Online consumer testing beyond central location tests: A case study for brewed coffee.

Due to the constraints of the COVID-19 pandemic, conducting sensory evaluations requiring direct interactions became challenging. In response, researchers have been motivated to devise non-face-to-face testing methods as alternatives. This study aimed to compare two non-face-to-face home-use tests (HUT) with the traditional face-to-face central location test (CLT). Both HUTs involved online recruitment and sample delivery to participants' homes. One HUT provided a written protocol with no direct interaction (contactless HUT; C-HUT), whereas the other included an online meeting with a researcher for live guidance (online HUT; O-HUT). Four coffee samples were evaluated on the basis of liking and sensory and emotional attributes. The comparison between CLT and O-HUT showed RV coefficients of 0.92, 0.93, and 0.98 (P < 0.05) for liking and sensory and emotional attributes, respectively. In addition, based on the RV coefficient, the CLT results showed a significantly greater similarity to those of O-HUT compared to those of C-HUT. The O-HUT also outperformed the C-HUT in its ability to significantly discriminate between samples. Hence, real-time interactions between researchers and participants, as facilitated by O-HUT, may be more suitable in certain scenarios compared to C-HUT, which relies solely on a written protocol. Overall, these findings suggest that C-HUT and O-HUT are suitable methods for collecting sensory data and overcoming geographic and face-to-face contact limitations, providing greater flexibility, and reducing the time and cost associated with traditional sensory evaluations.

Comparative transcriptome analysis of periodontitis and peri-implantitis in human subjects.

BACKGROUND: Peri-implantitis is similar to periodontitis, but there are some differences. For the effective control of peri-implantitis, it is necessary to clarify its similarities and differences with periodontitis in terms of gene expression. METHODS: This cross-sectional study included 20 participants (10 healthy subjects and 10 patients with periodontitis and peri-implantitis). Gingival tissue samples (10 healthy, 10 periodontitis, and 10 peri-implantitis tissues) were collected, RNAs were extracted, and RNA sequencing and analysis were performed. RESULTS: Differentially expressed gene (DEG) analysis identified 757 upregulated and 159 downregulated genes common between periodontitis and peri-implantitis. Periodontitis tissues uniquely showed 186 overexpressed and 22 suppressed genes compared with peri-implantitis and healthy tissues, while peri-implantitis had 1974 and 642, respectively. Each common and unique differential gene set showed distinct enriched biological features between periodontitis and peri-implantitis after the pathway enrichment analysis. The expression pattern of selected DEGs focused on the representability of the disease was validated by RT-qPCR. CONCLUSIONS: Although periodontitis and peri-implantitis showed common gene expression that was clearly differentiated from healthy conditions, there were also unique gene patterns that were differentially expressed only in peri-implantitis. These findings will help elucidate the mechanisms involved in the progression of peri-implantitis.

The effect of oral bacterial infection on DNA damage response in host cells.

Maintaining and transferring intact genomes from one generation to another plays a pivotal role in all living organisms. DNA damage caused by numerous endogenous and exogenous factors must be adequately repaired, as unrepaired and accumulated DNA mutations can cause severe deleterious effects, such as cell death and cancer. To prevent adverse consequences, cells have established DNA damage response mechanisms that address different forms of DNA damage, including DNA double-strand breaks, mismatches, nucleotide excision, and base excision. Among several sources of exogenous DNA damage, bacterial infections cause inflammation in the host, generating reactive oxygen species (ROS) and causing oxidative DNA damage. Recent studies have revealed the importance of the oral microbiome in inflammation and several systemic host diseases. Dysbiosis of oral bacteria can induce chronic inflammation, which enhances ROS-induced DNA damage, and improperly repaired damage can lead to carcinogenesis. This review describes the various DNA repair pathways that are affected by chronic inflammation and the discovery of the DNA damage response induced by oral bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum.

Consumer testing away from a sensory facility: Application of home-use test and no-contact home-use test.

COVID-19 pandemic-related restrictions for approximately three years have heavily influenced sensory evaluations. People have become accustomed to working remotely and communicating online. This has led to opportunities in sensory testing paired with logistics systems and information technologies, resulting in a wide application of the home-use test (HUT), wherein panelists evaluate samples from their homes or other off-site locations. This study aimed to compare three sensory evaluation conditions: a central location test (CLT, n = 104), a HUT (n = 120), and a no-contact HUT (N-HUT, n = 111). We recruited participants via the local community website, delivered samples using a delivery service, and conducted sensory testing using a smartphone for the N-HUT. Participants were requested to report the acceptance ratings, sensory profiles, and emotion responses to four coffee samples. Some differences in the acceptance ratings might be due to the different attitudes participating in the evaluation. In the sensory profiling of the samples, multi-factor analysis (MFA) revealed highly similar sensory characteristics across the three types of tests. All RV coefficients (RVs) among the test conditions were above 0.93. The emotion responses to coffee samples were similar among test conditions based on the MFA with RV values greater than 0.84. In conclusion, we found that N-HUT produced similar results regarding the descriptions of sensory profiles and emotions, indicating that N-HUT is a suitable test method for collecting sensory data and overcoming CLT and HUT's regional limitations. Modern logistics systems and information technologies make it possible to conduct nationwide sensory evaluations without in-person contact or participant attendance at sensory testing facilities.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m6A modifications and splicing factors.

N6-methyladenosine (m6A) modification in RNA affects various aspects of RNA metabolism and regulates gene expression. This modification is modulated by many regulatory proteins, such as m6A methyltransferases (writers), m6A demethylases (erasers), and m6A-binding proteins (readers). Previous studies have suggested that alterations in m6A regulatory proteins induce genome-wide alternative splicing in many cancer cells. However, the functional effects and molecular mechanisms of m6A-mediated alternative splicing have not been fully elucidated. To understand the consequences of this modification on RNA splicing in cancer cells, we performed RNA sequencing and analyzed alternative splicing patterns in METTL3-knockdown osteosarcoma U2OS cells. We detected 1,803 alternatively spliced genes in METTL3-knockdown cells compared to the controls and found that cell cycle-related genes were enriched in differentially spliced genes. A comparison of the published MeRIP-seq data for METTL14 with our RNA sequencing data revealed that 70-87% of alternatively spliced genes had an m6A peak near 1 kb of alternative splicing sites. Among the 19 RNA-binding proteins enriched in alternative splicing sites, as revealed by motif analysis, expression of SFPQ highly correlated with METTL3 expression in 12,839 TCGA pan-cancer patients. We also found that cell cycle-related genes were enriched in alternatively spliced genes of other cell lines with METTL3 knockdown. Taken together, we suggest that METTL3 regulates m6A-dependent alternative splicing, especially in cell cycle-related genes, by regulating the functions of splicing factors such as SFPQ.

Effect of Substitutional Oxygen on Properties of Ti3 C2 Tx MXene Produced Using Recycled TiO2 Source.

MXenes are an emerging class of 2D materials with unique properties including metallic conductivity, mechanical flexibility, and surface tunability, which ensure their utility for diverse applications. However, the synthesis of MXenes with high crystallinity and atomic stoichiometry in a low-cost process is still challenging because of the difficulty in controlling the oxygen substitute in the precursors and final products of MXenes, which limits their academic understanding and practical applications. Here, a novel cost-effective method is reported to synthesize a highly crystalline and stoichiometric Ti3 C2 Tx MXene with minimum substitutional oxygen impurities by controlling the amount of excess carbon and time of high-energy milling in carbothermal reduction of recycled TiO2 source. The highest used content (2 wt%) of excess-carbon yields TiC with the highest carbon content and minimal oxygen substitutes, which leads to the Ti3 AlC2 MAX phase with improved crystallinity and atomic stoichiometry, and finally Ti3 C2 Tx MXene with the highest electrical conductivity (11738 S cm-1 ) and superior electromagnetic shielding effectiveness. Additionally, the effects of carbon content and substitutional oxygen on the physical properties of TiC and Ti3 AlC2 are elucidated by density-functional-theory calculations. This inexpensive TiO2 -based method of synthesizing high-quality Ti3 C2 Tx MXene can facilitate large-scale production and thus accelerate global research on MXenes.

Role of Oxygen in the Ti3AlC2 MAX Phase in the Oxide Formation and Conductivity of Ti3C2-Based MXene Nanosheets.

Ti3C2Tx MXene, a two-dimensional transition metal carbide, has attracted substantial interest due to its unique physical properties and a wide range of potential applications. Although the properties of devices using MXene have been substantially enhanced in recent years, it is not fully understood how the oxygen concentration in Ti3AlC2 MAX affects oxide formation in Ti3C2-based MXene nanosheets and their fundamental properties. To this end, we compared two types of MAX phases: MAX with low oxygen content (LO-MAX) and MAX synthesized by a conventional process. Since the conventional MAX synthesis employs metal (Ti) as a primary material, it is referred to as metal-based MAX (MB-MAX) from here. The oxygen content of the LO-MAX was only 0.56 wt %, which was about 20% compared to that of MAX synthesized using conventional methods. We compared the properties of MXene nanosheets prepared from the LO-MAX with MXene nanosheets obtained from the MB-MAX. Microscopic and chemical analyses revealed smooth and wrinkle-free morphology and small amounts of oxygen in MXene nanosheets prepared from LO-MAX (LO-MXene). The LO-MXene nanosheet film exhibited an exceptionally high conductivity of 10,540 S/cm and an ultralow surface roughness of 1.7 nm, which originated from inhibited surface oxide formation. Moreover, the inhibition of oxide formation strengthened the function of -O or -OH groups on the surface of MXene, thereby facilitating strong hydrogen bonding to the polymer with hydroxyl groups. To clearly reveal these properties, we prepared a pressure sensor by coating these MXene nanosheets on nylon/polyester fibers. The fabricated sensor exhibited a high sensitivity of up to 85.6/kPa and excellent stretch stability and reliability. These results clearly revealed that lowering the oxygen content in MAX can make a decisive contribution to improving the fundamental properties of MXene nanosheets prepared therefrom.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Mesenchymal stem cells target microglia via galectin-1 production to rescue aged mice from olfactory dysfunction.

Olfactory loss has been considered as the earliest complication for the aging process while underlying mechanisms and therapeutic strategies remain unclear. Given the correlation between microglial activation and olfactory dysfunction, here we investigated whether the immunomodulatory action of mesenchymal stem cells (MSCs) can rescue the olfactory impairment in old mice. The intranasal delivery of MSCs limited microglial activation and neuronal apoptosis in the olfactory bulb (OB), leading to improvement in olfaction. MSCs down-regulated the proportion of CD86+ microglia and prevented the maturation of cathepsin S, one of the inflammatory mediators in olfactory impairment, via the suppression of p38 MAPK signaling. Notably, old astrocytes could not prevent excessive microgliosis because the endogenous production of Galectin-1 (Gal1), one of the key microglia regulators secreted by astrocytes, was not sufficiently upregulated in the aged brain despite the presence of reactive astrogliosis. Considering that Gal1 is known as a potent paracrine factor of MSCs, we investigated whether MSC-derived Gal1 could compensate for defective astrocyte function in terms of microglial regulation. MSCs and their culture supernatant (MSC-CM) could regulate the direction of microglial differentiation by impeding the polarization towards the pro-inflammatory M1 type; notably, a selective Gal1 inhibitor OTX008 could hinder this phenomenon, indicating that Gal1 is involved in immunomodulation exerted by MSCs. Also, acute microglial activation within the OB upon LPS infusion was attenuated by MSC-CM in a Gal1-dependent manner. Our study demonstrates the therapeutic benefit of MSCs on age-related olfactory dysfunction and suggests Gal1 as a key mediator of the anti-inflammatory action of MSCs.

Compact Smartphone-Based Laser Speckle Contrast Imaging Endoscope Device for Point-of-Care Blood Flow Monitoring.

Laser speckle contrast imaging (LSCI) is a powerful visualization tool for quantifying blood flow in tissues, providing simplicity of configuration, ease of use, and intuitive results. With recent advancements, smartphone and camera technologies are suitable for the development of smartphone-based LSCI applications for point-of-care (POC) diagnosis. A smartphone-based portable LSCI endoscope system was validated for POC diagnosis of vascular disorders. The endoscope consisted of compact LED and laser illumination, imaging optics, and a flexible fiberscope assembled in a 3D-printed hand-held cartridge for access to body cavities and organs. A smartphone's rear camera was mounted thereto, enabling endoscopy, LSCI image acquisition, and processing. Blood flow imaging was calibrated in a perfused tissue phantom consisting of a microparticle solution pumped at known rates through tissue-mimicking gel and validated in a live rat model of BBN-induced bladder cancer. Raw LSCI images successfully visualized phantom flow: speckle flow index showed linearity with the pump flow rate. In the rat model, healthy and cancerous bladders were distinguishable in structure and vasculature. The smartphone-based low-cost portable mobile endoscope for monitoring blood flow and perfusion shows promise for preclinical applications and may be suitable for primary diagnosis at home or as a cost-effective POC testing assay.

Induction and Maintenance Treatment of Lupus Nephritis: A Comprehensive Review of Meta-Analyses.

BACKGROUND: Lupus nephritis (LN) is present in over 50% of patients with systemic lupus erythematosus (SLE) which is managed with immunosuppressive and immunomodulatory therapies. However, several novel therapeutic approaches for LN are under investigation due to the adverse effects spectrum of conventional therapy; Methods: We performed a comprehensive review of meta-analyses aggregating the comparative efficacies of various pharmacotherapies for LN. We conducted a literature search and retrieved a total of 23 meta-analyses and network meta-analyses for summarization. Pharmacotherapies were evaluated across six major outcomes: remission, relapse, mortality, end stage kidney disease (ESKD) progression, infection, and malignancy. RESULT: Calcineurin inhibitors (CNI), particularly tacrolimus (TAC), in combination with glucocorticoids (GC) outperformed cyclophosphamide (CPA) with GC in the rate of remission, either complete or partial remission, and in terms of infectious complications. In maintenance therapy, MMF was superior to azathioprine (AZA) as the MMF-treated patients had lower relapse rate. INTERPRETATION: This review aggregates evidence of therapy for clinicians and sheds light on comparative efficacies of alternative LN treatments. As more promising agents are entering the market, such as voclosporin, belimumab, and obinutuzumab, LN management might undergo significant changes during the next years.

Crosstalk between different DNA repair pathways for DNA double strand break repairs.

DNA double strand breaks (DSBs) are the most threatening type of DNA lesions and must be repaired properly in order to inhibit severe diseases and cell death. There are four major repair pathways for DSBs: non-homologous end joining (NHEJ), homologous recombination (HR), single strand annealing (SSA) and alternative end joining (alt-EJ). Cells choose repair pathway depending on the cell cycle phase and the length of 3' end of the DNA when DSBs are generated. Blunt and short regions of the 5' or 3' overhang DNA are repaired by NHEJ, which uses direct ligation or limited resection processing of the broken DNA end. In contrast, HR, SSA and alt-EJ use the resected DNA generated by the MRN (MRE11-RAD50-NBS1) complex and C-terminal binding protein interacting protein (CtIP) activated during the S and G2 phases. Here, we review recent findings on each repair pathway and the choice of repair mechanism and highlight the role of mismatch repair (MMR) protein in HR.

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics.

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.

Determination of ethanol in foods and beverages by magnetic stirring-assisted aqueous extraction coupled with GC-FID: A validated method for halal verification.

An in-house method, employing magnetic stirring-assisted aqueous extraction combined with gas chromatography-flame ionization detector, for determination of ethanol in different foods and beverages was validated according to the ISO/IEC 17025 standard. Validation parameters, including selectivity, method limits of detection (approx. 0.006 mg/g) and quantification (approx. 0.02 mg/g), linearity (R2 of >0.999), trueness (relative biases of <3%), accuracy (recoveries of 96-105%), and precision (relative standard deviations of <5%), were satisfactory. The proposed method was as accurate and precise as and more sensitive than the AOAC method 2016.12, with estimated relative expanded uncertainties of around 8% for all samples. The validated method was successfully applied for determination of ethanol in 108 commercially processed foods and beverages, and it could be used for halal verification. Accordingly, this study provided a reliable method for routine quantitative analysis of ethanol in processed foods and beverages to ensure their halal integrity prior to halal certification.

Effect of Alternative Preservatives on the Quality of Rice Cakes as Halal Food.

The halal food market is steadily increasing. The use of alcohol for any reason is strictly prohibited in halal foods; however, ethanol is widely used as a preservative for commercial rice cakes (tteok). The purpose of this study was to investigate the use of natural substances as alternative preservatives for rice cakes. Four different solutions were tested: distilled water (control), ethanol, grapefruit seed extract (GSE), and a mixture of citric extracts and organic acids (MCO). We investigated the total plate count (TPC), yeast and mold counts, color, texture profile assays (TPA), and sensory evaluation. Significant reductions of 3.65 log CFU were observed in TPC in rice cake treated with MCO solution after 28 days of storage. However, mold and yeast counts were only reduced by ethanol treatment. Among the physical texture properties analysis, hardness was maintained for the 28 days in all samples. The total color difference values (ΔE) revealed no significant color changes in any rice cake compared to the controls. The ethanol-treated rice cake scored the lowest for overall preference and desired hardness.

RNF126 is a positive regulator of TRAF3 ubiquitination.

Ubiquitination and deubiquitination of signaling molecules are critical regulatory mechanisms in various biological contexts such as inflammatory signaling and the DNA damage response. Thus, finely tuned regulation of protein ubiquitination is essential for maintaining cellular homeostasis. Here, we showed that the RING finger protein RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination, which is a crucial step in the TRAF3-dependent antiviral response. We found that RNF126 also interacts with OTUB1, a deubiquitinating enzyme that negatively regulates K63-linked ubiquitination of TRAF3. RNF126 promotes ubiquitination of OTUB1, leading to reduced deubiquitinating activity toward TRAF3. Moreover, RNF126 promotes ubiquitination of OTUB1 on cysteine 91, which is reportedly required for its catalytic activity. Taken together, our results suggest that RNF126 positively regulates the antiviral response by directly promoting K63-linked polyubiquitination of TRAF3 and by reducing OTUB1 activity.

One-Way Continuous Deposition of Monolayer MXene Nanosheets for the Formation of Two Confronting Transparent Electrodes in Flexible Capacitive Photodetector.

MXenes based on titanium carbide are promising next-generation transparent electrode materials due to their high metallic conductivity, optical transparency, mechanical flexibility, and abundant hydrophilic surface functionality. MXene electrodes offer a much wider conductive surface coverage than metal nanowires, thereby gaining popularity as flexible electrode materials in supercapacitors and energy devices. However, given that monolayer MXene nanosheets are only a few nanometers thick, meticulous surface treatments and deposition technologies are required for a practical implementation of these transparent electrodes. Unfortunately, a capacitor produced by forming high-quality transparent MXene electrodes on both sides of a film has not yet been reported. We report the successful development of a one-way continuous deposition technology to form high-quality MXene nanosheet-based transparent electrodes on both surfaces of a polymer film without large physical stresses on the MXene nanosheets. One transparent electrode was formed by transferring MXene nanosheets predeposited on a temporary glass substrate to the film surface, while the other was directly deposited on the exposed film surface. The Ti3AlC2 precursor (MAX) was synthesized via a spark plasma sintering crystallization, and the MXene nanosheets were prepared via a subsequent Al-selective etching and delamination. We used this material to implement a capacitive photodetector consisting of two layers of opposing transparent electrodes. The flexible photodetector was based on poly(vinyl butyral) (PVB), which was solidly bonded with MXene nanosheets to serve as a free-standing binder for the Cu-doped ZnS semiconductor particles. The fabricated device exhibited excellent mechanical stability due to the high affinity between the MXene nanosheets and PVB. Furthermore, the device exhibited an initial capacitance of 2 nF, photosensitivity of 12.5 µF/W, and rise and decay times of 0.031 and 0.751 s, respectively. All these parameters were 1 to 2 orders of magnitude higher or faster than reported capacitive photodetectors. Overall, the proposed approach resolves the core issues associated with existing metal nanowire-based electrodes, and it is a breakthrough in the development of next-generation flexible devices comprising two layers of confronting transparent electrodes.

Effects of Ultra-high doserate FLASH Irradiation on the Tumor Microenvironment in Lewis Lung Carcinoma: Role of Myosin Light Chain.

PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.