A high-resolution imaging approach to investigate chromatin architecture in complex tissues.

We present ChromATin, a quantitative high-resolution imaging approach for investigating chromatin organization in complex tissues. This method combines analysis of epigenetic modifications by immunostaining, localization of specific DNA sequences by FISH, and high-resolution segregation of nuclear compartments using array tomography (AT) imaging. We then apply this approach to examine how the genome is organized in the mammalian brain using female Rett syndrome mice, which are a mosaic of normal and Mecp2-null cells. Side-by-side comparisons within the same field reveal distinct heterochromatin territories in wild-type neurons that are altered in Mecp2-null nuclei. Mutant neurons exhibit increased chromatin compaction and a striking redistribution of the H4K20me3 histone modification into pericentromeric heterochromatin, a territory occupied normally by MeCP2. These events are not observed in every neuronal cell type, highlighting ChromATin as a powerful in situ method for examining cell-type-specific differences in chromatin architecture in complex tissues.

The Genome-Wide Binding Profile for Human RE1 Silencing Transcription Factor Unveils a Unique Genetic Circuitry in Hippocampus.

Early studies in mouse neurodevelopment led to the discovery of the RE1 Silencing Transcription Factor (REST) and its role as a master repressor of neuronal gene expression. Recently, REST was reported to also repress neuronal genes in the human adult brain. These genes were found to be involved in pro-apoptotic pathways; and their repression, associated with increased REST levels during aging, were found to be neuroprotective and conserved across species. However, direct genome-wide REST binding profiles for REST in adult brain have not been identified for any species. Here, we apply this approach to mouse and human hippocampus. We find an expansion of REST binding sites in the human hippocampus that are lacking in both mouse hippocampus and other human non-neuronal cell types. The unique human REST binding sites are associated with genes involved in innate immunity processes and inflammation signaling which, on the basis of histology and recent public transcriptomic analyses, suggest that these new target genes are repressed in glia. We propose that the increases in REST expression in mid-adulthood presage the beginning of brain aging, and that human REST function has evolved to protect the longevity and function of both neurons and glia in human brain.SIGNIFICANCE STATEMENT The RE1 Silencing Transcription Factor (REST) repressor has served historically as a model for gene regulation during mouse neurogenesis. Recent studies of REST have also suggested a conserved role for REST repressor function across lower species during aging. However, direct genome-wide studies for REST have been lacking for human brain. Here, we perform the first genome-wide analysis of REST binding in both human and mouse hippocampus. The majority of REST-occupied genes in human hippocampus are distinct from those in mouse. Further, the REST-associated genes unique to human hippocampus represent a new set related to innate immunity and inflammation, where their gene dysregulation has been implicated in aging-related neuropathology, such as Alzheimer's disease.

The TAS Test project: a prospective longitudinal validation of new online motor-cognitive tests to detect preclinical Alzheimer's disease and estimate 5-year risks of cognitive decline and dementia.

BACKGROUND: The worldwide prevalence of dementia is rapidly rising. Alzheimer's disease (AD), accounts for 70% of cases and has a 10-20-year preclinical period, when brain pathology covertly progresses before cognitive symptoms appear. The 2020 Lancet Commission estimates that 40% of dementia cases could be prevented by modifying lifestyle/medical risk factors. To optimise dementia prevention effectiveness, there is urgent need to identify individuals with preclinical AD for targeted risk reduction. Current preclinical AD tests are too invasive, specialist or costly for population-level assessments. We have developed a new online test, TAS Test, that assesses a range of motor-cognitive functions and has capacity to be delivered at significant scale. TAS Test combines two innovations: using hand movement analysis to detect preclinical AD, and computer-human interface technologies to enable robust 'self-testing' data collection. The aims are to validate TAS Test to [1] identify preclinical AD, and [2] predict risk of cognitive decline and AD dementia. METHODS: Aim 1 will be addressed through a cross-sectional study of 500 cognitively healthy older adults, who will complete TAS Test items comprising measures of motor control, processing speed, attention, visuospatial ability, memory and language. TAS Test measures will be compared to a blood-based AD biomarker, phosphorylated tau 181 (p-tau181). Aim 2 will be addressed through a 5-year prospective cohort study of 10,000 older adults. Participants will complete TAS Test annually and subtests of the Cambridge Neuropsychological Test Battery (CANTAB) biennially. 300 participants will undergo in-person clinical assessments. We will use machine learning of motor-cognitive performance on TAS Test to develop an algorithm that classifies preclinical AD risk (p-tau181-defined) and determine the precision to prospectively estimate 5-year risks of cognitive decline and AD. DISCUSSION: This study will establish the precision of TAS Test to identify preclinical AD and estimate risk of cognitive decline and AD. If accurate, TAS Test will provide a low-cost, accessible enrichment strategy to pre-screen individuals for their likelihood of AD pathology prior to more expensive tests such as blood or imaging biomarkers. This would have wide applications in public health initiatives and clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05194787 , 18 January 2022. Retrospectively registered.

Characterizing the cortical pathways underlying visual trigger induced urinary urgency incontinence by functional MRI.

AIMS: Visual triggers have long been recognized clinically to stimulate urgency urinary incontinence (UUI). Current pathophysiology recognizes the importance of cortical control over micturition but there is no standardized methodology for clinicians to study the impact of visual triggers. Our aim was to develop an imaging protocol able to characterize the brain's response to personalized visual triggers, providing a methodology for evaluation on connectivity within the brain in patients with visually triggered urinary urgency. METHODS: A magnetic resonance imaging (MRI) methodology specific for urologic use was developed. A 3T-Elition Scanner was first used to acquire static structural images. These images were then used to define approximately 200 brain regions of interest (ROI) using a validated brain atlas. Then, real-time functional MRI (fMRI) scans were conducted during natural bladder filling, where study subjects were shown randomized block sequences of visual stimuli comprised of both subject-specific trigger images and neutral images. The fMRI scan data were merged to identify key ROI underlying UUI. RESULTS: Dynamic fMRI scans were conducted in 10 subjects, 4 with trigger-induced UUI, 2 with trigger-induced urgency, and 4 with no urgency or leakage to visual triggers. No subjects with UUI history lost continence during imaging, but all four subjects reported sensations of urgency in response to their own subject-specific trigger images. The ROI identified were the periaqueductal gray, anterior cingulate gyrus, pons, and prefrontal cortex. We found increased activity in the prefrontal cortex and limbic system ROI in response to subject-specific visual triggers of UUI. CONCLUSIONS: This information provides proof of principle for further exploration of subject-specific trigger image evaluation using fMRI to explore causation in patients with UUI.

Applications of artificial intelligence to aid early detection of dementia: A scoping review on current capabilities and future directions.

BACKGROUND & OBJECTIVE: With populations aging, the number of people with dementia worldwide is expected to triple to 152 million by 2050. Seventy percent of cases are due to Alzheimer's disease (AD) pathology and there is a 10-20 year 'pre-clinical' period before significant cognitive decline occurs. We urgently need, cost effective, objective biomarkers to detect AD, and other dementias, at an early stage. Risk factor modification could prevent 40% of cases and drug trials would have greater chances of success if participants are recruited at an earlier stage. Currently, detection of dementia is largely by pen and paper cognitive tests but these are time consuming and insensitive to the pre-clinical phase. Specialist brain scans and body fluid biomarkers can detect the earliest stages of dementia but are too invasive or expensive for widespread use. With the advancement of technology, Artificial Intelligence (AI) shows promising results in assisting with detection of early-stage dementia. This scoping review aims to summarise the current capabilities of AI-aided digital biomarkers to aid in early detection of dementia, and also discusses potential future research directions. METHODS & MATERIALS: In this scoping review, we used PubMed and IEEE Xplore to identify relevant papers. The resulting records were further filtered to retrieve articles published within five years and written in English. Duplicates were removed, titles and abstracts were screened and full texts were reviewed. RESULTS: After an initial yield of 1,463 records, 1,444 records were screened after removal of duplication. A further 771 records were excluded after screening titles and abstracts, and 496 were excluded after full text review. The final yield was 177 studies. Records were grouped into different artificial intelligence based tests: (a) computerized cognitive tests (b) movement tests (c) speech, conversion, and language tests and (d) computer-assisted interpretation of brain scans. CONCLUSIONS: In general, AI techniques enhance the performance of dementia screening tests because more features can be retrieved from a single test, there are less errors due to subjective judgements and AI shifts the automation of dementia screening to a higher level. Compared with traditional cognitive tests, AI-based computerized cognitive tests improve the discrimination sensitivity by around 4% and specificity by around 3%. In terms of speech, conversation and language tests, combining both acoustic features and linguistic features achieve the best result with accuracy around 94%. Deep learning techniques applied in brain scan analysis achieves around 92% accuracy. Movement tests and setting smart environments to capture daily life behaviours are two potential future directions that may help discriminate dementia from normal aging. AI-based smart environments and multi-modal tests are promising future directions to improve detection of dementia in the earliest stages.

Anti-Mycobacterial Activity of Flavonoid and Pyrimidine Compounds.

We evaluated the anti-mycobacterial effect of a flavonoid 5,7-dihydroxy-2-(4-hydroxyphenyl) 4H-chromen-4-one (1) and two pyrimidines, 4-hydroxy-2-dimethylamino-5-nitroso-6-aminopyrimidine (2) and 2-chloro-5-n-nonylpyrimidine (3) in vitro against Mycobacterium tuberculosis (M. tuberculosis, H37Ra) and Mycobacterium avium (M. avium), using a Microplate Alamar Blue Assay (MABA). The effects of the compounds 1-3 in combination with first- and second-line anti-TB drugs isoniazid, rifampicin, cycloserine, and clarithromycin on the growth of M. tuberculosis and M. avium were also evaluated in in vitro assays. As a single agent, compounds 1 and 2 exhibited modest activity while compound 3 was the most effective against M. tuberculosis and M. avium. When compounds 1-3 were evaluated at lower than 50% of their inhibitory concentrations in a two-drug combination with isoniazid or rifampicin, they showed additive to synergistic interactions. This inhibitory effect was improved when each of the three compounds was tested together in a three-drug combination with two of the first-line anti-TB drugs. Compounds 1-3 also demonstrated strong synergistic interaction in combination with cycloserine and clarithromycin in inhibiting the growth of M. tuberculosis and M. avium, respectively. This study demonstrated that compounds 1-3 have potential to be developed as effective anti-TB agents with combined use.

Assessment of in-cabin noise of wide-body aircrafts.

The aviation industry has seen dramatic growth over the decades till the recent disruption due to the COVID-19 pandemic. Moreover, long-haul routes with a distance of more than 4000 km are common for major airlines worldwide. Therefore, aircraft cabin noise assessment is essential, especially in long-haul flights, for passenger and flight crew health wellness. In this paper, the cabin noise of five wide-body aircraft, namely Airbus A330-300ER, A350-900, A380-800, and Boeing B777-200ER and B787-900, was recorded using a calibrated in-house developed smartphone application. The sound pressure levels of in-cabin noise have been measured on two different decibel scales, namely, A-weighted [dB(A)] and C-weighted scales [dB(C)]. The sound pressure levels of Airbus A380-800 were lowest among selected models, while the in-cabin pressure level values of Airbus A350-900 were maximum. However, the difference in decibel levels between the aircraft is minimal as it is within 3 dB.

A Micro-Level Compensation-Based Cost Model for Resource Allocation in a Fog Environment.

Fog computing aims to support applications requiring low latency and high scalability by using resources at the edge level. In general, fog computing comprises several autonomous mobile or static devices that share their idle resources to run different services. The providers of these devices also need to be compensated based on their device usage. In any fog-based resource-allocation problem, both cost and performance need to be considered for generating an efficient resource-allocation plan. Estimating the cost of using fog devices prior to the resource allocation helps to minimize the cost and maximize the performance of the system. In the fog computing domain, recent research works have proposed various resource-allocation algorithms without considering the compensation to resource providers and the cost estimation of the fog resources. Moreover, the existing cost models in similar paradigms such as in the cloud are not suitable for fog environments as the scaling of different autonomous resources with heterogeneity and variety of offerings is much more complicated. To fill this gap, this study first proposes a micro-level compensation cost model and then proposes a new resource-allocation method based on the cost model, which benefits both providers and users. Experimental results show that the proposed algorithm ensures better resource-allocation performance and lowers application processing costs when compared to the existing best-fit algorithm.

A role for glia in the progression of Rett's syndrome.

Rett's syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

Investigation of C-5 alkynyl (alkynyloxy or hydroxymethyl) and/or N-3 propynyl substituted pyrimidine nucleoside analogs as a new class of antimicrobial agents.

The resurgence of mycobacterial infections and the emergence of drug-resistant strains urgently require a new class of agents that are distinct than current therapies. A group of 5-ethynyl (6-10), 5-(2-propynyloxy) (16, 18, 20, 22, 24), 5-(2-propynyloxy)-3-N-(2-propynyl) (17, 19, 21, 23, 25) and 5-hydroxymethyl-3-N-(2-propynyl) (30-33) derivatives of pyrimidine nucleosides were synthesized and evaluated against mycobacteria [Mycobacterium tuberculosis (Mtb), Mycobacterium bovis (BCG) and Mycobacterium avium], gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and gram-negative bacteria (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) alone and in combination with existing drugs in in vitro assays. Although several compounds exhibited marked inhibitory activity at a higher concentration against Mtb, M. bovis, S. aureus and E. faecalis, they displayed unexpected synergistic and additive interactions at their lower concentrations with antitubercular drugs isoniazid and rifampicin, and antibacterial drug gentamicin. The active analogues were also found to inhibit intracellular Mtb in a human monocytic cell line infected with H37Ra. Oral administration of 5-hydroxymethyl-3-N-(2-propynyl)-3'-azido-2',3'-dideoxyuridine (32) and 5-hydroxymethyl-3-N-(2-propynyl)-2',3'-dideoxyuridine (33) at a dose of 100mg/kg for two weeks showed promising in vivo effects in mice infected with Mtb (H37Ra). No in vitro cytotoxicity of the test compounds was observed up to the highest concentration tested (CC50>300µg/mL).

Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome.

De novo mutations in the X-linked gene encoding the transcription factor methyl-CpG binding protein 2 (MECP2) are the most frequent cause of the neurological disorder Rett syndrome (RTT). Hemizygous males usually die of neonatal encephalopathy. Heterozygous females survive into adulthood but exhibit severe symptoms including microcephaly, loss of purposeful hand motions and speech, and motor abnormalities, which appear after a period of apparently normal development. Most studies have focused on male mouse models because of the shorter latency to and severity in symptoms, yet how well these mice mimic the disease in affected females is not clear. Very few therapeutic treatments have been proposed for females, the more gender-appropriate model. Here, we show that self-complementary AAV9, bearing MeCP2 cDNA under control of a fragment of its own promoter (scAAV9/MeCP2), is capable of significantly stabilizing or reversing symptoms when administered systemically into female RTT mice. To our knowledge, this is the first potential gene therapy for females afflicted with RTT.

Effect of Sarizotan, a 5-HT1a and D2-like receptor agonist, on respiration in three mouse models of Rett syndrome.

Disturbances in respiration are common and debilitating features of Rett syndrome (RTT). A previous study showed that the 5-HT1a receptor agonist (R)-(+)-8-hydroxy-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) significantly reduced the incidence of apnea and the irregular breathing pattern in a mouse model of the disorder. 8-OH-DPAT, however, is not available for clinical practice. Sarizotan, a full 5-HT1a agonist and a dopamine D2-like agonist/partial agonist, has been used in clinical trials for the treatment of l-dopa-induced dyskinesia. The purpose of this study was to evaluate the effects of sarizotan on respiration and locomotion in mouse models of RTT. Studies were performed in Bird and Jaenisch strains of methyl-CpG-binding protein 2--deficient heterozygous female and Jaenisch strain Mecp2 null male mice and in knock-in heterozygous female mice of a common nonsense mutation (R168X). Respiratory pattern was determined with body plethysmography, and locomotion was determined with open-field recording. Sarizotan or vehicle was administered 20 minutes before a 30-minute recording of respiratory pattern or motor behavior. In separate studies, a crossover design was used to administer the drug for 7 and for 14 days. Sarizotan reduced the incidence of apnea in all three RTT mouse models to approximately 15% of their pretreatment levels. The irregular breathing pattern was corrected to that of wild-type littermates. When administered for 7 or 14 days, apnea decreased to 25 to 33% of the incidence seen with vehicle. This study indicates that the clinically approved drug sarizotan is an effective treatment for respiratory disorders in mouse models of RTT.

Computer-Aided Analysis of the "Beautiful" Umbilicus.

BACKGROUND: The position, shape, size, and depth of the umbilicus influence the overall aesthetics of the abdomen. Hence, umbilicoplasty is a common adjunct to aesthetic and reconstructive surgery of the abdominal wall. Delineation of the position and shape of the "beautiful" umbilicus can aid in the planning of abdominoplasty and lipoabdominoplasty. OBJECTIVES: The authors aimed to identify key parameters of the beautiful umbilicus. METHODS: Previously, the authors developed software (the Aesthetic Analyzer) for marking and analyzing parameters from images of the nose, breast, and umbilicus. In the present study, the Aesthetic Analyzer was utilized to determine parameters of the beautiful umbilicus from images of 37 Playboy playmates. The vertical position, horizontal position, length, and shape of the umbilicus were assessed. RESULTS: Based on these images, the beautiful umbilicus possesses the following properties: a vertical ratio of 46:54 (with respect to the xiphoid process and lower limit of the vulvar cleft), a midline horizontal position, a length that is 5% of the length from the xiphoid process to the lower limit of the vulvar cleft, and an oval shape with no hooding (29.8%) or superior hooding (21.6%). CONCLUSIONS: Awareness of the ideal position, shape, and size of the umbilicus can be useful for achieving successful reconstruction of the umbilicus during abdominoplasty and lipoabdominoplasty.

Long-term monitoring of outdoor natural gamma absorbed dose rate in air in Bengaluru, Karnataka, India.

This research forms a part of the comprehensive Indian Environmental Radiation Monitoring Network program, focusing on the continuous measurement of absorbed dose rate in outdoor air due to natural gamma radiation (cosmic and terrestrial) in Bengaluru, Karnataka, India. Over the course of a decade (2013-2023), data were collected from 41 monitoring locations in the city using permanently field-installed Geiger-Mueller detector-based environmental radiation monitors. This paper presents an analysis of the extensive long-term monitoring results. The mean absorbed gamma dose rate in outdoor air across the monitoring locations ranged from 84 ± 9 to 156 ± 4 nGy.h-1, with a calculated mean value of 124 ± 15 nGy.h-1. The estimated mean annual effective dose due to outdoor natural gamma radiation varied from 0.10 ± 0.01 to 0.19 ± 0.01 mSv.y-1, with an overall mean of 0.15 ± 0.02 mSv.y-1.

Exercise-Related Physical Activity Relates to Brain Volumes in 10,125 Individuals.

BACKGROUND: The potential neuroprotective effects of regular physical activity on brain structure are unclear, despite links between activity and reduced dementia risk. OBJECTIVE: To investigate the relationships between regular moderate to vigorous physical activity and quantified brain volumes on magnetic resonance neuroimaging. METHODS: A total of 10,125 healthy participants underwent whole-body MRI scans, with brain sequences including isotropic MP-RAGE. Three deep learning models analyzed axial, sagittal, and coronal views from the scans. Moderate to vigorous physical activity, defined by activities increasing respiration and pulse rate for at least 10 continuous minutes, was modeled with brain volumes via partial correlations. Analyses adjusted for age, sex, and total intracranial volume, and a 5% Benjamini-Hochberg False Discovery Rate addressed multiple comparisons. RESULTS: Participant average age was 52.98±13.04 years (range 18-97) and 52.3% were biologically male. Of these, 7,606 (75.1%) reported engaging in moderate or vigorous physical activity approximately 4.05±3.43 days per week. Those with vigorous activity were slightly younger (p < 0.00001), and fewer women compared to men engaged in such activities (p = 3.76e-15). Adjusting for age, sex, body mass index, and multiple comparisons, increased days of moderate to vigorous activity correlated with larger normalized brain volumes in multiple regions including: total gray matter (Partial R = 0.05, p = 1.22e-7), white matter (Partial R = 0.06, p = 9.34e-11), hippocampus (Partial R = 0.05, p = 5.96e-7), and frontal, parietal, and occipital lobes (Partial R = 0.04, p≤1.06e-5). CONCLUSIONS: Exercise-related physical activity is associated with increased brain volumes, indicating potential neuroprotective effects.

Brief webcam test of hand movements predicts episodic memory, executive function, and working memory in a community sample of cognitively asymptomatic older adults.

INTRODUCTION: Low-cost simple tests for preclinical Alzheimer's disease are a research priority. We evaluated whether remote unsupervised webcam recordings of finger-tapping were associated with cognitive performance in older adults. METHODS: A total of 404 cognitively-asymptomatic participants (64.6 [6.77] years; 70.8% female) completed 10-second finger-tapping tests (Tasmanian [TAS] Test) and cognitive tests (Cambridge Neuropsychological Test Automated Battery [CANTAB]) online at home. Regression models including hand movement features were compared with null models (comprising age, sex, and education level); change in Akaike Information Criterion greater than 2 (ΔAIC > 2) denoted statistical difference. RESULTS: Hand movement features improved prediction of episodic memory, executive function, and working memory scores (ΔAIC > 2). Dominant hand features outperformed nondominant hand features for episodic memory (ΔAIC = 2.5), executive function (ΔAIC = 4.8), and working memory (ΔAIC = 2.2). DISCUSSION: This brief webcam test improved prediction of cognitive performance compared to age, sex, and education. Finger-tapping holds potential as a remote language-agnostic screening tool to stratify community cohorts at risk for cognitive decline.

Harnessing Innate Immunity to Treat Mycobacterium tuberculosis Infections: Heat-Killed Caulobacter crescentus as a Novel Biotherapeutic.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a serious and devastating infectious disease worldwide. Approximately a quarter of the world population harbors latent Mtb infection without pathological consequences. Exposure of immunocompetent healthy individuals with Mtb does not result in active disease in more than 90% individuals, suggesting a defining role of host immunity to prevent and/or clear early infection. However, innate immune stimulation strategies have been relatively underexplored for the treatment of tuberculosis. In this study, we used cell culture and mouse models to examine the role of a heat-killed form of a non-pathogenic microbe, Caulobacter crescentus (HKCC), in inducing innate immunity and limiting Mtb infection. We also examined the added benefits of a distinct chemo-immunotherapeutic strategy that incorporates concurrent treatments with low doses of a first-line drug isoniazid and HKCC. This therapeutic approach resulted in highly significant reductions in disseminated Mtb in the lungs, liver, and spleen of mice compared to either agent alone. Our studies demonstrate the potential of a novel innate immunotherapeutic strategy with or without antimycobacterial drugs in controlling Mtb infection in mice and open new avenues for the treatment of tuberculosis in humans.

Increased CD4/CD8 Lymphocyte ratio predicts favourable neoadjuvant treatment response in gastric cancer: A prospective pilot study.

BACKGROUND: Despite optimal neoadjuvant chemotherapy only 40% of gastric cancer tumours achieve complete or partial treatment response. In the absence of treatment response, neoadjuvant chemotherapy in gastric cancer contributes to adverse events without additional survival benefit compared to adjuvant treatment or surgery alone. Additional strategies and methods are required to optimize the allocation of existing treatment regimens such as FLOT chemotherapy (5-Fluorouracil, Leucovorin, Oxaliplatin and Docetaxel). Predictive biomarkers detected using immunohistochemistry (IHC) methods may provide useful data regarding treatment response. AIM: To investigate the utility of CD4, CD8, Galectin-3 and E-cadherin in predicting neoadjuvant FLOT chemotherapy tumour response in gastric adenocarcinoma. METHODS: Forty-three adult patients with gastric adenocarcinoma, of which 18 underwent neoadjuvant chemotherapy, were included in a prospective clinical cohort. Endoscopic biopsies were obtained from gastric cancer and normal adjacent gastric mucosa. Differences in expression of Galectin-3, E-cadherin, CD4+ and CD8+ molecules between tumours with and without treatment response to neoadjuvant chemotherapy were assessed with IHC. Treatment response was graded by clinical pathologists using the Tumour Regression Score according to the College of American Pathologists criteria. Treatment response was defined as complete or near complete tumour response, whereas partial or poor/no response was defined as incomplete. Digital IHC images were annotated and quantitatively assessed using QuPath 0.3.1. Biomarker expression between responsive and incomplete response tumours was assessed using a two-sided Wilcoxon test. Biomarker expression was also compared between normal and cancer tissue and between 15 paired tumour samples before and after chemotherapy. We performed a preliminary multivariate analysis and power analysis to guide future study. Statistical analyses were completed using R 4.1.2. RESULTS: The ratio between CD4+ and CD8+ lymphocytes was significantly greater in treatment responsive tumours (Wilcoxon, P = 0.03). In univariate models, CD4+/CD8+ ratio was the only biomarker that significantly predicted favourable treatment response (Accuracy 86%, P < 0.001). Using a glmnet multivariate model, high CD4+/CD8+ ratio and low Galectin-3 expression were the most influential variables in predicting a favourable treatment response. Analyses of paired samples found that FLOT chemotherapy also results in increased expression of CD4+ and CD8+ tumour infiltrating lymphocytes (Paired Wilcoxon, P = 0.002 and P = 0.008, respectively). Our power analysis suggests future study requires at least 35 patients in each treatment response group for CD8 and Galectin-3 molecules, whereas 80 patients in each treatment response group are required to assess CD4 and E-cadherin biomarkers. CONCLUSION: We demonstrate that an elevated CD4+/CD8+ Ratio is a promising IHC-based biomarker to predict favourable treatment response to FLOT neoadjuvant chemotherapy in locally advanced gastric cancer.

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Countrywide monitoring of absorbed dose rate in air due to outdoor natural gamma radiation in India.

The Indian Environmental Radiation Monitoring Network continuously monitors, throughout India, the absorbed dose rate in air due to outdoor natural gamma radiation, by using Geiger-Mueller detector-based standalone environmental radiation monitors. The network consists of 546 monitors spread across 91 monitoring locations distributed all over the country. In this paper, the countrywide long-term monitoring results are summarised. The measured mean dose rate of the monitoring locations followed a log-normal distribution and ranged from 50 to 535 nGy.h-1 with a median value of 91 nGy.h-1. Due to outdoor natural gamma radiation, the average annual effective dose was estimated to be 0.11 mSv.y-1.