The BET degrader ZBC260 suppresses stemness and tumorigenesis and promotes differentiation in triple-negative breast cancer by disrupting inflammatory signaling.

BACKGROUND: Breast cancer stem cells (BCSCs) are resistant to standard therapies, facilitate tumor dissemination, and contribute to relapse and progression. Super-enhancers are regulators of stemness, and BET proteins, which are critical for super-enhancer function, are a potential therapeutic target. Here, we investigated the effects of BET proteins on the regulation of breast cancer stemness using the pan-BET degrader ZBC260. METHODS: We evaluated the effect of ZBC260 on CSCs in TNBC cell lines. We assessed the effect of ZBC260 on cellular viability and tumor growth and measured its effects on cancer stemness. We used RNA sequencing and stemness index to determine the global transcriptomic changes in CSCs and bulk cells and further validated our findings by qPCR, western blot, and ELISA. RESULTS: ZBC260 potently inhibited TNBC growth both in vitro and in vivo. ZBC260 reduced stemness as measured by cell surface marker expression, ALDH activity, tumorsphere number, and stemness index while increasing differentiated cells. GSEA analysis indicated preferential downregulation of stemness-associated and inflammatory genes by ZBC260 in ALDH+ CSCs. CONCLUSIONS: The BET degrader ZBC260 is an efficient degrader of BET proteins that suppresses tumor progression and decreases CSCs through the downregulation of inflammatory genes and pathways. Our findings support the further development of BET degraders alone and in combination with other therapeutics as CSC targeting agents.

Multiple Infection and Human Immunodeficiency Virus Superinfection Among Persons who Inject Drugs in Indonesia and Ukraine.

BACKGROUND: The HIV Prevention Trials Network (HPTN) 074 study evaluated an integrated human immunodeficiency virus (HIV) treatment and prevention strategy among persons who inject drugs (PWID) in Indonesia, Ukraine, and Vietnam. We previously detected multiple HIV infection in 3 of 7 (43%) of seroconverters with 3-8 HIV strains per person. In this report, we analyzed multiple HIV infection and HIV superinfection (SI) in the HPTN 074 cohort. METHODS: We analyzed samples from 70 participants in Indonesia and Ukraine who had viral load >400 copies/mL at enrollment and the final study visit (median follow-up, 2.5 years). HIV was characterized with Sanger sequencing, next-generation sequencing, and phylogenetic analysis. Additional methods were used to characterize a rare case of triple-variant SI. RESULTS: At enrollment, multiple infection was detected in only 3 of 58 (5.2%) participants with env sequence data. SI was detected in only 1 of 70 participants over 172.3 person-years of follow-up (SI incidence, 0.58/100 person-years [95% confidence interval, .015-3.2]). The SI case involved acquisition of 3 HIV strains with rapid selection of a strain with a single pol region cluster. CONCLUSIONS: These data from a large cohort of PWID suggest that intrahost viral selection and other factors may lead to underestimation of the frequency of multiple HIV infection and SI events.

BACILLARY LAYER DETACHMENT IN ACUTE VOGT-KOYANAGI-HARADA DISEASE: A Novel Swept-Source Optical Coherence Tomography Analysis.

PURPOSE: To report the frequency, optical coherence tomography (OCT) findings, and visual and anatomic outcomes of patients with acute Vogt-Koyanagi-Harada disease presenting with the bacillary layer detachment (BLD) (intraretinal split at the photoreceptor inner segment myoid). METHODS: This was a retrospective analysis of a consecutive series of patients with Vogt-Koyanagi-Harada disease having a minimum follow-up of 6 months. All patients had swept-source OCT, fluorescein angiography, and indocyanine green angiography performed at baseline. The characteristics of serial swept-source OCT were recorded and analyzed. RESULTS: Sixty-two subjects (42 women; age: 34.2 ± 12 years) with Vogt-Koyanagi-Harada disease were included. 118 eyes (95.2%) had serous retinal detachment at presentation. 112 eyes (94.9%) showed the BLD at baseline. In 8 of 112 (7.1%) eyes with the BLD, the external limiting membrane at the anterior aspect of the BLD showed focal discontinuity. The interdigitation zone at the base of the BLD showed discontinuity in 53 of 112 (47.3%) eyes with the BLD. The ellipsoid zone could not be identified as a separate hyperreflective line at the base of the BLD in 102 of 112 eyes (91.1%). Bacillary layer detachments resolved within 3.4 ± 1.3 days after intravenous methylprednisolone therapy with improvement in the best-corrected visual acuity from 0.96 to 0.4 logarithm of the minimum angle of resolution (20/184 Snellen's equivalent) (P < 0.001). Resolution of serous retinal detachment was observed after 5.9 ± 2.6 days. CONCLUSION: In eyes with acute Vogt-Koyanagi-Harada disease, the BLD is a common finding and represents a split in the photoreceptor layer at the inner segment myoid and can be differentiated from serous retinal detachment using swept-source OCT. In addition, resolution of the BLD and photoreceptor recovery can be evaluated using serial swept-source OCT.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics.

Dioxins are a group of highly toxic environmental persistent organic pollutants, which are lipophilic in nature. 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic representative of this class. TCDD causes several human health effects like endocrine disruption, carcinogenesis and reproductive toxicity mediated by aryl-hydrocarbon receptor. Current detection methods of dioxins like gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry etc. are costly and time consuming. Therefore, the present study aims to develop a relatively faster and cheaper technique called reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to detect dioxins. Cultured granulosa cells used as a model system were treated with different doses (5, 10 and 15 pg/mL) of aryl hydrocarbon receptor (AhR)responsive xenobiotic, TCDD, in accordance with maximum residue limit values. Cells were treated for 6, 12 and 24 h, respectively to study the gene expression of TCDD receptor called AhR and AhR responsive genes, CYP1A1 and CYP1B1, in a dose and time dependent manner. All targeted genes expression significantly increased after 6 and 12 h by 1.3-8 folds. For the development of RT-LAMP assay, CYP1A1 gene was used with 6 h TCDD treatment. RT-LAMP assay was standardized with optimal color change at 30 min using 50 ng of cellular RNA. In all the cases, we could distinguish RT-LAMP-positive condition from one sample to another sample due to intensity of color. The method was also validated by spectrometric method. In conclusion, the developed method will be used to screen AhR receptor responsive xenobiotics by observing the color change in RT-LAMP assay like dioxin used in the present study.

Enhancement in brain uptake of vitamin D3 nanoemulsion for treatment of cerebral ischemia: formulation, gamma scintigraphy and efficacy study in transient middle cerebral artery occlusion rat models.

AIM: For the treatment of cerebral ischaemia, vitamin-D3 loaded nanoemulsions were developed. METHOD: Tween 20 and polyethylene glycol were chosen as surfactant/co-surfactant, while oleic acid as the oil phase. The formulation was characterised for various in-vitro parameters. Targeting efficiency was investigated through radiometry, gamma scintigraphy and efficacy was studied in transient middle cerebral artery occlusion (MCAo) rat model. RESULT: Vitamin D3-nanoemulsion showed a mean size range of 49.29 ± 10.28 nm with polydispersity index 0.17 ± 0.04 and zeta potential 13.77 mV. The formulation was found stable during thermodynamic stability study and permeated within 180 min through sheep nasal mucosa (permeation coefficient 7.873 ± 0.884 cm/h). Gamma scintigraphy and radiometry assay confirmed better percentage deposition (2.53 ± 0.17%) of 99mTc-vitamin D3-nanoemulsion through nasal route compared to IV administered 99mTc-vitamin D3 solution (0.79 ± 0.03%). Magnetic Resonance Imaging (MRI) of the ischaemic model confirmed better efficacy of vitamin D3-nanoemulsion. CONCLUSION: This work demonstrated better permeation, deposition, and efficacy of vitaminD3-nanoemulsion through the intranasal route.

Adherence capability and safety assessment of an indigenous probiotic strain Lactobacillus rhamnosus MTCC-5897.

With the growing interest in probiotic microorganisms based on their well established immense health benefits, the present investigation was aimed to assess the adhesion potential and safety of probiotic Lactobacillus rhamnosus MTCC- 5897 (LR) before it can be put into a probiotic formulations. L. rhamnosus showed an adhesion index of 166.7 ±â€¯11, which was further confirmed by scanning electron microscopy and relative expression of mucus binding protein (Mub) and mucus adhesion promoting protein (Map-A) genes. In vitro safety assessment by tetrazolium dye reduction, neutral red and lactate dehydrogenase (LDH) release assays revealed unchanged metabolic activity of Caco-2 cells even when incubated with L. rhamnosus ranged between 106-1010 cfu/mL for 24 h. Similarly, a moderate increase in bile salt hydrolase (bsh) expression (6.84 ±â€¯0.73 and 3.42 ±â€¯0.39 folds in 1% and 3% bile medium respectively) further proved its safety towards normal lipid digestion and absorption. Moreover, L. rhamnosus feeding to mice (107, 109, 1011 and 1013 cfu/animal/d) repetitively for 28 days revealed no adverse effects on parameters of general animal health status including body weight, organ indices, plasma glucose, liver malondialdehyde (MDA), serum aspartate amino transaminase (AST), cholesterol, triglycerides, high-density lipoprotein (HDL). Similarly, significant (p ≤ 0.05) reduced activities of serum alanine amino transaminase (ALT) and LDH on continuous probiotic feeding were also indicative of normal liver/kidney functions as they were in normal range for mice. Further, insignificant changes in macrophage chemoattractant protein (MCP-1) in intestinal fluid irrespective of bacterial dose fed along with significant reduction (p ≤ 0.05) of tumor necrosis factor-α (TNF-α) at much higher dose (1013 cfu/animal/d) also confirmed safe response of probotic L.rhamnosus against inflammation. To conclude, the results obtained under in vitro and in vivo studies has established the Lactobacillus rhamnosus as safe and non-toxic to weaning mice as well as human epithelial cells and thus may be used as a safe food additive.

Three-dimensional finite element analysis of the stress distribution in the endodontically treated maxillary central incisor by glass fiber post and dentin post.

INTRODUCTION: From the point of dental practice, the restoration of endodontically treated teeth has become an important aspect as it involves a range of treatment options of variable complexity. Restoring teeth with insufficient coronal tooth structure, it is always indicated to use the post to retain a core for definitive restoration. Fiber post has a modulus of elasticity in analogs to dentin structure, thus reducing the stress areas at the dowel dentin interface. However, the only material that can substantiate all these properties can be none other than dentin itself. MATERIALS AND METHODOLOGY: Three-dimensional (3D) models of the maxillary central incisor were developed incorporating all the nonlinearities. Continuum 3D elements were used in three dimensions. Maxillary central incisor was laser scanned, duplicated with the help of reverse engineering into STL format, and it was converted into 3D model for finite element analysis (FEA). For the model, fixed boundary conditions were applied at the outer bone, while 100 N static vertical occlusal loads were prescribed at 135° on the loading component of the simulated tooth. The stress distribution was evaluated using dentin and fiber post with prescribed materials, loading and boundary conditions in endontically treated teeth by 3D FEA. RESULTS: The analysis for von Misses stress for dentin post showed that the stress in the dentin post at the cervical area was 127 MPa. The displacement in the dentin post was <0.025 mm. Von Misses stress for the fiber post at the cervical area was approximately 182 MPa and the displacement was <0.035 mm. CONCLUSION: The FEA results showed that the stress in the cervical area of the dentin was more for fiber post when compared to dentin post, and maximum displacement values were less for dentin post in comparison to fiber post.

Long-Term Efficacy of Selective Laser Trabeculoplasty in Primary Angle-Closure Disease After Laser Peripheral Iridotomy.

PURPOSE: To evaluate the long-term efficacy of selective laser trabeculoplasty (SLT) in eyes with primary angle-closure (PAC) and primary angle-closure glaucoma (PACG) following a laser peripheral iridotomy (LPI). METHOD: In this prospective cross-sectional study, 45 eyes of 34 patients with PAC/PACG diagnosis, uncontrolled intraocular pressure (IOP), and visible pigmented trabecular-meshwork (TM) at least 180° on gonioscopy following a LPI were recruited. Following a detailed baseline ophthalmic evaluation, all eligible eyes underwent SLT, and the patients were examined on day1, at 1 week, 1-, 3-, and 6-months, and 1-, 2-, 3-, 4-, and 5-year subsequently. The main outcomes measured were IOP, number of IOP-lowering agents, and complications. RESULTS: The mean age of the cohort was 57.80 ± 6.44 years, the male-female ratio was 8:26, and 17 eyes were PACG, and 28 were PAC. The baseline IOP was 23.81 ± 1.78 mm Hg, and was significantly declined at all follow-ups (p < .0001). The cumulative probability of overall success was 91% and 84% at 2-, and 5-year, respectively. At 5-year SLT provided drug-freedom in 80% of PAC and 23% of PACG eyes. Six eyes had IOP spike at 1-week and two patients underwent repeat SLT after 1-year. No other complications, such as pain/discomfort, inflammation, an increase in peripheral anterior synechiae and cystoid-macular-edema, were noted. CONCLUSIONS: SLT appears a safe and cost-effective procedure in PAC/mild- moderate PACG eyes with uncontrolled IOP after laser iridotomy. The long-term effectiveness of SLT as adjuvant treatment was good, but need large sized randomized studies for more validation.

Natural Killer Cell Regulation of Breast Cancer Stem Cells Mediates Metastatic Dormancy.

Patients with breast cancer with estrogen receptor-positive tumors face a constant risk of disease recurrence for the remainder of their lives. Dormant tumor cells residing in tissues such as the bone marrow may generate clinically significant metastases many years after initial diagnosis. Previous studies suggest that dormant cancer cells display "stem-like" properties (cancer stem cell, CSC), which may be regulated by the immune system. To elucidate the role of the immune system in controlling dormancy and its escape, we studied dormancy in immunocompetent, syngeneic mouse breast cancer models. Three mouse breast cancer cell lines, PyMT, Met1, and D2.0R, contained CSCs that displayed short- and long-term metastatic dormancy in vivo, which was dependent on the host immune system. Each model was regulated by different components of the immune system. Natural killer (NK) cells were key for the metastatic dormancy phenotype in D2.0R cells. Quiescent D2.0R CSCs were resistant to NK cell cytotoxicity, whereas proliferative CSCs were sensitive. Resistance to NK cell cytotoxicity was mediated, in part, by the expression of BACH1 and SOX2 transcription factors. Expression of STING and STING targets was decreased in quiescent CSCs, and the STING agonist MSA-2 enhanced NK cell killing. Collectively, these findings demonstrate the role of immune regulation of breast tumor dormancy and highlight the importance of utilizing immunocompetent models to study this phenomenon. Significance: The immune system controls disseminated breast cancer cells during disease latency, highlighting the need to utilize immunocompetent models to identify strategies for targeting dormant cancer cells and reducing metastatic recurrence. See related commentary by Cackowski and Korkaya, p. 3319.

Exploring Computational Advancements in ADME: Essential Insights for Drug Disposition.

The physicochemical properties of the physiological makeup and the chemical componentof the system make this challenging throughout strenuous procedure. The current review concentrated on in silico modelling of drug disposition, involving absorption process, distribution process, and excretion process and includes thorough knowledge of various database expeditions, the development of a pharmacophore model, molecular docking studies, homology modelling supported sequence similarity and quantitative structure-activity relationships (QSAR)/ quantitative structure-property relationships (QSPR) evaluation along with all information about drug movement and related computational tools for understanding potential chemical and pathophysiological changes. The primary development in ADMET modeling in current times has been the clarification of the function and effective modeling of various transporters. In ADMET modelling, there is still work to be done on including the impact of these transporters into existing models. The present state of modelling different elements of drug disposal at the systemic level will then be discussed, along with recent developments in modelling a wide range of active transporters and their effects on drug pharmacokinetic profiles. A more thorough knowledge of the underlying processes governing different aspects of drug disposition should also lead to an increase in mechanism-based modelling methods that are simple to grasp and put into practice. These developments will hasten the transition of model construction from computational to experimental scientists.

An overview of immediate root analogue zirconia implants.

Owing to its excellent biomechanical characteristics, biocompatibility, and bright tooth-like color, zirconia has the potential to become a substitute for titanium as dental implant material and to be successfully used as root-analogue implants by reproducing the contours of the extracted tooth. This article presents an overview of the technique of using root analogue zirconia dental implants as an immediate implantation material. These implants are replicas of the extracted tooth and therefore truly anatomically correct and socket friendly.

A photonic artificial synapse with a reversible multifaceted photochromic compound.

Modern computational technology based on the von Neumann architecture physically partitions memory and the central processing unit, resulting in fundamental speed limitations and high energy consumption. On the other hand, the human brain is an extraordinary multifunctional organ composed of more than a billion neurons capable of simultaneously thinking, processing, and storing information. Neurons are interconnected with synapses that control information flow from pre-synaptic-to-post-synaptic neurons. Therefore, emulating synaptic functionalities and developing neuromorphic computational architecture has recently attracted much interest. Due to their high-speed, large bandwidth, and no interconnect-related power loss, photonic (all-optical) synapses can overcome the existing hurdles with electronic synapses. Here, we show an artificial photonic synapse by utilizing the well-established reversible, high-contrast photochromic organic compound, spiropyran, stimulated by optical pulses. Optical transmission of spiropyran significantly changes during spiropyran-merocyanine isomerization driven by UV-visible optical pulses. Such changes are equivalent to the biological synapses' inhibitory and excitatory synaptic actions. The slow relaxation to the initial state is considered as synaptic plasticity responsible for learning and memory formation. Short-term memory (STM), long-term memory (LTM), and transition from the STM to the LTM are demonstrated in all-optical synapses by modulating the stimuli's strength. The solvatochromic properties of spiropyran are further utilized to augment memory in synapses. Our work shows that photochromic organic compounds are excellent hosts for artificial photonic synapses and can be implemented in neuromorphic applications.

Assessment of Quality of Life after Hysterectomy using European Quality of Life Five Dimension Scale (EQ5D).

In several regions throughout the globe, caesarean sections constitute the most common nonobstetric surgery, followed by hysterectomy, which is the surgical excision of the uterus. While it is not the only solution for reproductive organ issues, it is the most effective technique to treat many illnesses over the long term. The uterus is a very critical reproductive organ for all age groups as this is not only essential for giving birth but also for hormonal-related physiology in women's life. The quality of life is impacted by a number of hysterectomy-related effects on females. Physical, psychological, environmental, and social relations are some of these impacts. All EuroQol five-dimensions (EQ5D) subscales significantly improved, as per the research 's findings. Preoperative psychosocial status, perioperative pain, indication of hysterectomy, complications occur during surgery, and mode of hysterectomy postoperative infection had been discovered as determinants of quality of life outcome following hysterectomy. In most of the subjects we noticed small, however, noticable improvements in all component of EQ5D Scale. The strengths of EQ5D questionnaire lie in its simplicity and moreover it is available in several languages.

Natural killer cell regulation of breast cancer stem cells mediates metastatic dormancy.

Breast cancer patients with estrogen receptor positive tumors face a constant risk of disease recurrence for the remainder of their lives. Dormant tumor cells residing in tissues such as the bone marrow may generate clinically significant metastases many years after initial diagnosis. Previous studies suggest that dormant cells display "stem like" properties (CSCs), which may be regulated by the immune system. Although many studies have examined tumor cell intrinsic characteristics of dormancy, the role of the immune system in controlling dormancy and its escape is not well understood. This scientific gap is due, in part, to a lack of immunocompetent mouse models of breast cancer dormancy with many studies involving human xenografts in immunodeficient mice. To overcome this limitation, we studied dormancy in immunocompetent, syngeneic mouse breast cancer models. We find that PyMT, Met-1 and D2.0R cell lines contain CSCs that display both short- and long-term metastatic dormancy in vivo, which is dependent on the host immune system. Natural killer cells were key for the metastatic dormancy phenotype observed for D2.0R and the role of NK cells in regulating CSCs was further investigated.Quiescent D2.0R CSC are resistant to NK cytotoxicity, while proliferative D2.0R CSC were sensitive to NK cytotoxicity both in vitro and in vivo. This resistance was mediated, in part, by the expression of Bach1 and Sox2 transcription factors. NK killing was enhanced by the STING agonist MSA-2. Collectively, our findings demonstrate the important role of immune regulation of breast tumor dormancy and highlight the importance of utilizing immunocompetent models to study this phenomenon.

Predictors of participant retention in a community-based HIV prevention cohort: perspectives from the HPTN 071 (PopART) study.

INTRODUCTION: In 2021, there were 38.4 million people living with HIV (PLHIV) globally, of which 20.6 million (54%) were living in Eastern and Southern Africa. Longitudinal studies, inclusive of community randomized trials (CRTs), provide critical evidence to guide a broad range of health care interventions including HIV prevention. In this study, we have used an individual-level cohort study design to evaluate the association between sex and other baseline characteristics and participant retention in the HPTN 071 (PopART) trial in Zambia and South Africa. METHODS: HPTN 071 (PopART) was a community randomized trial (CRT) conducted from 2013 to 2018, in 21 communities. The primary outcome was measured in a randomly selected population cohort (PC), followed up over 3 to 4 years at annual rounds. PC retention was defined as completion of an annual follow-up questionnaire. Baseline characteristics were described by study arm and Poisson regression analyses used to measure the association between baseline factors and retention. In addition, we present a description of researcher-documented reasons for study withdrawal by PC participants. RESULTS: Of the 38,474 participants enrolled during the first round of the trial (PC0), most were women (27,139, 71%) and 73% completed at least one follow-up visit. Retention was lower in men (adj RR: 0.90; 95% CI: 0.88, 0.91) and higher among older participants (adj RR: 1.23; 95% CI 1.20, 1.26) when comparing ages 35-44 to 18-24 years. Retention was higher among individuals with high socioeconomic status (SES) (adj RR 1.16; 95% CI 1.14, 1.19) and medium SES (adj RR 1.12; 95% CI 1.09, 1.14) compared to low SES. The most common reasons for study withdrawal were study refusal (23%) and relocation outside the CRT catchment area (66%). CONCLUSION: Despite challenges, satisfactory retention outcomes were achieved in PopART with limited variability across study arms. In keeping with other studies, younger age, male sex, and lower SES were associated with lower levels of retention. Relocation outside of catchment area was the most common reason for non-retention in this CRT.

Consequences of Arsenic Contamination on Plants and Mycoremediation-Mediated Arsenic Stress Tolerance for Sustainable Agriculture.

Arsenic contamination in water and soil is becoming a severe problem. It is toxic to the environment and human health. It is usually found in small quantities in rock, soil, air, and water which increase due to natural and anthropogenic activities. Arsenic exposure leads to several diseases such as vascular disease, including stroke, ischemic heart disease, and peripheral vascular disease, and also increases the risk of liver, lungs, kidneys, and bladder tumors. Arsenic leads to oxidative stress that causes an imbalance in the redox system. Mycoremediation approaches can potentially reduce the As level near the contaminated sites and are procuring popularity as being eco-friendly and cost-effective. Many fungi have specific metal-binding metallothionein proteins, which are used for immobilizing the As concentration from the soil, thereby removing the accumulated As in crops. Some fungi also have other mechanisms to reduce the As contamination, such as biosynthesis of glutathione, cell surface precipitation, bioaugmentation, biostimulation, biosorption, bioaccumulation, biovolatilization, methylation, and chelation of As. Arsenic-resistant fungi and recombinant yeast have a significant potential for better elimination of As from contaminated areas. This review discusses the relationship between As exposure, oxidative stress, and signaling pathways. We also explain how to overcome the detrimental effects of As contamination through mycoremediation, unraveling the mechanism of As-induced toxicity.

FAST-IT: Find A Simple Test - In TIA (transient ischaemic attack): a prospective cohort study to develop a multivariable prediction model for diagnosis of TIA through proteomic discovery and candidate lipid mass spectrometry, neuroimaging and machine learning-study protocol.

INTRODUCTION: Transient ischaemic attack (TIA) may be a warning sign of stroke and difficult to differentiate from minor stroke and TIA-mimics. Urgent evaluation and diagnosis is important as treating TIA early can prevent subsequent strokes. Recent improvements in mass spectrometer technology allow quantification of hundreds of plasma proteins and lipids, yielding large datasets that would benefit from different approaches including machine learning. Using plasma protein, lipid and radiological biomarkers, our study will develop predictive algorithms to distinguish TIA from minor stroke (positive control) and TIA-mimics (negative control). Analysis including machine learning employs more sophisticated modelling, allowing non-linear interactions, adapting to datasets and enabling development of multiple specialised test-panels for identification and differentiation. METHODS AND ANALYSIS: Patients attending the Emergency Department, Stroke Ward or TIA Clinic at the Royal Adelaide Hospital with TIA, minor stroke or TIA-like symptoms will be recruited consecutively by staff-alert for this prospective cohort study. Advanced neuroimaging will be performed for each participant, with images assessed independently by up to three expert neurologists. Venous blood samples will be collected within 48 hours of symptom onset. Plasma proteomic and lipid analysis will use advanced mass spectrometry (MS) techniques. Principal component analysis and hierarchical cluster analysis will be performed using MS software. Output files will be analysed for relative biomarker quantitative differences between the three groups. Differences will be assessed by linear regression, one-way analysis of variance, Kruskal-Wallis H-test, χ2 test or Fisher's exact test. Machine learning methods will also be applied including deep learning using neural networks. ETHICS AND DISSEMINATION: Patients will provide written informed consent to participate in this grant-funded study. The Central Adelaide Local Health Network Human Research Ethics Committee approved this study (HREC/18/CALHN/384; R20180618). Findings will be disseminated through peer-reviewed publication and conferences; data will be managed according to our Data Management Plan (DMP2020-00062).

Organochlorine pesticide dieldrin upregulate proximal promoter (PII) driven CYP19A1 gene expression and increases estrogen production in granulosa cells.

Organochlorine pesticides are highly persistent environmental pollutants, generally shown to act through estrogen receptor alpha and alter estrogen biosynthesis. However, the molecular mechanism of regulation of estrogen biosynthesis by these pesticides is not clear. Estrogen is main female fertility hormone regulated by rate-limiting enzyme aromatase. It is encoded by the CYP19A1 gene, which is expressed using specific promoters. In the present study, the attempt has been made to elucidate the effect of dieldrin on the promoter-specific CYP19A1 gene expression and estrogen hormone production in buffalo granulosa cells. The buffalo granulosa cells were cultured and treated with dieldrin in a dose (100,150 and 200 ng/mL) and time (6, 12, and 24 h) dependent manner, followed by analysis of CYP19A1, promoter-specific CYP19A1 transcript expression, and estrogen production. Results showed that dieldrin significantly increased the expression of the CYP19A1 gene after 6 and 12 h while its expression was decreased after 24 h. To understand the upregulation of CYP19A1 gene, promoters' specific CYP19A1 transcript analysis was done. The finding showed that dieldrin significantly increased the proximal promoter specific CYP19A1 transcript while there was no effect on distal promoter specific CYP19A1 transcripts. This specific-promoter activity was quantified by chromatin immunoprecipitation assay (ChIP). Results confirmed the involvement of the proximal promoter in the overexpression of CYP19A1 gene. Furthermore, a significant increase in estradiol-17ß level was also observed. Overall, the present study demonstrated the stimulatory effect of dieldrin on the CYP19A1 gene and will help to understand the toxicological role of dieldrin on the reproductive system.