The Use of Thermoporometry in the Study of Frost Resistance of Rocks.

From an engineering point of view, it is important to know the factors influencing the frost resistance of rocks with porosity above 2% due to their different frost resistance. The article focused on frost durability research using the thermoporometry method (TMP) and the assessment of water phase transition in the pore spaces of selected rocks. For this purpose, the differential scanning calorimetry method (DSC) was used with the adoption of an original algorithm for eliminating the thermal inertia of the measurement system. The results of the DSC method were supplemented with the results of pore size distribution using the mercury intrusion porosimetry method (MIP) and standard rock frost resistance tests. Based on the research carried out using the thermoporometry method, it was confirmed that the ability of water to freeze in the temperature range from -5 °C to -20 °C was important, as well as the ability of rocks to increase the degree of water saturation during freeze-thaw cycles. Based on calorimetric tests combined with thermoporometry, in the case of non-frost-resistant rocks, a significant (dominant) share of pores with a radius of under 10 nm (amounting to over 0.008 cm3/cm3) was found. Pore connections in the water freezing process do not influence the investigated rocks' frost resistance.

Comprehensive genomic exploration of class III peroxidase genes in guava unravels physiology, evolution, and postharvest storage responses.

Peroxidases (PRXs) play multifaceted roles in plant growth, development, and stress responses. Here, we present a comprehensive analysis of the PRX gene family in guava, a globally significant fruit. In the guava genome, we identified 37 PRX genes, a number lower than that of Arabidopsis, suggesting a distinctive gene family expansion pattern. Phylogenetic analysis unveiled close relationships with Arabidopsis PRXs, with 12 PgPRX genes forming ortholog pairs, indicating a specific expansion pattern. Predictions placed most PRX proteins in the chloroplast and extracellular regions. Structural analysis of PgPRX proteins revealed commonalities in domain structures and motif organization. Synteny analysis underscored the dynamic role of segmental duplication in the evolution of guava's PRX genes. We explored the dynamic expression of PgPRX genes across guava tissues, exposing functional diversity. Furthermore, we examined changes in peroxidase levels and gene expressions during postharvest fruit storage, providing insights for preserving fruit quality. This study offers an initial genome-wide identification and characterization of Class III peroxidases in guava, laying the foundation for future functional analyses.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India.

Apple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.

Rindera graeca (A. DC.) Boiss. & Heldr. (Boraginaceae) In Vitro Cultures Targeting Lithospermic Acid B and Rosmarinic Acid Production.

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC-PDA-ESI-HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds.

A Survey of Current Practice in Operative Management of Testicular Torsion in Poland.

The primary aim of this study is to evaluate heterogeneity in the current management of testicular torsion (TT). A secondary aim is to investigate incidences of recurrent torsion and the methods used for primary fixation. An online multiple-choice questionnaire comprising 10 questions was distributed to paediatric surgeons and urologists. There were a total of 99 questionnaires distributed to representatives from 39 paediatric surgery and urology departments in Poland. The majority of participants agreed on fixation of the torsed testicle (98%). Use of sutures was reported by 95% of surgeons: absorbable by 48%, non-absorbable by 42%, and 4% using both. There was no consensus on the number of sutures. The contralateral testicle was always fixed by 69%, with 28% fixing it only in case of necrosis and amputation of the torsed testicle, and the remaining 2% never fixing the contralateral side. In case of negative scrotal exploration, 18% of surgeons would still fix the testis. The recurrence of torsion after prior fixation was identified by eight of the participants. The most commonly reported primarily used technique was absorbable sutures. There is a general consensus on the fixation of torsed testicles; however, other aspects remain controversial. Based on the survey and the literature review, the use of non-absorbable sutures rather than absorbable sutures would be advisable.

Manipulation of light spectrum is an effective tool to regulate biochemical traits and gene expression in lettuce under different replacement methods of nutrient solution.

The use of light-emitting diode (LED) technology represents a promising approach to improve plant growth and metabolic activities. The aim of this study was to investigate the effect of different light spectra: red (656 nm), blue (450 nm), red/blue (3:1), and white (peak at 449 nm) on biochemical properties, photosynthesis and gene expression in two lettuce cultivars (Lollo Rossa and Lollo Bionda) grown under different methods of nutrient solution replacement in hydroponics. Complete replacement and EC-based replacement of nutrient solution increased content of proline and soluble sugars and activity of antioxidant enzymes (CAT, GPX and SOD) under the red/blue LED and red LED light treatments in both cultivars. In addition, the red/blue and the monochromatic red light increased the soluble protein content and the antioxidant activity in the Lollo Rosa cultivar under the replacement method according to the needs of the plant. An increase in flavonoid content in the EC-based method in the Lollo Rosa variety treated with a combination of red and blue light was also observed. The red/blue light had the greatest induction effect on anthocyanin content, expression of the UFGT, CHS, and Rubisco small subunit genes, and the net photosynthetic rate. Data presented here will directly contribute to the development of nutrient solution and LED spectrum management strategies to significantly improve plant growth and metabolism, while avoiding water and nutrient waste, and environmental pollution.

Prevalence of Dry Eye Symptoms and Associated Risk Factors among University Students in Poland.

Aim: The aim of the study was to demonstrate the prevalence and risk factors of dry eye symptoms (DES) among university students in Poland. Material and methods: A cross-sectional study survey was conducted among 312 Polish university students. The questionnaire consisted of the Ocular Surface Disease Index (OSDI), the 5-Item Dry Eye Questionnaire (DEQ-5) and questions regarding medical history and risk factors. Results: According to the OSDI, more than half of respondents (57.1%) have symptoms of ocular surface disease. Time spent using electronic devices is correlated with scores gathered in both OSDI and DEQ-5 (p < 0.001). There is a statistically significant dependence between psychotropics (p = 0.002), glucocorticosteroids usage (p = 0.026), the presence of depression (p < 0.001), diabetes (p = 0.01) or allergy (p = 0.008) and dry eye symptoms proved in both questionnaires. Respondents with refractive errors and those living in metropolitan areas have a statistically higher symptom intensity(p < 0.022). Stress felt by students is associated with higher DES risk. No correlation between DES and smoking habits was observed. The history of SARS-CoV-2 infection was associated with the severity of DES (p = 0.036). Conclusion: Pathogenesis of DES is multifactorial and its severity depends on several factors, both genetic and environmental. Its prevalence among the young population is underestimated. Determining risk factors will enable the implementation of appropriate prophylaxis and early diagnosis.

Effect of Cellulose Ether and Starch Ether on Hydration of Cement Processes and Fresh-State Properties of Cement Mortars.

The production of factory-made mortars is a multicomponent system. Viscosity-modifying admixtures (VMAs) are an inherent ingredient of these materials. The correct choice of the amount and type of these admixtures is important from the practical and scientific points of view. In this article, the use of cellulose ether (CE) and starch ether (SE) in cement pastes and mortars is studied. This research focuses on the hydration process and fresh-state properties of mortars because this subject determines the correct choice of the amount and type of admixture used, and the results determine the application and properties of hardened mortars. Polymers were added in the range from 0.056% to 0.22% in relation to the dry ingredients of the mortar. The research showed that cellulose ether had the greatest impact on the consistency, air content, bulk density, and water retention of ordinary dry-mix mortars. On the other hand, starch ether affected the hydration process, delaying the setting and hardening processes much more than cellulose ether. The action of these admixtures rose with the increase in the amount of polymer used in different ways (depending on the type of ether).

Photosynthetic and yield responses of rotating planting strips and reducing nitrogen fertilizer application in maize-peanut intercropping in dry farming areas.

Improving cropping systems together with suitable agronomic management practices can maintain dry farming productivity and reduce water competition with low N inputs. The objective of the study was to determine the photosynthetic and yield responses of maize and peanut under six treatments: sole maize, sole peanut, maize-peanut intercropping, maize-peanut rotation-intercropping, 20% and 40% N reductions for maize in the maize-peanut rotation-intercropping. Maize-peanut intercropping had no land-use advantage. Intercropped peanut is limited in carboxylation rates and electron transport rate (ETR), leading to a decrease in hundred-grain weight (HGW) and an increase in blighted pods number per plant (NBP). Intercropped peanut adapts to light stress by decreasing light saturation point (Isat) and light compensation point (Icomp) and increasing the electron transport efficiency. Intercropped maize showed an increase in maximum photosynthetic rate (Pnmax) and Icomp due to a combination of improved intercellular CO2 concentration, carboxylation rates, PSII photochemical quantum efficiency, and ETR. Compare to maize-peanut intercropping, maize-peanut rotation-intercropping alleviated the continuous crop barriers of intercropped border row peanut by improving carboxylation rates, electron transport efficiency and decreasing Isat, thereby increasing its HGW and NBP. More importantly, the land equivalent ratio of maize-peanut rotation-intercropping in the second and third planting years were 1.05 and 1.07, respectively, showing obvious land use advantages. A 20% N reduction for maize in maize-peanut rotation-intercropping does not affect photosynthetic character and yield for intercropped crops. However, a 40% N reduction decreased significantly the carboxylation rates, ETR, Icomp and Pnmax of intercropped maize, thereby reducing in a 14.83% HGW and 5.75% lower grain number per spike, and making land-use efficiency negative.

A Comparative Study on Hygric Properties and Compressive Strength of Ceramic Bricks.

This article analyzes the results of capillary rise, compressive strength and water absorption tests on solid ceramic bricks from existing structures and demolition materials taken from 11 different structures. In addition (for more extensive interpretation and evaluation of porosity), tests were performed for the selected series of bricks using a mercury porosimeter (MIP) and a micro computed tomography (micro-CT). Contemporary bricks (2 series) were also evaluated for comparison purposes. The conducted tests indicate that bricks obtained from different sources are characterized by "individual" relation of compressive strength and porosity, and "individual" relation of water absorption coefficient and porosity. In addition, on the basis of the results obtained in the study, compared with the literature data, it can be deduced that ceramic bricks with a water absorption coefficient of less than 50 g/m2s0.5 are characterized by a compressive strength of more than 80 MPa. As the research shows, the properties of bricks even from a single building can differ one from another, which can result in varying durability even within a single building. When choosing a material during the renovation or restoration of facilities, it is important to perform tests on the physical and mechanical properties of the original material, which will be reused.

The Role of Dysregulated miRNAs in the Pathogenesis, Diagnosis and Treatment of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is an eye disease causing damage to the macular region of the retina where most of the photoreceptors responsible for central visual acuity are located. MicroRNAs (miRNAs) are small single-stranded non-coding RNA molecules that negatively regulate genes by silent post-transcriptional gene expressions. Previous studies have shown that changes in specific miRNAs are involved in the pathogenesis of eye diseases, including AMD. Altered expressions of miRNAs are related to disturbances of regulating oxidative stress, inflammation, angiogenesis, apoptosis and phagocytosis, which are known factors in the pathogenesis of AMD. Moreover, dysregulation of miRNA is involved in drusen formation. Thus, miRNAs may be used as potential molecular biomarkers for the disease and, furthermore, tailoring therapeutics to particular disturbances in miRNAs may, in the future, offer hope to prevent irreversible vision loss. In this review, we clarify the current state of knowledge about the influence of miRNA on the pathogenesis, diagnosis and treatment of AMD. Our study material consisted of publications, which were found in PubMed, Google Scholar and Embase databases using "Age-related macular degeneration", "miRNA", "AMD biomarkers", "miRNA therapeutics" and "AMD pathogenesis" as keywords. Paper search was limited to articles published from 2011 to date. In the section "Retinal, circulating and vitreous body miRNAs found in human studies", we limited the search to studies with patients published in 2016-2021.

Method to analyze effects of low-level laser therapy on biological cells with a digital holographic microscope.

Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.

Are There Still Difficult-to-Treat Patients with Chronic Hepatitis C in the Era of Direct-Acting Antivirals?

Difficult-to-treat populations with chronic hepatitis C (CHC), in the era of interferon treatment, included patients with liver cirrhosis, kidney impairment, treatment-experienced individuals, and those coinfected with the human immunodeficiency virus. The current study aimed to determine whether, in the era of direct-acting antivirals (DAA), there are still patients that are difficult-to-treat. The study included all consecutive patients chronically infected with hepatitis C virus (HCV) who started interferon-free therapy between July 2015 and December 2020 in the Department of Infectious Diseases in Kielce. The analyzed real-world population consisted of 963 patients, and most of them were infected with genotype 1 (87.6%) with the predominance of subtype 1b and were treatment-naïve (78.8%). Liver cirrhosis was determined in 207 individuals (21.5%), of whom 82.6% were compensated. The overall sustained virologic response, after exclusion of non-virologic failures, was achieved in 98.4%. The univariable analysis demonstrated the significantly lower response rates in males, patients with liver cirrhosis, decompensation of hepatic function at baseline, documented esophageal varices, concomitant diabetes, body mass index ≥25, and previous ineffective antiviral treatment. Despite an overall very high effectiveness, some unfavorable factors, including male gender, genotype 3 infection, liver cirrhosis, and treatment experience, significantly reduce the chances for a virologic response were identified.

Numerical refractive index correction for the stitching procedure in tomographic quantitative phase imaging.

Tomographic quantitative phase imaging (QPI) lacks an absolute refractive index value baseline, which poses a problem when large dense objects extending over multiple fields of view are measured volume by volume and stitched together. Some of the measurements lack the natural baseline value that is provided by the mounting medium with a known refractive index. In this work, we discuss the problem of the refractive index (RI) baseline of individual reconstructed volumes that are deprived of access to mounting medium due to the extent of the object. The solution of this problem is provided by establishing the RI offsets based on the overlapping regions. We have proven that the process of finding the offset RI values may be justifiably reduced to the analogous procedure in the 2D baseline correction (2D-BC). Finally, we proposed the enhancement of the state-of-the-art 2D-BC procedure previously introduced in the context of 2D QPI. The processing is validated at the examples of a synthetic dataset and a liver organoid.

Genotype-specific versus pangenotypic regimens in patients infected with hepatitis C virus genotype 1b in real-world settings.

Introduction: The introduction of direct-acting antivirals (DAAs) has provided us with hope to eliminate hepatitis C virus (HCV) infection as a significant public health problem in the coming years. Objective: Our study aimed to compare the effectiveness and safety of genotype-specific and pangenotypic regimens in genotype 1b­infected patients treated in real-world settings. Patients and methods: Patients were selected from 990 HCV-infected individuals treated with DAAs in the Department of Infectious Diseases in Kielce, Poland, who had the therapy initiated between July 1, 2015 and December 31, 2020. Results: A total of 795 genotype 1b­infected patients with a median age of 51 years, female predominance (55%), and a 21.1% rate of cirrhosis were included in the analysis. A total of 69.9% of patients were treated with genotype-specific regimens. Those patients were significantly older, more often were treatment experienced, and had advanced liver fibrosis and cirrhosis compared with patients assigned to pangenotypic regimens. An overall sustained virologic response rate of 97.9% in the intention-to-treat (ITT) analysis and 99% after excluding nonvirologic nonresponders was achieved, with no significant difference between patients in the 2 treatment arms. Significantly higher proportions of men (P = 0.001) and DAA-experienced patients (P = 0.049) were documented among virologic nonresponders. Conclusions: We confirmed very high effectiveness and a good safety profile of both genotype-specific and pangenotypic regimens used in patients with genotype 1b HCV infection, and we found no differences between these 2 generations of medications. Male sex and previous treatment with DAAs were identified as negative predictors for therapy effectiveness.

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides.

Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo­electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.

Phosphorus-Induced Adaptation Mechanisms of Rye Grown on Post-Flotation Copper Tailings.

Although a considerable effort has been made over the last decades to develop cost-effective phytotechnologies as an alternative to conventional techniques for the management of contaminated lands, successful revegetation of the tailings still represents a major challenge. Here, we evaluate the potential of rye (Secale cereale L.) for growth and survival on the tailings after copper (Cu) ore processing. Four rye varieties were cultivated in a pot experiment on the post-flotation sediment with increasing phosphorus (P) doses (22, 44, 66, 88, and 110 mg·kg-1). The resistance of the studied rye genotypes to stress was assessed by observing the growth and development of plants, determining the dry mass accumulation, the Cu and P uptake and content, and a number of physiological parameters related mainly to P mobilisation. Exposure of tested rye varieties to high Cu concentrations in the tailings did not result in any significant plant mortality, with the intracellular Cu concentrations being below the critical toxic level. In contrast, the low availability of P due to alkaline properties of the tailings and the mechanisms involved in the mobilisation of sparingly soluble forms of this element (i.e., H+-ATPase-driven proton efflux in roots and organic acid exudation), were identified as main factor determining the level of tolerance. The efficiency of the photosynthetic activity was a key determinant for the P-mobilising capacity of rye. We further showed that rye varieties with more primitive genetic background might be potentially more suitable for growth on the post-flotation copper tailings. The results provide important and novel knowledge that will certainly support future works in developing strategies for successful revitalisation of degraded lands.

Effectiveness and Safety of Pangenotypic Regimens in the Most Difficult to Treat Population of Genotype 3 HCV Infected Cirrhotics.

There is still limited data available from real-world experience studies on the pangenotypic regimens in patients with genotype (GT) 3 hepatitis C virus (HCV) infection and liver cirrhosis. The current study aimed to evaluate the efficacy and safety of pangenotypic regimens in this difficult-to-treat population. A total of 236 patients with mean age 52.3 ± 11.3 years and male predominance (72%) selected from EpiTer-2 database were included in the analysis; 72% of them were treatment-naïve. The majority of patients (55%) received the combination of sofosbuvir/velpatasvir (SOF/VEL), 71 without and 58 with ribavirin (RBV), whereas the remaining 107 individuals were assigned to glecaprevir/pibrentasvir (GLE/PIB). The effectiveness of the treatment following GLE/PIB and SOF/VEL regimens (96% and 93%) was higher compared to SOF/VEL + RBV option (79%). The univariate analysis demonstrated the significantly lower sustained virologic response in males, in patients with baseline HCV RNA ≥ 1,000,000 IU/mL, and among those who failed previous DAA-based therapy. The multivariate logistic regression analysis recognized only the male gender and presence of ascites at baseline as the independent factors of non-response to treatment. It should be emphasized that despite the availability of pangenotypic, strong therapeutic options, GT3 infected patients with cirrhosis still remain difficult-to-treat, especially those with hepatic impairment and DAA-experienced.

Can di-4-ANEPPDHQ reveal the structural differences between nanodiscs and liposomes?

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.