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Abstract Bulk zinc oxide (ZnO-BPs) and its nanoparticles (ZnO-NPs) are frequently used in various products for humans. Helisoma duryi embryos can serve as effective model organisms for studying the toxicity of NPs. This study aimed to compare the teratogenic potency of ZnO-BPs and ZnO NPs in the embryonic stages of H. duryi to evaluate the utility of this snail as a bioindicator for ZnO-NPs in the aquatic environment. The mechanisms of teratogenesis were evaluated by determination of the LC50, studying the effect of sub-lethal concentrations of both ZnO forms on the embryos, and studying their enzyme activity, oxidative stress, and biochemical analysis. The SDS-PAGE electrophoresis was undertaken to assess the effect of ZnO-BPs and ZnO NPs on protein synthesis. The results revealed that the veliger stage of H. duryi is the specific stage for bulk and nano ZnO. ZnO-NPs proved to be more toxic to snails’ embryos than ZnO-BPs. Exposure to ZnO influences specific types of defects in development, which in the case of BPs are far less drastic than those caused by NPs. Thus, the toxicity of ZnO-NPs in embryonic development is due to their unique physicochemical properties. The observed malformations include mainly hydropic malformation, exogastrulation, monophthalmia, shell misshapen, and cell lyses. Almost all tested oxidative biomarkers significantly changed, revealing that ZnONPs display more oxidative stress than ZnO-BPs. Also, the low concentration of ZnO induces many disturbances in the organic substances of veliger larvae, such as a decrease in the total protein and total lipid levels and an increase in the glycogen level. The results indicated that ZnO-BPs increase the number of protein bands. Conversely, ZnO-NPs concealed one band from treated egg masses, which was found in the control group. Embryos of snail are an appropriate model to control freshwater snails. This study demonstrates that H. duryi embryos can serve as effective model organisms to study the toxicity of ZnO-NPs.

Abstract Background Genetic diversity, population structure, agro-morphological traits, and molecular characteristics, are crucial for either preserving genetic resources or developing new cultivars. Due to climate change, water availability for agricultural use is progressively diminishing. This study used 100 molecular markers (25 TRAP, 22 SRAP, 23 ISTR, and 30 SSR). Additionally, 15 morphological characteristics were utilized to evaluate the optimal agronomic traits of 12 different barley genotypes under arid conditions. Results Substantial variations, ranging from significant to highly significant, were observed in the 15 agromorphological parameters evaluated among the 12 genotypes. The KSU-B101 barley genotype demonstrated superior performance in five specific traits: spike number per plant, 100-grain weight, spike number per square meter, harvest index, and grain yield. These results indicate its potential for achieving high yields in arid regions. The Sahrawy barley genotype exhibited the highest values across five parameters, namely leaf area, spike weight per plant, spike length, spike weight per square meter, and biological yield, making it a promising candidate for animal feed. The KSU-B105 genotype exhibited early maturity and a high grain count per spike, which reflects its early maturity and ability to produce a high number of grains per spike. This suggests its suitability for both animal feed and human food in arid areas. Based on marker data, the molecular study found that the similarity coefficients between the barley genotypes ranged from 0.48 to 0.80, with an average of 0.64. The dendrogram constructed from these data revealed three distinct clusters with a similarity coefficient of 0.80. Notably, the correlation between the dendrogram and its similarity matrix was high (0.903), indicating its accuracy in depicting the genetic relationships. The combined analysis revealed a moderate correlation between the morphological and molecular analysis, suggesting alignment between the two characterization methods. Conclusions The morphological and molecular analyses of the 12 barley genotypes in this study effectively revealed the varied genetic characteristics of their agro-performance in arid conditions. KSU-B101, Sahrawy, and KSU-B105 have emerged as promising candidates for different agricultural applications in arid regions. Further research on these genotypes could reveal their full potential for breeding programs.

Fuchs endothelial corneal dystrophy (FECD), the leading indication for corneal transplantation in the U.S., causes loss of corneal endothelial cells (CECs) and corneal edema leading to vision loss. FECD pathogenesis is linked to impaired response to oxidative stress and environmental ultraviolet A (UVA) exposure. Although UVA is known to cause nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation, ferroptosis has not been characterized in FECD. We investigated the roles of genetic background and UVA exposure in causing CEC degeneration in FECD. Using ungenotyped FECD patient surgical samples, we found increased levels of cytosolic ferrous iron (Fe2+) and lipid peroxidation in end-stage diseased tissues compared with healthy controls. Using primary and immortalized cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found altered gene and protein expression involved in ferroptosis compared to controls including elevated levels of Fe2+, basal lipid peroxidation, and the ferroptosis-specific marker transferrin receptor 1. Increased cytosolic Fe2+ levels were detected after physiologically relevant doses of UVA exposure, indicating a role for ferroptosis in FECD disease progression. Cultured cells were more prone to ferroptosis induced by RSL3 and UVA than controls, indicating ferroptosis susceptibility is increased by both FECD genetic background and UVA. Finally, cell death was preventable after RSL3 induced ferroptosis using solubilized ubiquinol, indicating a role for anti-ferroptosis therapies in FECD. This investigation demonstrates that genetic background and UVA exposure contribute to iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated disease progression in FECD.

BackgroundSmartphone apps are a convenient, low-cost approach to delivering smoking cessation support to large numbers of individuals. Yet, the apps are susceptible to low rates of user engagement and retention. ObjectiveThis study aims to test the effects of a new game module (called Inner Dragon) integrated into Smoke Free (23 Limited), a leading smoking cessation app with established efficacy. The primary outcomes measured user engagement with the app. MethodsA 2-arm, parallel-group, randomized controlled trial was conducted in the United States with an 8-week follow-up. Adult individuals who smoked ≥1 cigarettes daily and planned to quit smoking within 7 days were recruited and randomized (N=500), with equal allocation. Both groups received free access to the original Smoke Free app with “core” features of its smoking cessation program (eg, a diary and craving log). The treated group received additional access to the integrated Inner Dragon game that incorporated several game mechanics designed to increase user engagement. User engagement outcomes were the number of unique app sessions, average minutes per session, days with a session, and program adherence. Self-reported and verified smoking abstinence and app satisfaction were also assessed. The main analysis estimated the intention-to-treat effect of access to Inner Dragon on each outcome. Further analyses assessed effect modification by participant characteristics and the association of intensity of game use with program adherence and abstinence. ResultsOverall, user engagement was greater for treated versus control participants: they had 5.3 more sessions of Smoke Free (mean 29.6, SD 36.5 sessions vs mean 24.3, SD 37.9 sessions; P=.06), 0.8 more minutes per session (mean 6.9, SD 5.4 min vs mean 6.1, SD 5.2 min; P=.047), and 3.4 more days with a session (mean 14.3, SD 15.3 days vs mean 11.9, SD 14.3 days; P=.03). Program adherence, based on the number of times core features of the original Smoke Free app were used, was higher for treated versus control participants (mean 29.4, SD 41.3 times vs mean 22.6, SD 35.6 times; P=.03). Self-reported 7-day and 30-day point-prevalence abstinence and verified 7-day point-prevalence abstinence at 8 weeks did not significantly differ by study group. The mean repeated 1-day prevalence of quitting was higher among the treated group versus the control group (mean 17.3%, SD 25.6 vs mean 12.4%, SD 21.3; P=.01). App satisfaction and the motivation to (stay) quit did not differ by study group. Higher intensity of game use was associated with increased program adherence and self-reported abstinence. ConclusionsFindings suggest that the Inner Dragon game increased user engagement and program adherence. Additional refinements to the game design may clarify whether the game increases abstinence rates. Overall, it is feasible to deploy games and gamification to enhance user engagement in existing smoking cessation interventions. Trial RegistrationClinicalTrials.gov NCT05227027; https://clinicaltrials.gov/study/NCT05227027

Abstract Using the particular nature of melanoma mutanomes to develop medicines that activate the immune system against specific mutations is a game changer in immunotherapy individualisation. It offers a viable solution to the recent rise in resistance to accessible immunotherapy alternatives, with some patients demonstrating innate resistance to these drugs despite past sensitisation to these agents. However, various obstacles stand in the way of this method, most notably the practicality of sequencing each patient's mutanome, selecting immunotherapy targets, and manufacturing specific medications on a large scale. With the robustness and advancement in research techniques, artificial intelligence (AI) is a potential tool that can help refine the mutanome-based immunotherapy for melanoma. Mutanome-based techniques are being employed in the development of immune-stimulating vaccines, improving current options such as adoptive cell treatment, and simplifying immunotherapy responses. Although the use of AI in these approaches is limited by data paucity, cost implications, flaws in AI inference capabilities, and the incapacity of AI to apply data to a broad population, its potential for improving immunotherapy is limitless. Thus, in-depth research on how AI might help the individualisation of immunotherapy utilising knowledge of mutanomes is critical, and this should be at the forefront of melanoma management.

Abstract Cancer is the world's second-leading cause of death. Drug development efforts frequently focus on medicinal plants since they are a valuable source of anticancer medications. A phytochemical investigation of the edible Ziziphus spina-christi (F. Rhamnaceae) leaf extract afforded two new dammarane type saponins identified as christinin E and F (1, 2), along with the known compound christinin A (3). Different cancer cell lines, such as lung cancer (A549), glioblastoma (U87), breast cancer (MDA-MB-231), and colorectal carcinoma (CT-26) cell lines, were used to investigate the extracted compounds' cytotoxic properties. Our findings showed significant effects on all the tested cell lines at varying concentrations (1, 5, 10, and 20 µg/mL). The three compounds exhibited potent activity at low concentrations (

Abstract Background Cancer continues to be one of the biggest causes of death that affects human health. Chemical resistance is still a problem in conventional cancer treatments. Fortunately, numerous natural compounds originating from different microbes, including fungi, possess cytotoxic characteristics that are now well known. This study aims to investigate the anticancer prospects of five fungal strains that were cultivated and isolated from the Red Sea soft coral Paralemnalia thyrsoides. The in vitro cytotoxic potential of the ethyl acetate extracts of the different five isolates were evaluated using MTS assay against four cancer cell lines; A549, CT-26, MDA-MB-231, and U87. Metabolomics profiling of the different extracts using LC-HR-ESI-MS, besides molecular docking studies for the dereplicated compounds were performed to unveil the chemical profile and the cytotoxic mechanism of the soft coral associated fungi. Results The five isolated fungal strains were identified as Penicillium griseofulvum (RD1), Cladosporium sphaerospermum (RD2), Cladosporium liminiforme (RD3), Penicillium chrysogenum (RD4), and Epicoccum nigrum (RD5). The in vitro study showed that the ethyl acetate extract of RD4 exhibited the strongest cytotoxic potency against three cancer cell lines A549, CT-26 and MDA-MB-231 with IC50 values of 1.45 ± 8.54, 1.58 ± 6.55 and 1.39 ± 2.0 µg/mL, respectively, also, RD3 revealed selective cytotoxic potency against A549 with IC50 value of 6.99 ± 3.47 µg/mL. Docking study of 32 compounds dereplicated from the metabolomics profiling demonstrated a promising binding conformation with EGFR tyrosine kinase that resembled its co-crystallized ligand albeit with better binding energy score. Conclusion Our results highlight the importance of soft coral-associated fungi as a promising source for anticancer metabolites for future drug discovery.

ObjectiveMost of the work in terms of liquid biopsies in patients with solid tumors is focused on circulating tumor DNA (ctDNA). Our aim was to evaluate the feasibility of using circulating tumor cells (CTCs) in peripheral blood samples from patients with advanced or metastatic gastrointestinal (GI) cancers.MethodsIn this prospective study, blood samples were collected from each patient in 2 AccuCyte® blood collection tubes and each tube underwent CTC analysis performed utilizing the RareCyte® platform. The results from both tubes were averaged and a total of 150 draws were done, with 281 unique reported results. The cadence of sampling was based on convenience sampling and piggybacked onto days of actual clinical follow-ups and treatment visits. The CTC results were correlated with patient- and tumor-related variables.ResultsData from a total of 59 unique patients were included in this study. Patients had a median age of 58 years, with males representing 69% of the study population. More than 57% had received treatment prior to taking blood samples. The type of GI malignancy varied, with more than half the patients having colorectal cancer (CRC, 54%) followed by esophageal/gastric cancer (17%). The least common cancer was cholangiocarcinoma (9%). The greatest number of CTCs were found in patients with colorectal cancer (Mean: 15.8 per 7.5 ml; Median: 7.5 per 7.5 ml). In comparison, patients with pancreatic cancer (PC) had considerably fewer CTCs (Mean: 4.2 per 7.5 ml; Median: 3 per 7.5 ml). Additionally, we found that patients receiving treatment had significantly fewer CTCs than patients who were not receiving treatment (Median 2.7 versus 0.7). CTC numbers showed noteworthy disparities between patients with responding/stable disease in comparison to those with untreated/progressive disease (Median of 2.7 versus 0). When CTCs were present, biomarker analyses of the four markers human epidermal growth factor receptor 2 (HER2)/programmed death-ligand 1 (PD-L1)/Kiel 67 (Ki-67)/epidermal growth factor receptor (EGFR) was feasible. Single cell sequencing confirmed the tumor of origin.ConclusionOur study is one of the first prospective real-time studies evaluating CTCs in patients with GI malignancies. While ctDNA-based analyses are more common in clinical trials and practice, CTC analysis provides complementary information from a liquid biopsy perspective that is of value and worthy of continued research.

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC50s ranged from 2.53-8.67 µM, 8.67–62.47 µM, and 4.19–24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC50 values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC50 values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC50 = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.

BackgroundSeveral stand-alone smartphone apps have used serious games to provide an engaging approach to quitting smoking. So far, the uptake of these games has been modest, and the evidence base for their efficacy in promoting smoking cessation is still evolving. The feasibility of integrating a game into a popular smoking cessation app is unclear. ObjectiveThe aim of this paper was to describe the design and iterative development of the Inner Dragon game within Smoke Free, a smartphone app with proven efficacy, and the results of a single-arm feasibility trial as part of a broad program that seeks to assess the effectiveness of the gamified app for smoking cessation. MethodsIn phase 1, the study team undertook a multistep process to design and develop the game, including web-based focus group discussions with end users (n=15). In phase 2, a single-arm study of Smoke Free users who were trying to quit (n=30) was conducted to assess the feasibility and acceptability of the integrated game and to establish the feasibility of the planned procedures for a randomized pilot trial. ResultsPhase 1 led to the final design of Inner Dragon, informed by principles from psychology and behavioral economics and incorporating several game mechanics designed to increase user engagement and retention. Inner Dragon users maintain an evolving pet dragon that serves as a virtual avatar for the users’ progress in quitting. The phase-2 study established the feasibility of the study methods. The mean number of app sessions completed per user was 13.8 (SD 13.1; median 8; range 1-46), with a mean duration per session of 5.8 (median 1.1; range 0-81.1) minutes. Overall, three-fourths (18/24, 75%) of the participants entered the Inner Dragon game at least once and had a mean of 2.4 (SD 2.4) sessions of game use. The use of Inner Dragon was positively associated with the total number of app sessions (correlation 0.57). The mean satisfaction score of participants who provided ratings (11/24, 46%) was 4.2 (SD 0.6) on a 5-point scale; however, satisfaction ratings for Inner Dragon were only completed by 13% (3/24) of the participants. ConclusionsFindings supported further development and evaluation of Inner Dragon as a beneficial feature of Smoke Free. The next step of this study is to conduct a randomized pilot trial to determine whether the gamified version of the app increases user engagement over a standard version of the app.

Abstract Background It has been shown that copper oxide nanoparticles (CuO NPs) induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NPs from the respiratory tract. The purposes of this study were to observe the kinetics of pulmonary inflammation during and after CuO NP sub-acute inhalation exposure and to investigate copper (Cu) biodistribution and clearance rate from the exposure site and homeostasis of selected trace elements in secondary organs of BALB/c mice. Results Sub-acute inhalation exposure to CuO NPs led to pulmonary inflammation represented by increases in lactate dehydrogenase, total cell counts, neutrophils, macrophages, inflammatory cytokines, iron levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed Cu concentration increased steadily during exposure and gradually declined after exposure. Cu elimination from the lung showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart indicating that inhaled Cu could be translocated into the bloodstream and heart tissue, and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs; increase in the kidneys and decrease in the spleen. Furthermore, concentrations of selenium in kidneys and iron in spleen were decreased, pointing to disruption of trace element homeostasis. Conclusions Sub-acute inhalation exposure of CuO NPs induced pulmonary inflammation, which was correlated to Cu concentrations in the lungs and started to resolve once exposure ended. Dosimetry analysis showed that Cu in the lungs was translocated into the bloodstream and heart tissue. Secondary organs affected by CuO NPs exposure were kidneys and spleen as they showed the disruption of trace element homeostasis and organ weight changes.

Abstract Background Interleukin‐1 alpha (IL‐1α) is a pro‐inflammatory cytokine that can activate immune effector cells and trigger anti‐tumor immune responses. However, dose‐limiting toxicities including cytokine storm and hypotension has limited its use in the clinic as a cancer therapy. We propose that polymeric microparticle (MP)‐based delivery of IL‐1α will suppress the acute pro‐inflammatory side effects by allowing for slow and controlled release of IL‐1α systemically, while simultaneously triggering an anti‐tumor immune response. Methods Polyanhydride copolymers composed of 1,6‐bis‐(p‐carboxyphenoxy)‐hexane:sebacic 20:80 (CPH:SA 20:80) was utilized to fabricate MPs. Recombinant IL‐1α (rIL‐1α) was encapsulated into CPH:SA 20:80 MPs (IL‐1α‐MPs) and the MPs were characterized by size, charge, loading efficiency, and in‐vitro release and activity of IL‐1α. IL‐1α‐MPs were injected intraperitonially into head and neck squamous cell carcinoma (HNSCC)‐bearing C57Bl/6 mice and monitored for changes in weight, tumor growth, circulating cytokines/chemokines, hepatic and kidney enzymes, blood pressure, heart rate, and tumor‐infiltrating immune cells. Results CPH:SA IL‐1α‐MPs demonstrated sustained release kinetics of IL‐1α (100% protein released over 8–10 days) accompanied by minimal weight loss and systemic inflammation compared to rIL‐1α‐treated mice. Blood pressure measured by radiotelemetry in conscious mice demonstrates that rIL‐1α‐induced hypotension was prevented in IL‐1α‐MP‐treated mice. Liver and kidney enzymes were within normal range for all control and cytokine‐treated mice. Both rIL‐1α and IL‐1α‐MP‐treated mice showed similar delays in tumor growth and similar increases in tumor‐infiltrating CD3+ T cells, macrophages, and dendritic cells. Conclusions CPH:SA‐based IL‐1α‐MPs generated a slow and sustained systemic release of IL‐1α resulting in reduced weight loss, systemic inflammation, and hypotension accompanied by an adequate anti‐tumor immune response in HNSCC‐tumor bearing mice. Therefore, MPs based on CPH:SA formulations may be promising as delivery vehicles for IL‐1α to achieve safe, effective, and durable antitumor responses for HNSCC patients.

Background and Aim: Tooth eruption is a multifaceted physiological process, which can be delayed by factors such as malnutrition. This study aimed to compare the serum levels of vitamin D and calcium in infants with a longer than normal eruption time of the first deciduous tooth with the control group (normal eruption). Materials and Methods: In this case-control study, serum levels of vitamin D in 61 children with a mean age of normal dental eruption in Iranian children (8 months and less) were compared with 61 children with eruption age of more than 8 months. The children were matched in terms of age, sex, place of birth and residence, age of birth, birth weight, and other eruption-related items. Student's t-test, analysis of variance (ANOVA), and chi-square test were used for statistical analysis. Results: The first erupted tooth in all infants was the mandibular incisor. The mean serum level of vitamin D was 38.1±24.1 ng/ml in infants with eruption age of 8 months or less and 40.0±21.5 ng/ml in the group of eruption age over 8 months (P=0.63). Serum calcium level was 10.03±0.55 mg/dl in the first group and 9.92±0.57 mg/dl in the second group (P=0.28). There was no significant relationship between gender, maternal education, A+D supplementation, infant nutrition (breast milk, formula, and combination), and different levels of vitamin D (deficient, inadequate, adequate, and toxic) with the average age of eruption of the first deciduous tooth (P>0.05). Conclusion: It seems that serum vitamin D and calcium levels in this age group do not affect the eruption time of deciduous teeth.

Tissue fibrosis is a major health issue that impacts millions of people and is costly to treat. However, few effective anti-fibrotic treatments are available. Due to their central role in fibrotic tissue deposition, fibroblasts and myofibroblasts are the target of many therapeutic strategies centered primarily on either inducing apoptosis or blocking mechanical or biochemical stimulation that leads to excessive collagen production. Part of the development of these drugs for clinical use involves in vitro prescreening. 2D screens, however, are not ideal for discovering mechanobiologically significant compounds that impact functions like force generation and other cell activities related to tissue remodeling that are highly dependent on the conditions of the microenvironment. Thus, higher fidelity models are needed to better simulate in vivo conditions and relate drug activity to quantifiable functional outcomes. To provide guidance on effective drug dosing strategies for mechanoresponsive drugs, we describe a custom force-bioreactor that uses a fibroblast-seeded fibrin gels as a relatively simple mimic of the provisional matrix of a healing wound. As cells generate traction forces, the volume of the gel reduces, and a calibrated and embedded Nitinol wire deflects in proportion to the generated forces over the course of 6 days while overhead images of the gel are acquired hourly. This system is a useful in vitro tool for quantifying myofibroblast dose-dependent responses to candidate biomolecules, such as blebbistatin. Administration of 50 μM blebbistatin reliably reduced fibroblast force generation approximately 40% and lasted at least 40 h, which in turn resulted in qualitatively less collagen production as determined via fluorescent labeling of collagen.

The COVID-19 pandemic has caused tremendous damage to the world. In order to quickly and accurately diagnose the virus and contain the spread, there is a need for rapid, sensitive, accurate, and cost-effective SARS-CoV-2 biosensors. In this paper, we report on a novel biosensor based on angiotensin converting enzyme 2 (ACE-2)-conjugated vertically-oriented silicon nanowire (vSiNW) arrays that can detect the SARS-CoV-2 spike protein with high sensitivity and selectivity relative to negative controls. First, we demonstrate the efficacy of using ACE-2 receptor to detect the SARS-CoV-2 spike protein via a capture assay test, which confirms high specificity of ACE-2 against the mock protein, and high affinity between the spike and ACE-2. We then report on results for ACE-2-conjugated vSiNW arrays where the biosensor device architecture is based on a p-n junction transducer. We confirm via analytical modeling that the transduction mechanism of the biosensor involves induced surface charge depletion of the vSiNWs due to negative electrostatic surface potential induced by the spike protein after binding with ACE-2. This vSiNW surface charge modulation is measured via current-voltage characteristics of the functionalized biosensor. Calibrated concentration dependent electrical response of the vSiNW sensor confirms the limit-of-detection for virus spike concentration of 100 ng/ml (or 575 pM). The vSiNW sensor also exhibits highly specific response to the spike protein with respect to negative controls, offering a promising point-of-care detection method for SARS-CoV-2.

Abstract The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.

Borcherding and Vishwakarma et al. characterize the tumor immune microenvironment in treatment-naïve clear cell renal carcinoma patients using single-cell RNA- and T-cell receptor sequencing. They find CD8 + T cells and macrophages are enriched in tumor tissue, and identify a proliferative CD8 + T-cell population, which may be relevant for anti-cancer responses.

The study is part of a comprehensive work to investigate impact of fish enemies on cultivated species. It has taken in consideration some vital abiotic characteristics necessary for cultivated fishes. Three fish farms were selected in two provinces, namely Al-Manahel fish farm (presently called Al-Belad) in Babylon province; Basrah University fish farm (Marine Science Center) and Al-Mutawaa fish farm situated south to city of Basrah. Water samples were monitored on monthly basis for the period from January to October 2002. Several parameters namely, temperature, pH, salinity, total alkalinity and total hardness were measured. The highest water temperature (31ºC) was recorded in July and August from Al-Mutawaa fish farm and the maximum pH value 8.0 was encountered during January from Basrah university fish farm and the lowest pH= 7.2 in May from the same farm. Results indicate a notable rise in salinity in Al-Mutawaa fish farm and the highest (6.0 g/L) encountered in June and the lowest (2.8 g/L) in March. The maximum concentration of total alkalinity (172 mg/L) was from Al-Mutawaa as well recorded in September. Values of total hardness were almost similar in Al- Manahel and Basrah university fish farms, but the latter revealed a bit higher value in May (581 mg/L), whereas Al-Mutawaa fish farm showed, by no means, the highest concentrations all the year round, but the maximum (880 mg/L) was detected in March.

The present investigation concerned with a thorough survey on the serious impact of the predaceous water birds from three selected fish farms in the country, namely Al- Manahel fish farm (presently called Al-Beilad) in Babylon province, Basrah University/ Marine Science Center fish farm and Al-Mutawaa fish farm. The latter two are situated in Basrah governorate. Samples were collected on monthly basis for the period from January to October 2002. Impacts of water birds on stocks of cultivated fish species and other faunal components was detected. Stomach of seven species of water birds, namely, herring gull (Larus argentatus), black headed gull (L. ridibundus), slender billed gull (L. genei), little gull (L. minutus), great white heron (Egretta alba ), little egret (E. garzetta) and cormorant (Phalacrocorax carbo sinensis) were examined to calculate food consumption. The study reveals that herring gull occupied first rank in Al–Manahel fish farm followed by cormorant which occupied first rank in Basrah University farm. Fishes formed 100% in food of cormorant, herring gull, black headed gull and slender billed gull. Shrimp was detected in food of little gull and little egret while frogs were consumed by great white heron and little egret. Cormorant occupied first rank in food menu for each birds followed by herring gull and slender billed gull.

Melanoma is the most lethal form of skin cancer and surgery remains the preferred and most effective treatment. Nevertheless, there are cases where surgery is not a viable method and alternative treatments are therefore adopted. One such treatment that has been tested is topical 5% imiquimod (IMQ) cream, which, although showing promise as a treatment for melanoma, has been found to have undesirable off-target effects. Resiquimod (RSQ) is an immunomodulatory molecule that can activate immune responses by binding to Toll-like receptors (TLR) 7 and 8 and may be more effective than IMQ in the context of melanoma treatment. RSQ can cross the stratum corneum (SC) easily without requiring pretreatment of the skin. In a gel formulation, RSQ has been studied as a monotherapy and adjuvant for melanoma treatment in pre-clinical studies and as an adjuvant in clinical settings. Although side effects of RSQ in gel formulation were also reported, they were never severe enough for the treatment to be suspended. In this review, we discuss the potential use of RSQ as an adjuvant for melanoma treatment.

Background CMP-001, also known as vidutolimod, is a virus-like particle containing a TLR9 agonist that is showing promise in early clinical trials. Our group previously demonstrated that the immunostimulatory effects of CMP-001 are dependent on an anti-Qβ antibody response which results in opsonization of CMP-001 and uptake by plasmacytoid dendritic cells (pDCs) that then produce interferon (IFN)-α. IFN-α then leads to an antitumor T-cell response that is responsible for the in vivo efficacy of CMP-001. Here, we explore mechanisms by which the initial effects of CMP-001 on pDCs activate other cells that can contribute to development of an antitumor T-cell response.Methods Uptake of CMP-001 by various peripheral blood mononuclear cell (PBMC) populations and response to anti-Qβ-coated CMP-001 were evaluated by flow cytometry and single-cell RNA sequencing. Purified monocytes were treated with anti-Qβ-coated CMP-001 or recombinant IFN-α to evaluate direct and secondary effects of anti-Qβ-coated CMP-001 on monocytes.Results Monocytes had the highest per cell uptake of anti-Qβ-coated CMP-001 with lower levels of uptake by pDCs and other cell types. Treatment of PBMCs with anti-Qβ-coated CMP-001 induced upregulation of IFN-responsive genes including CXCL10, PDL1, and indoleamine-2,3-dioxygenase (IDO) expression by monocytes. Most of the impact of anti-Qβ-coated CMP-001 on monocytes was indirect and mediated by IFN-α, but uptake of anti-Qβ-coated CMP-001 altered the monocytic response to IFN-α and resulted in enhanced expression of PDL1, IDO, and CD80 and suppressed expression of CXCL10. These changes included an enhanced ability to induce autologous CD4 T-cell proliferation.Conclusions Anti-Qβ-coated CMP-001 induces IFN-α production by pDCs which has secondary effects on a variety of cells including monocytes. Uptake of anti-Qβ-coated CMP-001 by monocytes alters their response to IFN-α, resulting in enhanced expression of PDL1, IDO and CD80 and suppressed expression of CXCL10. Despite aspects of an immunosuppressive phenotype, these monocytes demonstrated increased ability to augment autologous CD4 T-cell proliferation. These findings shed light on the complexity of the mechanism of action of anti-Qβ-coated CMP-001 and provide insight into pathways that may be targeted to further enhance the efficacy of this novel approach to immunotherapy.

Copper oxide (CuO) nanoparticles (NPs) are abundant in manufacturing processes, but they are an airway irritant. In vitro pulmonary toxicity of CuO NPs has been modeled using cell lines such as human bronchial epithelial cell line BEAS‐2B. In 2D in vitro culture, BEAS‐2B undergoes squamous differentiation due to the presence of serum. Differentiation is part of the repair process of lung cells in vivo that helps to preserve the epithelial lining of the respiratory tract. Herein, the effects of serum on the hydrodynamic diameter, cellular viability, cellular differentiation, and cellular uptake of 5 and 35 nm CuO NPs are investigated, and the mean cell area is used as the differentiation marker for BEAS‐2B cells. The results demonstrate that the hydrodynamic diameter decreases with the addition of serum to the culture medium. Serum also increases the mean cell area, and only affects dose‐dependent cytotoxicity of 35 nm CuO NPs, while simultaneously having no effect on intracellular Cu2+. This study presents evidence that both NP size and the presence of serum in culture media influence the relative viability of BEAS‐2B cells following CuO NP exposure and highlights a critical need for carefully designed experiments and accurately reported conditions.

Estrogens exert their physiological and pathophysiological effects via cellular receptors, named ERα, ERβ, and G-protein coupled estrogen receptor (GPER). Estrogen-regulated physiology is tightly controlled by factors that regulate estrogen bioavailability and receptor sensitivity, while disruption of these control mechanisms can result in loss of reproductive function, cancer, cardiovascular and neurodegenerative disease, obesity, insulin resistance, endometriosis, and systemic lupus erythematosus. Restoration of estrogen physiology by modulating estrogen bioavailability or receptor activity is an effective approach for treating these pathological conditions. Therapeutic interventions that block estrogen action are employed effectively for the treatment of breast and prostate cancer as well as for precocious puberty and anovulatory infertility. Theoretically, treatments that block estrogen biosynthesis should prevent estrogen action at ERs and GPER, although drug resistance and ligand-independent receptor activation may still occur. In addition, blockade of estrogen biosynthesis does not prevent activation of estrogen receptors by naturally occurring or man-made exogenous estrogens. A more complicated scenario is provided by anti-estrogen drugs that antagonize ERs since these drugs function as GPER agonists. Based upon its association with metabolic dysregulation and advanced cancer, GPER represents a therapeutic target with promise for the treatment of several critical health concerns facing Western society. Selective ligands that specifically target GPER have been developed and may soon serve as pharmacological agents for treating human disease. Here, we review current forms of estrogen therapy and the implications that GPER holds for these therapies. We also discuss existing GPER targeted drugs, additional approaches towards developing GPER-targeted therapies and how these therapies may complement existing modalities of estrogen-targeted therapy.

Background CMP-001 is a novel Toll-like receptor-9 agonist that consists of an unmethylated CpG-A motif-rich G10 oligodeoxynucleotide (ODN) encapsulated in virus-like particles. In situ vaccination of CMP-001 is believed to activate local tumor-associated plasmacytoid dendritic cells (pDCs) leading to type I interferon secretion and tumor antigen presentation to T cells and systemic antitumor T cell responses. This study is designed to investigate if CMP-001 would enhance head and neck squamous cell carcinoma (HNSCC) tumor response to anti-programmed cell death protein-1 (anti-PD-1) therapy in a human papilloma virus-positive (HPV+) tumor mouse model.Methods Immune cell activation in response to CMP-001±anti-Qβ was performed using co-cultures of peripheral blood mononuclear cells and HPV+/HPV- HNSCC cells and then analyzed by flow cytometry. In situ vaccination with CMP-001 alone and in combination with anti-PD-1 was investigated in C57BL/6 mice-bearing mEERL HNSCC tumors and analyzed for anti-Qβ development, antitumor response, survival and immune cell recruitment. The role of antitumor immune response due to CMP-001+anti-PD-1 treatment was investigated by the depletion of natural killer (NK), CD4+ T, and CD8+ T cells.Results Results showed that the activity of CMP-001 on immune cell (pDCs, monocytes, CD4+/CD8+ T cells and NK cells) activation depends on the presence of anti-Qβ. A 2-week ‘priming’ period after subcutaneous administration of CMP-001 was required for robust anti-Qβ development in mice. In situ vaccination of CMP-001 was superior to unencapsulated G10 CpG-A ODN at suppressing both injected and uninjected (distant) tumors. In situ vaccination of CMP-001 in combination with anti-PD-1 therapy induced durable tumor regression at injected and distant tumors and significantly prolonged mouse survival compared with anti-PD-1 therapy alone. The antitumor effect of CMP-001+anti-PD-1 was accompanied by increased interferon gamma (IFNγ)+ CD4+/CD8+ T cells compared with control-treated mice. The therapeutic and abscopal effect of CMP-001+ anti-PD-1 therapy was completely abrogated by CD8+ T cell depletion.Conclusions These results demonstrate that in situ vaccination with CMP-001 can induce both local and abscopal antitumor immune responses. Additionally, the antitumor efficacy of CMP-001 combined with α-PD-1 therapy warrants further study as a novel immunotherapeutic strategy for the treatment of HNSCC.

Background Obesity is a major risk factor for renal cancer, yet our understanding of its effects on antitumor immunity and immunotherapy outcomes remains incomplete. Deciphering these associations is critical, given the growing clinical use of immune checkpoint inhibitors for metastatic disease and mounting evidence for an obesity paradox in the context of cancer immunotherapies, wherein obese patients with cancer have improved outcomes.Methods We investigated associations between host obesity and anti-programmed cell death (PD-1)-based outcomes in both renal cell carcinoma (RCC) subjects and orthotopic murine renal tumors. Overall survival (OS) and progression-free survival (PFS) were determined for advanced RCC subjects receiving standard of care anti-PD-1 who had ≥6 months of follow-up from treatment initiation (n=73). Renal tumor tissues were collected from treatment-naive subjects categorized as obese (body mass index, ‘BMI’ ≥30 kg/m2) or non-obese (BMI

Using a three-dimensional numerical simulation, the dependencies of the electron cloud (virtual cathode) on the distance between the coils in the Polywell fusion reactor were examined. In the Polywell, the role of a stable and energetic virtual cathode is crucial for fusion. It is shown that by increasing the spacing coils, the electron confinement time increases initially and then remains constant. Using the simulation results, an optimum range for the spacing of coils was suggested, which leads to a longer and more effective confinement. The results obtained can be used to design future devices in order to have a more effective virtual cathode.

Background and aim: The effect of different milk consumption patterns in infants on the formation of dental occlusion is one of the issues under discussion. Due to the lack of specific results in this regard, the purpose of this study was to determine the occlusion of deciduous teeth in breastfed and bottle-fed infants. Materials and methods: This is a descriptive-analytical study on 316 children aged 4-5 years old. This study was based on clinical examination and filling questionnaires. The examination was done at kindergartens and in sitting position using a dental mirror and with exposure to natural light. The occlusal relationship, overjet, overbite, and crossbite were examined through direct observation in the centric occlusion. The questionnaires were completed by the mothers. The basis of this study was the responses that mothers gave in the questionnaire based on the type and duration of breastfeeding or bottle-feeding. The collected data were statistically analyzed by Chi-square test. Significance was considered at the level of P

Background: Hyper-Immunoglobulin M (IgM) syndrome (HIGM) is a rare primary immunodeficiency in which defective B-cell isotype switching results in a phenotype characterized by elevated or normal serum IgM levels and low levels of other Ig classes, leading to an increased susceptibility to infection, neutropenia, autoimmune disorders, and malignancies. In this disease, a mutation occurs in the CD40 gene, leading to combined B-cell and T-cell immunodeficiencies. The oral manifestations include wound-like lesions, oral candidiasis, gingivitis, periodontitis, and enamel defects. Theoretically, systemic conditions affecting ameloblastic activity during enamel mineralization, such as abnormal oxygen levels resulting from hypoventilation in various respiratory diseases, result in enamel defects. Case presentation: We report a 10-year-old male with hyper-IgM immunodeficiency. The patient had suffered from frequent infections, respiratory problems, and bronchopneumonia from the age of 2 years. At 4 years of age, type 1 diabetes mellitus was diagnosed. During dental examinations, enamel defects were found in seven permanent teeth. Conclusion: A meticulous dental evaluation of children with systemic diseases is mandatory in order to discover possible developmental dental defects and to plan early interventions.

Vidutolimod, also known as CMP-001, is a virus-like particle composed of the Qβ bacteriophage coat protein encasing a TLR9 agonist. Vidutolimod injected intratumorally is showing promise in early phase clinical trials based on its ability to alter the tumor microenvironment and induce an anti-tumor immune response. We previously demonstrated that the in vivo efficacy of vidutolimod is dependent on the presence of anti-Qβ antibodies that enhance opsonization and uptake of vidutolimod by TLR9-expressing plasmacytoid dendritic cells (pDCs). Here, we evaluated the effect of immune complexes, including anti-Qβ-coated vidutolimod, on induction of Type 1 Interferon production by peripheral blood mononuclear cells in response to vidutolimod and soluble TLR9 agonists. Immune complexes, including but not limited to anti-Qβ-coated vidutolimod, indirectly suppressed TLR9-mediated Type 1 Interferon production by pDCs in a monocyte-dependent manner. These findings indicate that anti-Qβ-coated vidutolimod has effects in addition to those mediated by TLR9 that could have important clinical implications for understanding the mechanism of action of this exciting new approach to in situ immunization and cancer immunotherapy.

Background and aim: First permanent molars with poor prognosis may be candidate for timely extraction and replacement by second permanent molars. The presence of third molars should be considered in this treatment plan. The aim of the present study was to evaluate the critical developmental stages of permanent second molar and the status of third molar bud in an Iranian population. Materials and methods: Four hundred panoramic radiographs of 7 to 11-year-old children were evaluated in this descriptive study. Data were collected from patients’ files and through phone interview with parents. The stage of tooth development in each age group was determined according to the modified Demirjian method. Statistical analysis was performed using Wilcoxon signed Rank test and Chi-Square test. Results: Stage E (beginning of root formation) in permanent second molars had the highest prevalence among 8 and 9-year-olds in the mandible, and among 9 and 10-year-olds in the maxilla. The predominant stage of development in third molar buds was stage 0 (no evidence of bud formation) in the corresponding age groups. There was no significant difference between boys and girls in terms of developmental stages. Mandibular second and third molars were more advanced than maxillary molars in terms of development, with no gender predilection. Conclusion: The beginning of calcification in the furcation area of permanent second molar (Stage E, between 8 to 10 years of age) is the most proper stage to coincide with first molar extraction. However, during this time period, the signs of third molar bud formation are not detectable in many individuals, especially in the maxillary arch.

Bullfrog oil, an animal oil extracted from the adipose tissue of Rana catesbeiana Shaw, showed promising cytotoxic activity against melanoma cells and, therefore, has the potential to become a pharmaceutical active compound. However, there is a lack of information regarding the pathways involved in its pharmacological activity. Thus, the aim of this study was to investigate and elucidate the cytotoxic effect of this oil against A2058 human melanoma cells. The cytotoxic potential was evaluated by the MTT assay, the cell cycle analysis and the cell death assay. In addition, the apoptotic potential was investigated by (i) the DNA fragmentation using propidium iodide staining analysis, (ii) the evaluation of mitochondrial membrane potential and (iii) the determination of intracellular Reactive Oxygen Species (ROS) level. The results showed that the bullfrog oil was able to promote a time-dependent cytotoxic effect, decreasing cell viability to 38% after 72 h of treatment without affecting the cell cycle. Additionally, the bullfrog oil induced the apoptosis in A2058 cells, increasing up to 50 ± 13% of the intracellular ROS level, maintaining the DNA integrity and promoting an approximate decrease of 35 ± 5% in the mitochondrial membrane potential. It can be concluded that the in vitro cytotoxic effect of the bullfrog oil in A2058 human melanoma cells is mediated by oxidative stress that induces mitochondrial dysfunction, triggering the apoptosis. These unprecedented results highlight the pharmacological potential of bullfrog oil and provide important information to support studies on the development of new pharmaceutical products for complementary and alternative treatments for melanoma.