Light Phase Research Articles

Comparison of forced and voluntary exercise types on male rat brain monoamine levels, anxiety-like behaviour, and physiological indexes under light and dark phases

Comparison of forced and voluntary exercise types on male rat brain monoamine levels, anxiety-like behaviour, and physiological indexes under light and dark phases

Automated home cage monitoring of an aging colony of mice—Implications for welfare monitoring and experimentation

IntroductionOur understanding of laboratory animal behavior and the implications of husbandry activities on their wellbeing remains incomplete. This is especially relevant with an aging colony as their activity patterns may change as they mature. Home Cage Monitoring (HCM) provides valuable insights into mouse activity within the animal's own environment and can shed light on acclimatization periods and responses to husbandry activities such as cage changing. The aim of this study was to monitor and explore changes in the activity and rest disturbance (RDI) patterns of an aging colony of male and female C57/BL6 mice.MethodsThe mice were housed in the Digitally Ventilated Cage® system, for up to 18 months of age. Data was then downloaded to investigate how the activity patterns and RDI of the mice changed over time. Habituation, aging and cage change assessments were conducted using linear mixed models, while cage separation and stereotypic behavior investigations were conducted by visual inspection of the data.ResultsAs expected during the study, mice were less active during the light phase compared to the dark phase. However, on arrival mice displayed heightened activity and RDI during the light phase and reduced activity and RDI during the dark phase, taking several days to adjust to baseline “acclimatized” patterns. With age, overall activity significantly decreased from 5 months until 14 months of age, after which it increased back toward baseline levels. We also observed activity spikes during our monitoring of this colony. Prolonged housing can lead to alarming stereotypic behaviors in animals. Cages of mice flagged for potential stereotypy displayed sustained activity spikes in the light and dark phases. Spikes in activity during the dark phase were much more pronounced than in the light phase. Cage changing led to an increase in the light phase activity and RDI compared to the previous day, with no observed difference in the dark phase post-cage change. This effect remained consistent as the animals aged.DiscussionThis study explores changes in the activity patterns of an aging colony of male and female C57/BL6 mice housed in the Digitally Ventilated Cage® system. We identified distinct aging phases concerning activity and RDI differences and a potential new welfare application for the DVC®, specifically for early detection of stereotypy. In conclusion, the adoption of HCM systems should be considered for long-term animal housing from both a welfare and behavioral perspective.

DNA-Mediated Formation of Phase-Separated Coacervates of the Nucleic Acid-Binding Domain of TAR DNA-Binding Protein (TDP-43) Prevents Its Amyloid-Like Misfolding.

Sequestration of protein molecules and nucleic acids to stress granules is one of the most promising strategies that cells employ to protect themselves from stress. In vitro, studies suggest that the nucleic acid-binding domain of TDP-43 (TDP-43tRRM) undergoes amyloid-like aggregation to β-sheet-rich structures in low pH stress. In contrast, we observed that the TDP-43tRRM undergoes complex coacervation in the presence of ssDNA to a dense and light phase, preventing its amyloid-like aggregation. The soluble light phase consists of monomeric native-like TDP-43tRRM. The microscopic data suggest that the dense phase consists of spherical coacervates with limited internal dynamics. We performed multiparametric analysis by employing various biophysical techniques and found that complex coacervation depends on the concentration and ratio of the participating biomolecules and is driven by multivalent interactions. The modulation of these forces due to environmental conditions or disease mutations regulates the extent of coacervation, and the weakening of interactions between TDP-43tRRM and ssDNA leads to amyloid-like aggregation of TDP-43tRRM. Our results highlight a competition among the native state, amyloid-like aggregates, and complex coacervates tuned by various environmental factors. Together, our results illuminate an alternate function of TDP-43tRRM in response to pH stress in the presence of the ssDNA.

Breaking of reciprocity and the Pancharatnam-Berry phase for light scattered by a disordered cold-atom cloud

Breaking of reciprocity and the Pancharatnam-Berry phase for light scattered by a disordered cold-atom cloud

Effect of Water Content on Light Nonaqueous Phase Fluid Migration in Sandy Soil

Contamination from light nonaqueous phase fluids (LNAPLs) and their derivatives during mining, production, and transportation has become a concern. Scholars have extensively studied LNAPL contamination, but the role of water content variation on its migration process in the unsaturated zone has not been sufficiently researched. The specific issue addressed in this study is the impact of water content on the migration of light nonaqueous phase liquids (LNAPLs) in sandy soils, a critical yet under-researched aspect of subsurface contamination. To tackle this, we employed indoor simulated vertical, one-dimensional, multiphase flow soil column experiments, utilizing the orthogonal experimental method to systematically evaluate the effects of varying water contents on the occurrence state and migration rate of LNAPLs. The experimental results indicate the following: (1) The migration rate of LNAPL exhibits an L-shaped trend during subsurface imbibition and a nonlinear relationship with migration time. The migration rate and migration time of surface infiltration have a linear growth relationship. (2) The residual rate of LNAPL is negatively correlated with water content and positively correlated with oil content in the homogeneous non-saturated state. With the increase in the amount of leaked oil, 40% of the leaked LNAPL is sorbed within the soil. (3) When the water content of the test medium is below 14%, and the oil content is below 11%, LNAPL appears in the unsaturated zone in a solid phase. As the water content increases, the adsorption rate of the oil phase gradually decreases and eventually reaches the oil saturation point. (4) When the water content of the medium exceeds 8%, over time, LNAPL will be subject to oil–water interfacial tension, and the rate of LNAPL movement first decreases and then increases, displaying nonlinear growth. The innovation of this work lies in the comprehensive analysis of LNAPL migration under controlled laboratory conditions, providing results that enhance the understanding of LNAPL behavior in sandy soils. These quantitative insights are crucial for developing targeted remediation strategies for LNAPL-induced pollution in the unsaturated zone.

Spatial mRNA expression patterns of orexin receptors in the dorsal hippocampus.

Orexins are wake-promoting neuropeptides that originate from hypothalamic neurons projecting to widespread brain areas throughout the central nervous system. They modulate various physiological functions via their orexin 1 (OXR1) and 2 (OXR2) receptors, including sleep-wake rhythm but also cognitive functions such as memory formation. Here, we provide a detailed analysis of OXR1 and OXR2 mRNA expression profiles in the dorsal hippocampus as a key region for memory formation, using RNAscope multiplex in situ hybridization. Interconnected subareas relevant for cognition and memory such as the medial prefrontal cortex and the nucleus reuniens of the thalamus were assessed as well. Both receptor types display distinct profiles, with the highest percentage of OXR1 mRNA-positive cells in the hilus of the dentate gyrus. Here, the content of OXR1 mRNA per cell was slightly modulated at selected time points over a 12h light/ 12 dark light phase. Using RNAScope and quantitative polymerase chain reaction approaches, we began to address a cell-type specific expression of OXR1 in hilar GABAergic interneurons. The distinct expression profiles of both receptor subtypes within hippocampal subareas and circuits provide an interesting basis for future interventional studies on orexin receptor function in spatial and contextual memory.

Resolution Improvement for Coherent Illumination Microscopy via Incident Light Phase Modulation

Resolution Improvement for Coherent Illumination Microscopy via Incident Light Phase Modulation

Persistent Na + current couples spreading depolarization to seizures in Scn8a gain of function mice.

Spreading depolarization (SD) is a slowly propagating wave of massive cellular depolarization that transiently impairs the function of affected brain regions. While SD typically arises as an isolated hemispheric event, we previously reported that reducing M-type potassium current (I KM ) by ablation of Kcnq2 in forebrain excitatory neurons results in tightly coupled spontaneous bilateral seizure-SD complexes in the awake mouse cortex. Here we find that enhanced persistent Na + current due to gain-of-function (GOF) mutations in Scn8a (N1768D/+, hereafter D/+) produces a similar compound cortical excitability phenotype. Chronic DC-band EEG recording detected spontaneous bilateral seizure-SD complexes accompanied by seizures with a profound tonic component, which occurs predominantly during the light phase and were detected in the mutant mice across ages between P40-100. Laser speckle contrast imaging of cerebral blood flow dynamics resolved SD as bilateral wave of hypoperfusion and subsequent hour-lasting hypoperfusion in Scn8a D/+ cortex in awake head-restrained mice subjected to a subconvulsive PTZ. Subcortical recordings in freely moving mice revealed that approximately half of the spontaneous cortical seizure-SD complexes arose with concurrent SD-like depolarization in the thalamus and delayed depolarization in the striatum. In contrast, SD-like DC potential shifts were rarely detected in the hippocampus or upper pons. Consistent with the high spontaneous incidence in vivo , cortical slices from Scn8a D/+ mice showed a raised SD susceptibility, and pharmacological inhibition of persistent Na + current (I NaP ), which is enhanced in Scn8a D/+ neurons, inhibited SD generation in cortical slices ex vivo , indicating that I NaP contributes to SD susceptibility. Ex vivo Ca 2+ imaging studies using acute brain slices expressing genetic Ca 2+ sensor (Thy1-GCAMP6s) demonstrated that pharmacological activation of I KM suppressed Ca 2+ spikes and SD, whereas I KM inhibitor drastically increased the frequency of Ca 2+ spikes in the hippocampus of Scn8a D/+ mice, but not in WT, suggesting that I KM restrains the hyperexcitability resulting from Scn8a GOF mutation. Together, our study identifies a cortical SD phenotype in Scn8a GOF mice shared with the Kcnq2 - cKO model of developmental epileptic encephalopathy and reveals that an imbalance of non-inactivating inward and outward membrane currents bidirectionally modulates spatiotemporal SD susceptibility.

Binary amplitude holograms for shaping complex light fields with digital micromirror devices

Abstract Digital micromirror devices are a popular type of spatial light modulators for wavefront shaping applications. While they offer several advantages when compared to liquid crystal modulators, such as polarization insensitivity and rapid-switching, they only provide a binary amplitude modulation. Despite this restriction, it is possible to use binary holograms to modulate both the amplitude and phase of the incoming light, thus allowing the creation of complex light fields. Here, a didactic exploration of various types of binary holograms is presented. A particular emphasis is placed on the fact that the finite number of pixels coupled with the binary modulation limits the number of complex values that can be encoded into the holograms. This entails an inevitable trade-off between the number of complex values that can be modulated with the hologram and the number of independent degrees of freedom available to shape light, both of which impact the quality of the shaped field. Nonetheless, it is shown that by appropriately choosing the type of hologram and its parameters, it is possible to find a suitable compromise that allows shaping a wide range of complex fields with high accuracy. In particular, it is shown that choosing the appropriate alignment between the hologram and the micromirror array allows for maximizing the number of complex values. Likewise, the implications of the type of hologram and its parameters on the diffraction efficiency are also considered.

Development and analysis of a traffic model in the AnyLogic environment

The Federal Work Program on Informatics for the 11th grade (advanced level) indicates the need to consider the issues of constructing simulation models, provides the practical works on motion modeling and processing of experimental results. Development and analysis of simulation models related to road traffic management is interesting and understandable for schoolchildren. These models can be implemented in the AnyLogic environment, for which it is advisable to offer schoolchildren to create a model of an intersection they are familiar with and analyze at what traffic intensity there is no need to install a traffic light, when a traffic light is necessary, what time phases of the traffic light provide the highest capacity of the intersection. The article describes algorithms for creating models of unregulated and regulated intersections; presents tasks for analyzing the capacity of the intersection for different values of the number of cars in each direction of traffic per hour; describes tasks for optimizing the phases of the traffic light to increase the capacity of the intersection; provides examples of project topics for organizing independent work of students.

Reprogrammable metasurface holographic image encryption technology based on a three-dimensional discrete hyperchaotic system

The emergence of metasurfaces provides a secure and efficient platform for optical encryption technology as they have broad prospects in the field of information security. However, the limited number of channels available on metasurfaces and the insufficient security of keys make them vulnerable to attacks by eavesdroppers. In this work, a reprogrammable metasurface optical encryption scheme based on a three-dimensional hyperchaotic system is proposed. The three-dimensional discrete hyperchaotic system has strong ergodicity, initial value sensitivity, and pseudorandomness compared to previous chaotic systems that can pass NIST randomness testing well. Additionally, based on this hyperchaotic property, we designed a metasurface encryption structure based on the geometric phase. The research results show that the introduction of the hyperchaotic system greatly improves the randomness and flexibility of key generation. This scheme can encrypt multiple images with high security. Decryption is only possible when the attacker steals the complete chaotic system and parameters, as well as over 70% of the correct incident light phase information. Our research results have great potential applications in the field of metasurface optical encryption.

Large-scale fabrication of meta-axicon with circular polarization on CMOS platform

Abstract Metasurfaces, consisting of arrays of subwavelength structures, are lightweight and compact while being capable of implementing the functions of traditional bulky optical components. Furthermore, they have the potential to significantly improve complex optical systems in terms of space and cost, as they can simultaneously implement multiple functions. The wafer-scale mass production method based on the CMOS (complementary metal oxide semiconductor) process plays a crucial role in the modern semiconductor industry. This approach can also be applied to the production of metasurfaces, thereby accelerating the entry of metasurfaces into industrial applications. In this study, we demonstrated the mass production of large-area meta-axicons with a diameter of 2 mm on an 8-inch wafer using DUV (Deep Ultraviolet) photolithography. The proposed meta-axicon designed here is based on PB (Pancharatnam–Berry) phase and is engineered to simultaneously modulate the phase and polarization of light. In practice, the fabricated meta-axicon generated a circularly polarized Bessel beam with a depth of focus (DoF) of approximately 2.3 mm in the vicinity of 980 nm. We anticipate that the mass production of large-area meta-axicons on this CMOS platform can offer various advantages in optical communication, laser drilling, optical trapping, and tweezing applications.

Ultrathin metasurface for super multi-view 3D display with linear and circular polarization control

Multiview 3D display technologies are advantageous due to their smooth motion parallax, visual discomfort alleviation, and wide depth of focus. However, the potential applications of this technology are constrained by its bulky size and limited light modulation capability. Metasurfaces offer diverse wavefront control by manipulating the phase, amplitude, and polarization of light. They provide design freedom and high image quality for multiview 3D display technologies. Here, we propose an ultrathin metasurface-based super multiview 3D display that can effectively modulate the flexibility of linearly polarized (LP) and circularly polarized (CP) electromagnetic waves. Numerical results demonstrate that it can produce 24 views together for LP and CP waves and high-quality reconstructed 3D images are obtained. The suggested metasurface for super multiview 3D display is promising to underpin the advancement of meta-optics-based operations in 3D display, imaging and integrated optics.

Differential Effects of Light and Dark Phase Modifications on Jet Lag Adaptability in Mice

ABSTRACTIn chronobiology, shifting light/dark cycles is a common method to disrupt circadian rhythms. While the direction and magnitude of a phase shift (e.g., +6 denoting a 6‐h advanced shift) dictate the temporal change before and after the shift, little attention has been paid to the duration and relative proportion of daytime and nighttime during the shift, leading to a critical, unexamined variable in circadian research. In this study, we introduce the concepts of “L‐shift” (longer light phase on the shift day) and “D‐shift” (longer dark phase), and investigate how these variations impact the adaptability of mice to jet lag. By examining multiple phase shifts (12L vs. 12D, +6L vs. +6D, −6L vs. −6D), we demonstrate that L‐shifts not only facilitate faster adaptation but also significantly reduce the severity of sepsis in a jet lag‐sensitive lipopolysaccharide‐induced sepsis model. Further investigations with additional phase shifts at 1‐h intervals (+8 to +11) reinforced the enhanced fitness of mice under L‐shifts. Mechanistically, L‐shifts were found to increase sleep duration, thereby improving circadian entrainment, with sleep deprivation nullifying the adaptability differences between lighting protocols. These findings underscore a previously unrecognized factor in circadian biology and suggest that optimizing lighting protocols could profoundly improve adaptability to circadian disruptions. This research opens new avenues for enhancing therapeutic strategies and refining experimental designs in the field of chronobiology.

Time of day affects MrgD-dependent modulation of cardiomyocyte contractility.

The renin-angiotensin system (RAS) is composed of a series of peptides, receptors, and enzymes that play a pivotal role in maintaining cardiovascular homeostasis. Among the most important players in this system are the angiotensin-II and angiotensin-(1-7) peptides. Our group has recently demonstrated that alamandine (ALA), a peptide with structural and functional similarities to angiotensin-(1-7), interacts with cardiomyocytes, enhancing contractility via the Mas-related G protein-coupled receptor member D (MrgD). It is currently unknown whether this modulation varies along the distinct phases of the day. To address this issue, we assessed the ALA-induced contractility response of cardiomyocytes from mice at four Zeitgeber times (ZTs). At ZT2 (light phase), ALA enhanced cardiomyocyte shortening in an MrgD receptor-dependent manner, which was associated with nitric oxide (NO) production. At ZT14 (dark phase), ALA induced a negative modulation on the cardiomyocyte contraction. β-Alanine, an MrgD agonist, reproduced the time-of-day effects of ALA on myocyte shortening. NG-nitro-l-arginine methyl ester, an NO synthase inhibitor, blocked the increase in fractional shortening induced by ALA at ZT2. No effect of ALA on myocyte shortening was observed at ZT8 and ZT20. Our results show that ALA/MrgD signaling in cardiomyocytes is subject to temporal modulation. This finding has significant implications for pharmacological approaches that combine chronotherapy for cardiac conditions triggered by disruption of circadian rhythms and hormonal signaling.NEW & NOTEWORTHY Alamandine, a member of the renin-angiotensin system, serves critical roles in cardioprotection, including the modulation of cardiomyocyte contractility. Whether this effect varies along the day is unknown. Our results provide evidence that alamandine via receptor MrgD exerts opposing actions on cardiomyocyte shortening, enhancing, or reducing contraction depending on the time of day. These findings may have significant implications for the development and effectiveness of future cardiac therapies.

From [Ba3S][GeS₄] to [Ba₃CO₃][MS₄] (M = Ge, Sn): Enhancing optical anisotropy in IR birefringent crystals via functional group implantation

Birefringent crystals are crucial for manipulating light's phase and polarization, making them vital components in various optical devices. Traditionally, strategies for designing high-performance birefringent crystals have focused on modifying the parent structure. However, there are limited examples demonstrating how changing functional groups can effectively enhance birefringence (Δn), as such changes often significantly alter the crystal structure. In this study, we propose a “functional group implantation” strategy aimed at significantly improving birefringent performance within the chalcogenide system. This involves replacing the isotropic [S]2⁻ ions with anisotropic π-conjugated [CO₃]2⁻ groups. We validated this approach through comprehensive comparisons between the chalcogenide [Ba₃S][GeS₄] and the oxychalcogenide [Ba₃CO₃][MS₄] (where M ​= ​Ge and Sn), both of which adopt the same space group and feature the same arrangements of functional groups. Experimental characterization and theoretical calculations confirm that the [CO₃]2⁻ groups exhibit significantly greater polarization anisotropy than the [S]2⁻ groups. This difference leads to a marked increase in Δn in [Ba₃CO₃][MS₄] (ranging from 0.088 to 0.112 ​at 546 ​nm) compared to [Ba₃S][GeS₄] (0.021 ​at 546 ​nm). This finding not only broadens the structural chemistry of π-conjugated chalcogenides but also illustrates the potential of functional group implantation for designing infrared birefringent crystals with enhanced optical anisotropy.

Lavender improves sleep through olfactory perception and GABAergic neurons of the central amygdala

Ethnopharmacological relevanceThe use of lavender as sleep aid or hypnotic agent can be traced back as early as ancient Romans and Greeks. Yet, objective experimental data on whether and how lavender enhances sleep duration or/and sleep quality remain lacking. Aim of the studyWe aimed to characterize the sleep-wake regulating effects of lavender in the mouse and to demonstrate the brain targets and neural circuits involved. Materials and methodsA self-made precise odor delivery system combined with chronic polysomnographic recordings was employed to assess the sleep-wake effects of inhalation with lavender essential oil (LEO, extracted from lavender) and its different constituents during the light and dark phases in free-moving C57BL/6J mice. Neuroviral labeling, in situ hybridization and pharmacogenetics were combined to identify the neural circuits and targets involved. Finally, an insomniac model of DL-4-Chlorophenylalanine (PCPA)-treated mice was established to examine the sleep-inducing potential of LEO. ResultsWe found that inhalation of LEO with a concentration at 25.0% during the light (inactive) phase significantly shortened the latency to non-rapid eye movement (NREM) sleep, increased the total amount of NREM sleep at the expense of wakefulness (W), and enhanced cortical EEG slow wave activities, notably delta power spectra density. We further identified linalool, d-limonene, 1,8-cineole, linalyl acetate and terpinene-4-ol as the major effective sleep-promoting monomer components. Importantly, we found that LEO no longer produced any of the above sleep-promoting effect following either nasal injection of zinc sulfate which interrupts the olfactory pathway, or pharmacogenetics silencing of central amygdala GABAergic neurons. Finally, LEO reestablished NREM sleep with short latency in PCPA-treated insomniac mice, effects comparable with those induced by a potent sedative diazepam. ConclusionsWe have characterized the quantitative and qualitative sleep-promoting effects of LEO and its effective components via the olfactory pathway and central amygdala GABA neuronal targets. The hypnotic property of LEO is reinforced by its ability to restore sleep in insomnia. Our study thus establishes a neurobiological basis for aromatherapy of sleep disorders using lavender.

Altered Patterns of Maternal Behavior Transitions in Rats Exposed to Limited Bedding and Nesting Material Paradigm.

Maternal care plays a fundamental role in early life, and the alteration of its patterns can negatively affect the developmental course of the offspring in a myriad of domains in both rats and humans. The limited bedding and nesting (LBN) protocol is an extensively used paradigm in rodents to address the impact of altered maternal behavior patterns on infants' neurodevelopment. Here, we explore the altered patterns of maternal care in rats in LBN conditions by describing sequences of transition between maternal behavior components using network analysis. Using this technique, we capture how often maternal behavior transitions take place during the LBN period and which behaviors play central roles in those transitions over time. Female rats and their pups were placed in standard and LBN housing conditions from Postpartum Days 2 to 9, during which maternal behavior was observed during the light and dark phases. We used inferential statistical analysis to compare the maternal behavior profiles of control and LBN dams, and network analysis was used to capture the altered sequence of maternal behavior transitions during the period of LBN. Compared to control dams, LBN dams significantly increased their high crouch nursing posture during light/dark phases (p=0.018), and the number of behavioral transitions increased only during the dark phase (p=0.0004). Network analysis revealed specific altered patterns of behavioral transitions in LBN dams, characterized by the predominance of switches between active nursing postures during the first five days of the LBN protocol. Nursing behavior was the most disrupted component of maternal behavior under the LBN protocol, mainly during the dark phase. Network analysis can complement and extend traditional methods to gain a more thorough understanding of maternal care strategies and behavioral patterns in LBN conditions and potential consequences for the offspring.

A diel multi-tissue genome-scale metabolic model of Vitis vinifera.

Vitis vinifera, also known as grapevine, is widely cultivated and commercialized, particularly to produce wine. As wine quality is directly linked to fruit quality, studying grapevine metabolism is important to understand the processes underlying grape composition. Genome-scale metabolic models (GSMMs) have been used for the study of plant metabolism and advances have been made, allowing the integration of omics datasets with GSMMs. On the other hand, Machine learning (ML) has been used to analyze and integrate omics data, and while the combination of ML with GSMMs has shown promising results, it is still scarcely used to study plants. Here, the first GSSM of V. vinifera was reconstructed and validated, comprising 7199 genes, 5399 reactions, and 5141 metabolites across 8 compartments. Tissue-specific models for the stem, leaf, and berry of the Cabernet Sauvignon cultivar were generated from the original model, through the integration of RNA-Seq data. These models have been merged into diel multi-tissue models to study the interactions between tissues at light and dark phases. The potential of combining ML with GSMMs was explored by using ML to analyze the fluxomics data generated by green and mature grape GSMMs and provide insights regarding the metabolism of grapes at different developmental stages. Therefore, the models developed in this work are useful tools to explore different aspects of grapevine metabolism and understand the factors influencing grape quality.

Soil gas gradient method for estimating natural source zone depletion rates of LNAPL and specific chemicals of concern

This paper presents a simplified approach for the soil gas gradient method for estimating natural source zone depletion (NSZD) rates of specific contaminants of concern (COCs) at sites contaminated by light non-aqueous phase liquids (LNAPL). Traditional approaches to quantify COC-specific NSZD rates often rely on numerical or analytical reaction-transport models that require detailed site-specific data. In contrast, the proposed method employs simple analytical solutions, making it more accessible to practitioners. Specifically, it requires only the maximum soil gas concentration, the effective diffusion coefficient, and the diffusive reaction length calculated from vertical soil gas concentration profiles. The simplified approach was validated against a reactive transport numerical model reported in the literature, showing consistent results within the same order of magnitude for BTEX NSZD rates at a gasoline spill site in South Carolina. Further validation using a larger dataset involved comparing NSZD rate estimates for benzene and total petroleum hydrocarbons (TPH) against those obtained using BioVapor, utilizing empirical soil gas data from the USEPA Petroleum Vapor Intrusion Database. Results demonstrated a strong correlation between NSZD rates and maximum soil gas concentrations, allowing the development of a rapid screening approach based only on the measured soil gas concentrations and literature values for diffusion coefficients and diffusive reaction lengths. This approach aligned well with previous modeling studies and was consistent with literature values for TPH NSZD rates. Overall, both the simplified and screening approaches offer practical, easy-to-use tools for evaluating temporal variability in natural attenuation rates, supporting baseline assessments and ongoing performance evaluations of remediation at LNAPL sites.