Ultra-Thin GaAs Single-Junction Solar Cells for Self-Powered Skin-Compatible Electrocardiogram Sensors.

GaAs thin-film solar cells have high efficiency, reliability, and operational stability, making them a promising solution for self-powered skin-conformal biosensors. However, inherent device thickness limits suitability for such applications, making them uncomfortable and unreliable in flexural environments. Therefore, reducing the flexural rigidity becomes crucial for integration with skin-compatible electronic devices. Herein, this study demonstrated a novel one-step surface modification bonding methodology, allowing a streamlined transfer process of ultra-thin (2.3 µm thick) GaAs solar cells on flexible polymer substrates. This reproducible technique enables strong bonding between dissimilar materials (GaAs-polydimethylsiloxane, PDMS) without high external pressures and temperatures. The fabricated solar cell showed exceptional performance with an open-circuit voltage of 1.018 V, short-circuit current density of 20.641 mA cm-2, fill factor of 79.83%, and power conversion efficiency of 16.77%. To prove the concept, the solar cell is integrated with a skin-compatible organic electrochemical transistor (OECT). Competitive electrical outputs of GaAs solar cells enabled high current levels of OECT under subtle light intensities lower than 50 mW cm-2, which demonstrates a self-powered electrocardiogram sensor with low noise (signal-to-noise ratio of 32.68 dB). Overall, this study presents a promising solution for the development of free-form and comfortable device structures that can continuously power wearable devices and biosensors.

Inflachromene ameliorates Parkinson's disease by targeting Nrf2-binding Keap1.

Parkinson's disease (PD) is the most common neurodegenerative disease characterized by movement disorder. Despite current therapeutic efforts, PD progression and the loss of dopaminergic neurons in the substantia nigra remain challenging to prevent due to the complex and unclear molecular mechanism involved. We adopted a phenotype-based drug screening approach with neuronal cells to overcome these limitations. In this study, we successfully identified a small molecule with a promising therapeutic effect for PD treatment, called inflachromene (ICM), through our phenotypic screening strategy. Subsequent target identification using fluorescence difference in two-dimensional gel electrophoresis (FITGE) revealed that ICM ameliorates PD by targeting a specific form of Keap1. This interaction led to upregulating various antioxidants, including HO-1, NQO1, and glutathione, ultimately alleviating PD symptoms. Furthermore, ICM exhibited remarkable efficacy in inhibiting the loss of dopaminergic neurons and the activation of astrocytes and microglia, which are critical factors in PD pathology. Our findings suggest that the phenotypic approach employed in this study identified that ICM has potential for PD treatment, offering new hope for more effective therapeutic interventions in the future.

NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

Electrospun Poly L-Lactic Acid Nanofiber Webs Presenting Enhanced Piezoelectric Properties.

There has been extensive research on electrospun ferroelectric nanoparticle-doped poly L-lactic acid (PLA) nanofiber web piezoelectric devices. In this study, BaTiO3 nanoparticles (BTNPs) were incorporated into the PLA to enhance the piezoelectric properties. The composite nanofiber webs were characterized using field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. The piezoelectric behavior was analyzed by measuring the peak-to-peak output voltage (Vp-p) of the samples. The sensors fabricated from the PLA/BTNP nanofiber webs exhibited higher Vp-p values than the conventional electrospun PLA sensors. Furthermore, the corona-poled PLA/BTNP nanofiber web sensors exhibited even higher Vp-p values than the non-corona-poled sensors. Lastly, the effect of stacking nanofiber webs in terms of enhancing the sensor performance was also evaluated.

Physical Unclonable Functions Employing Circularly Polarized Light Emission from Nematic Liquid Crystal Ordering Directed by Helical Nanofilaments.

This study proposes the use of physical unclonable functions employing circularly polarized light emission (CPLE) from nematic liquid crystal (NLC) ordering directed by helical nanofilaments in a mixed system composed of a calamitic NLC mixture and a bent-core molecule. To achieve this, an intrinsically nonemissive NLC is blended with a high concentration of a luminescent rod-like dye, which is miscible up to 10 wt % in the calamitic NLC without a significant decrease in the degree of alignment. The luminescence dissymmetry factor of CPLEs in the mixed system strongly depends on the degree of alignment of the dye-doped NLCs. Furthermore, the mixed system prepared in this study exhibits two randomly generated chiral domains with CPLEs of opposite signs. These chiral domains are characterized not only by their CPLE performances but also by their ability to generate random patterns up to several millimeters, making them promising candidates for high-performance secure authentication applications.

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability.

Plasmid-based transfection can be used in many applications such as transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome-mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE-based mAb production in HEK293 cells by up to 2.82-fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

Circularly Polarized Light Emission from Nonchiral Perovskites Incorporated into Nanoporous Cholesteric Polymer Templates.

Chiral perovskites have garnered significant attention, owing to their chiroptical properties and emerging applications. Current fabrication methods often involve complex chemical synthesis routes. Herein, an alternative approach for introducing chirality into nonchiral hybrid organic-inorganic perovskites (HOIPs) using nanotemplates composed of cholesteric polymeric networks is proposed. This method eliminates the need for additional molecular design. In this process, HOIP precursors are incorporated into a porous cholesteric polymer film, and two-dimensional (2D) HOIPs grow inside the nanopores. Circularly polarized light emission (CPLE) was observed even though the selective reflection band of the cholesteric polymer films containing a representative HOIP deviated from the emission wavelength of the 2D HOIP. This effect was confirmed by the induced circular dichroism (CD) observed in the absorbance band of the HOIP. The observed CPLE and CD are attributed to the chirality induced by the template in the originally nonchiral 2D HOIP. Additionally, the developed 2D HOIP exhibited a long exciton lifetime and good stability under harsh conditions. These findings provide valuable insights into the development and design of innovative optoelectronic materials.

One-Week-Ahead Prediction of Cyanobacterial Harmful Algal Blooms in Iowa Lakes.

Cyanobacterial harmful algal blooms (CyanoHABs) pose serious risks to inland water resources. Despite advancements in our understanding of associated environmental factors and modeling efforts, predicting CyanoHABs remains challenging. Leveraging an integrated water quality data collection effort in Iowa lakes, this study aimed to identify factors associated with hazardous microcystin levels and develop one-week-ahead predictive classification models. Using water samples from 38 Iowa lakes collected between 2018 and 2021, feature selection was conducted considering both linear and nonlinear properties. Subsequently, we developed three model types (Neural Network, XGBoost, and Logistic Regression) with different sampling strategies using the nine selected variables (mcyA_M, TKN, % hay/pasture, pH, mcyA_M:16S, % developed, DOC, dewpoint temperature, and ortho-P). Evaluation metrics demonstrated the strong performance of the Neural Network with oversampling (ROC-AUC 0.940, accuracy 0.861, sensitivity 0.857, specificity 0.857, LR+ 5.993, and 1/LR- 5.993), as well as the XGBoost with downsampling (ROC-AUC 0.944, accuracy 0.831, sensitivity 0.928, specificity 0.833, LR+ 5.557, and 1/LR- 11.569). This study exhibited the intricacies of modeling with limited data and class imbalances, underscoring the importance of continuous monitoring and data collection to improve predictive accuracy. Also, the methodologies employed can serve as meaningful references for researchers tackling similar challenges in diverse environments.

Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cells.

CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5'2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5'2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.

Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples.

Analysis of microbial communities in the epiphytic phyllosphere can be challenging, especially when applying sequencing-based techniques, owing to the interference of plant-derived biomolecules such as nucleic acids. A review of recent studies on the epiphytic microbiome revealed that both mechanical and enzymatic lysis methods are widely used. Here, we evaluated the effects of the two lysis methods on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number per ng of template genomic DNA under different extraction conditions. Furthermore, the effect on bacterial community composition, diversity, and reproducibility was examined using 16S rRNA gene amplicon sequencing. The enzymatic lysis method yielded one to two orders of magnitude more DNA, but the DNA quality was suboptimal. Conversely, the samples prepared using the mechanical method showed high DNA purity albeit lower yield. Unexpectedly, mechanical lysis showed a higher DNA integrity number (DIN) than enzymatic lysis. The 16S rRNA amplicon sequencing results demonstrated that the samples prepared via mechanical disruption exhibited reproducibly similar microbial community compositions regardless of the extraction conditions. In contrast, the enzymatic lysis method resulted in inconsistent taxonomic compositions under different extraction conditions. This study demonstrates that mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.

Effect of Nematogen Doping in Bent-Core Molecular Systems with a Helical Nanofilament and Dark Conglomerate.

Two types of binary mixtures were prepared. One consisted of a calamitic nematogen and bent-core molecule with a helical nanofilament, whereas the other contained a calamitic nematogen and bent-core molecule with a dark conglomerate. The chiroptical features of these two mixtures were investigated using polarized optical microscopy and circular dichroism. In addition, X-ray diffraction analysis was performed on the two binary mixtures. The chiroptical features of the two mixtures were remarkably different. One mixture showed enhanced chiroptical features, whereas the other did not show chiroptical features. This method may help in distinguishing between helical nanofilaments and dark conglomerates which originate from bent-core molecular systems.

Highly efficient thin-film 930 nm VCSEL on PDMS for biomedical applications.

Recently, biocompatible optical sources have been surfacing for new-rising biomedical applications, allowing them to be used for multi-purpose technologies such as biological sensing, optogenetic modulation, and phototherapy. Especially, vertical-cavity surface-emitting laser (VCSEL) is in the spotlight as a prospective candidate for optical sources owing to its low-driving current performance, low-cost, and package easiness in accordance with two-dimensional (2D) arrays structure. In this study, we successfully demonstrated the actualization of biocompatible thin-film 930 nm VCSELs transferred onto a Polydimethylsiloxane (PDMS) carrier. The PDMS feature with biocompatibility as well as biostability makes the thin-film VCSELs well-suited for biomedical applications. In order to integrate the conventional VCSEL onto the PDMS carrier, we utilized a double-transfer technique that transferred the thin-film VCSELs onto foreign substrates twice, enabling it to maintain the p-on-n polarity of the conventional VCSEL. Additionally, we employed a surface modification-assisted bonding (SMB) using an oxygen plasma in conjunction with silane treatment when bonding the PDMS carrier with the substrate-removed conventional VCSELs. The threshold current and maximum output power of the fabricated 930 nm thin-film VCSELs are 1.08 mA and 7.52 mW at an injection current of 13.9 mA, respectively.

Chiroptical Performances in Self-Assembled Hierarchical Nanosegregated Chiral Intermediate Phases Composed of Two Different Achiral Bent-Core Molecules.

In this paper, chiral intermediate phases composed of two achiral molecules are fabricated by utilizing nanophase separation and molecular hierarchical self-organization. An achiral bent-core guest molecule, exhibiting a calamitic nematic and a dark conglomerate phase according to the temperature, is mixed with another achiral bent-core host molecule possessing a helical nanofilament to separate the phases between them. Two nanosegregated phases are identified, and considerable chiroptical changes, such as circular dichroism and circularly polarized luminescence, are detected at the transition temperatures between the different nanophase-separated states. The nanosegregated chiral phase-wherein the helical nanofilament and dark conglomerate phases are phase-separated-exhibits the highest chiroptical intensities. The luminescence dissymmetry factor, |glum|, in this phase is amplified by an order of magnitude compared with that of another nanosegregated phase, wherein the helical nanofilament and nematic phases are phase-separated.

Neural network ensemble model for prediction of erythrocyte sedimentation rate (ESR) using partial least squares regression.

The erythrocyte sedimentation rate (ESR) is a non-specific blood test for determining inflammatory conditions. However, the long measurement time (60 min) to obtain ESR is an obstacle for a prompt evaluation. In this study, to reduce the measurement time of ESR, deep neural networks (DNNs) were applied to the sedimentation tendency of blood samples. DNNs using multilayer perceptron (MLP), long short-term memory (LSTM), and gated recurrent unit (GRU) were assessed and compared to determine a suitable length of time for the input sequence. To avoid overfitting, a stacking ensemble learning was adopted, which combines multiple models by using a meta model. Four meta models were compared: mean, median, least absolute shrinkage and selection operator, and partial least squares regression (PLSR) schemes. From the empirical results, LSTM and GRU models have better prediction than MLP over sequence lengths of 5 to 20 min. The decrease in [Formula: see text] and [Formula: see text] of GRU and LSTM was attenuated after a sequence length of 15 min, so the input sequence length is determined as 15 min. In terms of the meta model, the statistical comparison suggests that GRU combined with PLSR (GRU-PLSR) is the best case. Then, the GRU-PLSR was tested for prediction of ESR data obtained from periodontitis patients to check its applicability to a specific disease. The Bland-Altman plot shows acceptable agreement between measured and predicted ESR values. Based on the results, the GRU-PLSR can predict ESR with improved performance within 15 min and has potential applicability to ESR data with inflammatory and non-inflammatory conditions.

Chiroptical Characteristics of Nanosegregated Phases in Binary Mixture Consisting of Achiral Bent-Core Molecule and Bent-Core Base Main-Chain Polymer.

In this paper, a binary mixture system consisting of an achiral bent-core molecule and a bent-core base main-chain polymer is described. The mixture exhibits an intriguing nanosegregated phase generated by the phase separation of the helical nanofilament B4 phase (originating from the bent-core molecule) and the dark conglomerate phase (originating from the bent-core base main-chain polymer). This nanosegregated phase was identified using polarized optical microscopy, differential scanning calorimetry, and X-ray diffraction analysis. In this nanosegregated phase, the enantiomeric domains grew to a few millimeters and a giant circular dichroism was observed. The structural chirality of the helical nanofilament B4 phase affected the conformation of the bent-core base main-chain polymer embedded within the helical nanofilament networks of bent-core molecules.

Does a Customized 3D Printing Plate Based on Virtual Reduction Facilitate the Restoration of Original Anatomy in Fractures?

The purpose of this study was to evaluate the restoration of original anatomy after fixation of sawbone fractures using case-specific 3D printing plates based on virtual reduction (VR). Three-dimensional models of 28 tibia sawbones with cortical marking holes were obtained. The sawbones were fractured at various locations of the shaft and 3D models were obtained. The fractured models were reduced virtually and customized non-locking metal plates that fit the reduced model were produced via 3D printing. The fractured sawbones were actually fixed to the customized plate with nonlocking screws and 3D models were generated. With the proximal fragments of the 3D models overlapped, the changes in length, 3D angulation, and rotation of the distal fragment were evaluated. Compared to the intact model (IN), the virtual reduction model (VR) and the actual fixation model (AF) showed no significant differences in length. Compared to the IN, the VR and the AF had mean 3D angulations of 0.39° and 0.64°, respectively. Compared to the IN model, the VR and the AF showed mean rotations of 0.89° and 1.51°, respectively. A customized plate based on VR facilitates the restoration of near-original anatomy in fractures of tibial sawbone shaft.

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

Evaluation of anatomical and histological characteristics of human peripheral nerves: as an effort to develop an efficient allogeneic nerve graft.

Management of peripheral nerve defects is a complicated problem in clinical contexts. Autologous nerve grafting, a gold standard for surgical treatment, has been well known to have several limitations, such as donor site morbidity, a limited amount of available donor tissue, and size mismatches. Acellular nerve allografts (ANAs) have been developed as an alternative and have been applied clinically with favorable outcomes. However, because of the limited availability of commercialized ANAs due to supplier-related issues and high costs, efforts continue to produce alternative sources for ANAs. The present study evaluated the anatomical and histological characteristics of human peripheral nerves using 25 donated human cadavers. The length, diameter, and branching points of various peripheral nerves (median, ulnar, tibial, lateral femoral cutaneous, saphenous, and sural nerves) in both the upper and lower extremities were evaluated. The cross-sectional area (CSA), ratio of fascicular area, and numbers of fascicles were also evaluated via histologic analysis. CSA, the ratio of fascicular area, and the number of fascicles were analyzed statistically in correlation with demographic data (age, sex, height, weight, BMI). The mean length of all evaluated nerves ranged from 17.1 to 41.4 cm, and the mean diameter of all evaluated nerves ranged from 1.2 to 4.9 mm. Multiple regression analysis revealed correlations between the ratio of fascicular area and sex (p = 0.005) and BMI (p = 0.024) (R2 = 0.051). The results of the present study will be helpful in selecting necessary nerve allograft sources while considering the characteristics of each nerve in the upper and lower extremities during ANAs production.

Regulator of RNase E activity modulates the pathogenicity of Salmonella Typhimurium.

RNase E-mediated RNA processing and degradation are involved in bacterial adaptation to environmental changes. The RraA regulatory protein, which is highly conserved in γ-proteobacteria, differentially modulates RNase E activity. Recent studies have revealed the association of Salmonella enterica serovar Typhimurium RNase E (STRNase E) with bacterial pathogenicity; however, the molecular mechanisms are unknown. Here, we show that the expression levels of STRraA, a protein regulator of STRNase E activity, affect S. Typhimurium pathogenicity. RNA-sequencing and RT-PCR analyses indicated positive effects of STRraA levels on the abundance of mRNA species from class II flagellar operons. Primer extension analysis further identified STRraA-regulated STRNase E cleavage in the 5' untranslated region of fliDST mRNA. The cleavage affected the stability of this polycistronic mRNA, suggesting that STRraA protects fliDST mRNA from STRNase E cleavage, leading to enhanced flagellar assembly. Accordingly, STRraA positively regulated flagellar assembly and motility. In addition, STrraA-deleted cells showed decreased invasion ability and cytotoxicity in infection of human cervical epithelial carcinoma cells and reduced mortality in a mouse infection model compared to wild-type cells. These results support an active role of STRraA in RNase E-mediated modulation of pathogenesis in S. Typhimurium.

Characterization of Noise in a Single-Molecule Fluorescence Signal.

Single-molecule fluorescence experiments allow monitoring of the structural change and dynamics of a single biomolecule in real time using dye molecules attached to the molecule. Often, the molecules are immobilized on the surface to observe a longer molecular dynamics, yet the finite photon budget available from an individual dye molecule before photobleaching sets the limit to the relatively poor signal-to-noise level. To increase the accuracy of these single-molecule experiments, it is necessary to study the cause of noise in the fluorescence signal from the single molecules. To find the origin of this noise, the lifetime of the fluorescent dye molecules labeled on surface-immobilized DNA was measured by using time-correlation single photon counting. The standard deviation of the fluorescence lifetimes obtained from repeated measurements of a single dye molecule with the total photon number N decreased as 1/N, thus following a shot noise of the Poisson statistics. On the other hand, an additional constant noise source, which is independent of the photon number, was observed from the lifetime uncertainties from many molecules and became more dominant after a certain photon number N. This trend was also followed in the uncertainties of the single-molecule FRET signals obtained from single and many molecules. This additional noise is considered to come from the inhomogeneous environment of each DNA immobilized on the surface.