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This paper presents the development of near-infrared (NIR) fluorescent probes, A and B, engineered from hemicyanine dyes with 1,8-naphthalic and rhodamine derivatives for optimized photophysical properties and precise mitochondrial targeting. Probes A and B exhibit absorption peaks at 737 nm and low fluorescence in phosphate-buffered saline (PBS) buffer. Notably, their fluorescence intensities, peaking at 684 (A) and 702 nm (B), increase significantly with viscosity, as demonstrated through glycerol-to-PBS ratio experiments. This increase is attributed to restricted rotational freedom in the fluorophore and its linkages to rhodamine or 1,8-naphthalic groups. Theoretical modeling suggests nonplanar configurations for both probes, with primary absorptions in the rhodamine and hemicyanine cores (A: 543; B: 536 nm), and additional transitions to 1,8-naphthalic (A: 478 nm) and rhodamine (B: 626 nm) groups. Probe A is also responsive to human serum albumin (HSA), a key biomarker, with fluorescence increasing in HeLa cells as HSA concentrations rise. In contrast, probe B shows no response to HSA, likely due to steric hindrance from its bulky rhodamine group, illustrating a selectivity difference between the probes. Probe B, however, excels in mitochondrial imaging, confirmed through cellular and in vivo studies. In HeLa cells, it tracked viscosity changes following treatment with monensin, nystatin, and lipopolysaccharide (LPS), with fluorescence increasing in a dose-dependent manner. In fruit flies, probe B effectively detected monensin-induced viscosity changes, demonstrating its stability and in vivo applicability. These findings highlight the versatility and sensitivity of probes A and B as tools in biological research, with potential applications in monitoring mitochondrial health, detecting biomarkers like HSA, and investigating mitochondrial dynamics in disease. 

Mitochondria, central organelles pivotal for eukaryotic cell function, extend their influence beyond ATP production, encompassing roles in apoptosis, calcium signaling, and biosynthesis. Recent studies spotlight two emerging determinants of mitochondrial functionality: intramitochondrial viscosity and sulfur dioxide (SO2) levels. While optimal mitochondrial viscosity governs molecular diffusion and vital processes like oxidative phosphorylation, aberrations are linked with neurodegenerative conditions, diabetes, and cancer. Similarly, SO2, a gaseous signaling molecule, modulates energy pathways and oxidative stress responses; however, imbalances lead to cytotoxic sulfite and bisulfite accumulation, triggering disorders such as cancer and cardiovascular anomalies. Our research focused on development of a dual-channel fluorescent probe, applying electron-withdrawing acceptors within a coumarin dye matrix, facilitating monitoring of mitochondrial viscosity and SO2 in live cells. This probe distinguishes fluorescence peaks at 650 nm and 558 nm, allowing ratiometric quantification of SO2 without interference from other sulfur species. Moreover, it enables near-infrared viscosity determination, particularly within mitochondria. The investigation employed theoretical calculations utilizing Density Functional Theory (DFT) methods to ascertain molecular geometries and calculate rotational energies. Notably, the indolium segment of the probe exhibited the lowest rotational energy, quantified at 7.38 kcals/mol. The probe featured heightened mitochondrial viscosity dynamics when contained within HeLa cells subjected to agents like nystatin, monensin, and bacterial lipopolysaccharide (LPS). Overall, our innovative methodology elucidates intricate mitochondrial factors, presenting transformative insights into cellular energetics, redox homeostasis, and therapeutic avenues for mitochondrial-related disorders. 

Fluorescent probes play a crucial role in elucidating cellular processes, with NAD(P)H sensing being pivotal in understanding cellular metabolism and redox biology. Here, the development and characterization of three fluorescent probes, A, B, and C, based on the coumarin platform for monitoring of NAD(P)H levels in living cells are described. Probes A and B incorporate a coumarin-cyanine hybrid structure with vinyl and thiophene connection bridges to 3-quinolinium acceptors, respectively, while probe C introduces a dicyano moiety for replacement of the lactone carbonyl group of probe A which increases the reaction rate of the probe with NAD(P)H. Initially, all probes exhibit subdued fluorescence due to intramolecular charge transfer (ICT) quenching. However, upon hydride transfer by NAD(P)H, fluorescence activation is triggered through enhanced ICT. Theoretical calculations confirm that the electronic absorption changes upon the addition of hydride to originate from the quinoline moiety instead of the coumarin section and end up in the middle section, illustrating how the addition of hydride affects the nature of this absorption. Control and dose–response experiments provide conclusive evidence of probe C’s specificity and reliability in identifying intracellular NAD(P)H levels within HeLa cells. Furthermore, colocalization studies indicate probe C’s selective targeting of mitochondria. Investigation into metabolic substrates reveals the influence of glucose, maltose, pyruvate, lactate, acesulfame potassium, and aspartame on NAD(P)H levels, shedding light on cellular responses to nutrient availability and artificial sweeteners. Additionally, we explore the consequence of oxaliplatin on cellular NAD(P)H levels, revealing complex interplays between DNA damage repair, metabolic reprogramming, and enzyme activities. In vivo studies utilizing starved fruit fly larvae underscore probe C’s efficacy in monitoring NAD(P)H dynamics in response to external compounds. These findings highlight probe C’s utility as a versatile tool for investigating NAD(P)H signaling pathways in biomedical research contexts, offering insights into cellular metabolism, stress responses, and disease mechanisms. 

ProbesAandBwere developed for NAD(P)H sensing, exhibiting responsive near-infrared emissions with minimal photodamage and effective tissue penetration. ProbesC,D, andEshowed reduced responsiveness to NAD(P)H. 

Abstract Certain mycophagous Drosophila species are the only known eukaryotes that can tolerate some highly potent mycotoxins. This association between mycophagy and mycotoxin tolerance is well established because Drosophila species that switch hosts from mushrooms to other food sources lose their mycotoxin tolerance trait without any evolutionary lag. These findings suggest that mycotoxin tolerance may be a costly trait to maintain. In this study, we attempted to identify whether mycotoxin tolerance has a fitness cost. Larval competitive ability is a vital fitness trait, especially in holometabolous insects, where the larvae cannot move to a new host. Furthermore, larval competitive ability is known to be associated with many critical life-history traits. Here we studied whether mycotoxin tolerance adversely affects larval competitive ability on isofemale lines from 2 distinct locations. We observed that the extent of mycotoxin tolerance affected larval competitive ability, but only in isofemale lines from one location. Additionally, we observed that the high mycotoxin-tolerant isofemale lines from the same location showed poor survival to eclosion. This study shows that mycotoxin tolerance is associated with fitness costs and provides preliminary evidence of an association between local adaptation and mycotoxin tolerance. 

Nutrition and growth are strongly linked, but not much is known about how nutrition leads to growth. To understand the connection between nutrition through the diet, growth, and proliferation, we need to study the phenotypes resulting from the activation and inhibition of central metabolic pathways. One of the most highly conserved metabolic pathways across eukaryotes is the Target of Rapamycin (TOR) pathway, whose primary role is to detect the availability of nutrients and to either induce or halt cellular growth. Here we used the model organism Drosophila melanogaster ( D . mel .) and three non-model Drosophila species with different dietary needs, Drosophila guttifera ( D . gut .), Drosophila deflecta ( D . def .), and Drosophila tripunctata ( D . tri .), to study the effects of dietary amino acid availability on fecundity and longevity. In addition, we inhibited the Target of Rapamycin (TOR) pathway, using rapamycin, to test how the inhibition interplays with the nutritional stimuli in these four fruit fly species. We hypothesized that the inhibition of the TOR pathway would reverse the phenotypes observed under conditions of overfeeding. Our results show that female fecundity increased with higher yeast availability in all four species but decreased in response to TOR inhibition. The longevity data were more varied: most species experienced an increase in median lifespan in both genders with an increase in yeast availability, while the lifespan of D . mel . females decreased. When exposed to the TOR inhibitor rapamycin, the life spans of most species decreased, except for D . tri , while we observed a major reduction in fecundity across all species. The obtained data can benefit future studies on the evolution of metabolism by showing the potential of using non-model species to track changes in metabolism. Particularly, our data show the possibility to use relatively closely related Drosophila species to gain insight on the evolution of TOR signaling. 

Abstract Codon usage bias, where certain codons are used more frequently than their synonymous counterparts, is an interesting phenomenon influenced by three evolutionary forces: mutation, selection, and genetic drift. To better understand how these evolutionary forces affect codon usage bias, an extensive study to detect how codon usage patterns change across species is required. This study investigated 668 single-copy orthologous genes independently in 29 Drosophila species to determine how the codon usage patterns change with phylogenetic distance. We found a strong correlation between phylogenetic distance and codon usage bias and observed striking differences in codon preferences between the two subgenera Drosophila and Sophophora. As compared to the subgenus Sophophora, species of the subgenus Drosophila showed reduced codon usage bias and a reduced preference specifically for codons ending with C, except for codons with G in the second position. We found that codon usage patterns in all species were influenced by the nucleotides in the codon’s 2nd and 3rd positions rather than the biochemical properties of the amino acids encoded. We detected a concordance between preferred codons and preferred dinucleotides (at positions 2 and 3 of codons). Furthermore, we observed an association between speciation, codon preferences, and dinucleotide preferences. Our study provides the foundation to understand how selection acts on dinucleotides to influence codon usage bias.