report version. RESULTS: All 62 patients who started treatment were available at 4-week follow-up and no significant adverse effects were reported. SAD symptoms of both groups improved similarly and considerably, in line with previous results. Exploratory analyses indicate that a higher illuminance (lux) is associated with a larger symptom improvement in the BROAD light therapy group. CONCLUSIONS: BROAD light therapy is feasible and seems similarly effective as the standard of care while not confining the participants to 30 min in front of a light box. In follow-up trials, BROAD light therapy could be modified for increased illuminance, which would likely improve its effectiveness.

Construction of luminescent Escherichia coli via expressing lux operons and their application on toxicity test.

Recombinant luminescent Escherichia coli strains could be used to detect the toxicity of pure or mixed contaminants as a light-off sensor. In this work, the lux operon of Photobacterium phosphoreum T3 was identified for the first time. Recombinant luminescent E. coli strains were constructed via expressing the lux operons of P. phosphoreum T3 and Vibrio qinghaiensis Q67 in E. coli MG1655, and the optimal protectant containing 10% (w/v) trehalose and 4% sucrose was used to prepare the freeze-dried recombinant luminescent E. coli cells. Then, these freeze-dried E. coli cells were subjected to acute toxicity detection. The results showed that luminescent E. coli strains displayed sensitive toxic responses to BPA, nFe2O3, Cd, Pb, As, and Hg, for example, the EC50 values of BPA and nFe2O3 to luminescent E. coli strains ranged from 1.54 to 50.19 mg/l and 17.50 to 21.52 mg/l, respectively. Indeed, luminescent E. coli strains exhibited more sensitive responses to Cd, Pb, and Hg than the natural strain Q67. The results suggested that recombinant luminescent E. coli strains could be used for the detection of acute toxicity. Furthermore, the combined toxicities of BPA and nFe2O3, Hg, and Pb were measured, and the joint effects of these mixtures were evaluated with luminescent E. coli. The results indicated that the joint effects of BPA and nFe2O3 suggested to be synergistic or additive to luminescent E. coli, while the joint effects of heavy metals and nFe2O3 exhibited additivities. The cellular endocytosis for Fe2O3 nanoparticles was not observed, which could explain the additive instead of synergistic effects between heavy metals and nFe2O3. KEY POINTS: • Sequence of the lux operon from P. phosphoreum T3 was reported for the first time. • Recombinant luminescent E. coli was more sensitive to Cd, Pb, and Hg than Q67. • Joint effects of BPA and nFe2O3 were synergistic or additive to luminescent E. coli.

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system.

Bioluminescence-based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has, so far, not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.

Simultaneous Determination of Escitalopram Impurities including the R-enantiomer on a Cellulose tris(3,5-Dimethylphenylcarbamate)-Based Chiral Column in Reversed-Phase Mode.

A high-performance liquid chromatographic method was developed for the simultaneous determination of the related substances-three potential synthesis-related chemical impurities and the distomer-of escitalopram. The separation capacity of seven different polysaccharide-type chiral columns, including three amylose-based (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2) and four cellulose-based columns (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, and Lux Cellulose-4) were screened in the polar organic and reversed-phase modes. Lux Cellulose-1, based on cellulose tris(3,5-dimethylphenylcarbamate) as the chiral selector with an acetonitrile-water mixture containing 0.1% diethylamine was identified as the most promising separation system. Using the "one factor at a time" optimization approach, the effect of column temperature, flow rate, and mobile phase constituents on separation performance was evaluated, and the critical resolution values were determined. A U-shaped retention pattern was obtained when plotting the retention factors of the citalopram enantiomers versus the water content of the binary mobile phases on the Lux Cellulose-1 column. A thermodynamic analysis revealed enthalpy-driven enantioseparation in both the polar organic and reversed-phase modes. For further method optimizations, an L9 orthogonal array table was employed. Using the optimized parameters (Lux Cellulose-1 column with 0.1% (v/v) diethylamine in water/acetonitrile 55/45 (v/v); 0.8 mL/min flow rate at 25 °C), baseline separations were achieved between all compounds. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested with a commercially available pharmaceutical formulation. The method proved to be suitable for routine quality control of related substances and the enantiomeric purity of escitalopram.

The plant circadian clock regulates autophagy rhythm through transcription factor LUX ARRHYTHMO.

Autophagy is an evolutionarily conserved degradation pathway in eukaryotes; it plays a critical role in nutritional stress tolerance. The circadian clock is an endogenous timekeeping system that generates biological rhythms to adapt to daily changes in the environment. Accumulating evidence indicates that the circadian clock and autophagy are intimately interwoven in animals. However, the role of the circadian clock in regulating autophagy has been poorly elucidated in plants. Here, we show that autophagy exhibits a robust circadian rhythm in both light/dark cycle (LD) and in constant light (LL) in Arabidopsis. However, autophagy rhythm showed a different pattern with a phase-advance shift and a lower amplitude in LL compared to LD. Moreover, mutation of the transcription factor LUX ARRHYTHMO (LUX) removed autophagy rhythm in LL and led to an enhanced amplitude in LD. LUX represses expression of the core autophagy genes ATG2, ATG8a, and ATG11 by directly binding to their promoters. Phenotypic analysis revealed that LUX is responsible for improved resistance of plants to carbon starvation, which is dependent on moderate autophagy activity. Comprehensive transcriptomic analysis revealed that the autophagy rhythm is ubiquitous in plants. Taken together, our findings demonstrate that the LUX-mediated circadian clock regulates plant autophagy rhythms.

Serotonin receptor oligomerization regulates cAMP-based signaling.

Protein-protein interaction is often investigated using quantitative molecular microscopy with Förster resonant energy transfer (FRET). Here, we combined 'linear unmixing FRET' (lux-FRET) with the simultaneous application of a FRET-based biosensor for cAMP to investigate the oligomerization between the 5-HT7 receptor (5-HT7R, also known as HTR7) and the 5-HT1A receptor (5-HT1AR, also known as HTR1A) and its importance for cAMP signaling. We found that the 5-HT7R not only stimulates cAMP production, but also forms hetero-oligomers with 5-HT1AR, which blocks the inhibitory effect of the latter. 5-HT7R signaling, however, is not affected by this hetero-oligomerization. By modeling the kinetics of intracellular cAMP level changes in relation to the 5-HT7R:5-HT1AR stoichiometry, we were able to decipher the complex signaling characteristics of endogenous serotonin receptors in cultured hippocampal neurons. Our findings indicate that serotonergic signaling is not only modulated by the concentration of an individual receptor but also by its specific interaction with other receptors in endogenous systems. We conclude that the regulated ratio of serotonin receptors in immature and mature neurons may be critically involved in both the onset and response to treatments of psychiatric diseases, such as anxiety and depression.

Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces.

BACKGROUND: Identifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest in Streptomyces. RESULTS: A method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria. CONCLUSIONS: Our approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.

Modulations in irradiance directed at melanopsin, but not cone photoreceptors, reliably alter electrophysiological activity in the suprachiasmatic nucleus and circadian behaviour in mice.

Intrinsically photosensitive retinal ganglion cells convey intrinsic, melanopsin-based, photoreceptive signals alongside those produced by rods and cones to the suprachiasmatic nucleus (SCN) circadian clock. To date, experimental data suggest that melanopsin plays a more significant role in measuring ambient light intensity than cone photoreception. Such studies have overwhelmingly used diffuse light stimuli, whereas light intensity in the world around us varies across space and time. Here, we investigated the extent to which melanopsin or cone signals support circadian irradiance measurements in the presence of naturalistic spatiotemporal variations in light intensity. To address this, we first presented high- and low-contrast movies to anaesthetised mice whilst recording extracellular electrophysiological activity from the SCN. Using a mouse line with altered cone sensitivity (Opn1mwR mice) and multispectral light sources we then selectively varied irradiance of the movies for specific photoreceptor classes. We found that steps in melanopic irradiance largely account for the light induced-changes in SCN activity over a range of starting light intensities and in the presence of spatiotemporal modulation. By contrast, cone-directed changes in irradiance only influenced SCN activity when spatiotemporal contrast was low. Consistent with these findings, under housing conditions where we could independently adjust irradiance for melanopsin versus cones, the period lengthening effects of constant light on circadian rhythms in behaviour were reliably determined by melanopic irradiance, regardless of irradiance for cones. These data add to the growing evidence that modulating effective irradiance for melanopsin is an effective strategy for controlling the circadian impact of light.

Aerosol is the optimal route of respiratory tract infection to induce pathological lesions of colibacillosis by a lux-tagged avian pathogenic Escherichia coli in chickens.

Pathogenesis of colibacillosis caused by avian pathogenic Escherichia coli (APEC) in poultry is unclear and experimental studies reveal substantial inconsistency. In this study, the impact of three infection routes differing in the site of deposition of inoculum in the respiratory tract, were investigated. Two-weeks-old chickens were infected with a lux-tagged APEC strain via aerosol, intranasally or intratracheally, and sequentially sampled along with uninfected birds. At 1 and 3 days post infection (dpi), liver or spleen to body-weight ratios in all infected groups were significantly higher than in negative control, while at 7 dpi, such differences were significant in both organs in the aerosol-infected group. The infection-strain colonized tracheas and lungs in infected birds at 1 dpi and persisted until 7 dpi. Among infected groups, in lungs, bacterial load at 1 dpi was significantly lower in intranasally-inoculated birds. Histology revealed that, independent of infection route, lesions were mostly seen in the lower respiratory organs (lungs and air sacs) characterized by bronchitis/pneumonia and airsacculitis. Birds infected via aerosol showed the highest mean lesion score in lungs while intranasal application caused the mildest pathological changes, and difference between the two groups was significant at 1 dpi. In spleen, heterophilic infiltrations were prominent in affected birds. Interestingly, tracheas were pathologically unaffected. Altogether, the results demonstrated the importance of infection route, with aerosol being the most suitable to induce pathological lesions of colibacillosis without predisposing factors. Furthermore, the lux-tagged APEC strain was discriminated from native isolates enabling exact differentiation and enumeration.RESEARCH HIGHLIGHTS Lux-tagged APEC strain was used for infection to differentiate from native E. coli.Pathologically, lungs, air sacs and spleen but not trachea were affected.The route of infection strongly impacts the pathological outcome with APEC.The infection with APEC via aerosol caused the most severe lesions in chickens.

Twenty-Four-Month Outcomes of Drug-Coated Balloon in Diabetic Patients in the BIOLUX P-III Registry: A Subgroup Analysis.

OBJECTIVES: This study aims to assess the use of drug-coated balloon (DCB) in a large patient population under real-world conditions and, specifically, analyse the impact of diabetes mellitus on long term outcomes following DCB utilisation. METHODS: BIOLUX P-III is a prospective, international, multicentre, registry that was conducted at 41 centres. The present study is a 24-month subgroup analysis of patients with diabetes mellitus having infrainguinal lesions treated with the Passeo-18 Lux DCB. The primary endpoints were freedom from major adverse events (MAEs) within 6 months of intervention and freedom from clinically driven target lesion revascularisation (CD-TLR) within 12 months of intervention. RESULTS: Of the 882 patients in the registry, 418 had diabetes (516 lesions). Most diabetics had concomitant hypertension (88.8%) and hyperlipidaemia (70.3%). Insulin dependence was observed in 48.8% of diabetics. Moreover, smoking (62.2%) and chronic renal insufficiency (41.9%) were also found to be common in this cohort. Chronic limb threatening ischemia (Rutherford class ≥4) was present in 53.1% of all patients. 22.9% of lesions were infrapopliteal, while 22.5% of lesions were treated for in-stent restenosis. The mean target lesion length was 85.6 ± 73.2 mm, and 79.4% of lesions were calcified (of which 17.9% were heavily calcified). Overall, device success was 99.7%. Freedom from MAEs was 90.5% (95% confidence interval (95% CI): 87.2-93.0) at 6 months, 85.4% (95% CI: 81.5-88.6) at 12 months and 80% (95% CI: 75.5-83.8) at 24 months. Freedom from CD-TLR was 95.9% (95% CI: 93.8-97.4), 91.6% (95% CI: 88.7-93.8), and 87.1% (95% CI: 83.5-89.9) at 6, 12, and 24 months, respectively. All-cause mortality at 24 months in diabetics was 16.0% (95% CI: 12.6-20.2), and major target limb amputation was 6.1% (95% CI: 4.1-8.9), which was significantly higher than in non-diabetics (8.4% (95% CI: 6.0-11.6), P = 0.0005 and 1.2% (95% CI: 0.5-2.9), P <0.0001, respectively). At 24 months, 82.0% of patients had improved by ≥1 Rutherford class. CONCLUSION: Treatment of a real-world diabetic patient population with the Passeo-18 Lux DCB resulted in high efficacy and low complication rates, despite the fact that diabetic patients usually suffer from a multitude of concomitant comorbidities. CLINICAL TRIAL REGISTRATION: NCT02276313.

Quorum-sensing induced transitions between bistable steady-states for a cell-bulk ODE-PDE model with lux intracellular kinetics.

Intercellular signaling and communication are used by bacteria to regulate a variety of behaviors. In a type of cell-cell communication known as quorum sensing (QS), which is mediated by a diffusible signaling molecule called an autoinducer, bacteria can undergo sudden changes in their behavior at a colony-wide level when the density of cells exceeds a critical threshold. In mathematical models of QS behavior, these changes can include the switch-like emergence of intracellular oscillations through a Hopf bifurcation, or sudden transitions between bistable steady-states as a result of a saddle-node bifurcation of equilibria. As an example of this latter type of QS transition, we formulate and analyze a cell-bulk ODE-PDE model in a 2-D spatial domain that incorporates the prototypical LuxI/LuxR QS system for a collection of stationary bacterial cells, as modeled by small circular disks of a common radius with a cell membrane that is permeable only to the autoinducer. By using the method of matched asymptotic expansions, it is shown that the steady-state solutions for the cell-bulk model exhibit a saddle-node bifurcation structure. The linear stability of these branches of equilibria are determined from the analysis of a nonlinear matrix eigenvalue problem, called the globally coupled eigenvalue problem. The key role on QS behavior of a bulk degradation of the autoinducer field, which arises from either a Robin boundary condition on the domain boundary or from a constant bulk decay, is highlighted. With bulk degradation, it is shown analytically that the effect of coupling identical bacterial cells to the bulk autoinducer diffusion field is to create an effective bifurcation parameter that depends on the population of the colony, the bulk diffusivity, the membrane permeabilities, and the cell radius. QS transitions occur when this effective parameter passes through a saddle-node bifurcation point of the Lux ODE kinetics for an isolated cell. In the limit of a large but finite bulk diffusivity, it is shown that the cell-bulk system is well-approximated by a simpler ODE-DAE system. This reduced system, which is used to study the effect of cell location on QS behavior, is easily implemented for a large number of cells. Predictions from the asymptotic theory for QS transitions between bistable states are favorably compared with full numerical solutions of the cell-bulk ODE-PDE system.

Real-time tracking of stem cell viability, proliferation, and differentiation with autonomous bioluminescence imaging.

BACKGROUND: Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. RESULTS: We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20-30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. CONCLUSIONS: Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.