Enhancing scaffold-free spheroid models: 3D cell bioprinting method for metastatic HSC3-Oral squamous carcinoma cell line.

3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).

The dissection of a despotic society: exploration, dominance and hormonal traits.

Naked mole-rats (Heterocephalus glaber) live in large colonies with one breeding female (queen), one to three breeding males (BMs) and the remainder are non-reproductive subordinates. The animals have a linear dominance rank with the breeders at the top of the hierarchy. We investigated how dominance rank in naked mole-rats differs with exploration (the propensity to explore a novel environment) and related endocrine markers. Exploration behaviour, faecal progestagen metabolite (fPM), faecal glucocorticoid metabolite (fGCM), faecal androgen metabolite (fAM) and plasma prolactin concentrations were quantified in breeding, high-, middle- and low-ranked females and males from five naked mole-rat colonies. There were no significant differences between the dominance rank and exploration behaviour. Interestingly, the queens and high-ranking females had higher fGCM and fAM concentrations compared with middle- and low-ranked females. The queens had significantly higher fPM concentrations than all other ranked females, since they are responsible for procreation. In the males, the BMs had higher fGCM concentrations compared with high- and low-ranked males. In addition, BMs and middle-ranking males had overall higher prolactin levels than all other ranked males, which could be linked to cooperative care. Overall, the results suggest that physiological reproductive suppression is linked to high dominance rank.

Non-Invasive Quantification of Faecal and Urine Reproductive Hormone Metabolites in the Naked Mole-Rat (Heterocephalus glaber).

The naked mole-rat (Heterocephalus glaber) occurs in colonies with a distinct dominance hierarchy, including one dominant, breeding female (the queen), 1-3 breeding males, and non-reproductive subordinates of both sexes that are reproductively suppressed while in the colony. To non-invasively evaluate reproductive capacity in the species, we first had to examine the suitability of enzyme immunoassays (EIAs) for determining progestogen and androgen metabolite concentrations in the naked mole-rat, using urine and faeces. A saline control and gonadotrophin-releasing hormone (GnRH) were administered to twelve (six males and six females) naked mole-rats which were previously identified as dispersers and housed singly. The results revealed that urine is possibly not an ideal matrix for progestogen and androgen metabolite quantification in naked mole-rats as no signal was detected in the matrix post GnRH administration. A 5α-Progesterone EIA and an Epiandrosterone EIA were identified as suitable for quantifying faecal progesterone metabolites (fPMs) and faecal androgen metabolites (fAMs) in males and females, respectively. The results suggest that there are individual variations in baseline fPM and fAM concentrations, and only two out of six females and no males exhibited an increase in fPM concentrations greater than 100% (-20% SD) post GnRH administration. Conversely, only four out of six females and three out of six males had an increase in fAM concentrations greater than 100% (-20% SD) following GnRH administration. These results imply that some naked mole-rat individuals have a reduced reproductive capacity even when they are separated from the queen.

Retention and Predictors of Attrition Among People Living With HIV on Antiretroviral Therapy in Guinea: A 13-Year Historical Cohort Study in Nine Large-Volume Sites.

Objectives: The objective of this study was to estimate the retention rate of patients in an ART program and identify the predictors of attrition. Methods: This was a historical cohort study of HIV patients who started ART between September 2007 and April 2020, and were followed up on for at least 6 months in nine large-volume sites. Kaplan Meier techniques were used to estimate cumulative retention and attrition probabilities. Cox proportional hazards models were used to identify predictors of attrition. Results: The cumulative probability of retention at 12 and 24 months was 76.2% and 70.2%, respectively. The attrition rate after a median follow-up time of 3.1 years was 35.2%, or an incidence of 11.4 per 100 person-years. Having initiated ART between 2012 and 2015; unmarried status; having initiated ART with CD4 count <100 cells/µL; and having initiated ART at an advanced clinical stage were factors significantly associated with attrition. Conclusion: The retention rate in our study is much lower than the proposed national target (90%). Studies to understand the reasons for loss to follow-up are needed.

Validation of Enzyme Immunoassays via an Adrenocorticotrophic Stimulation Test for the Non-Invasive Quantification of Stress-Related Hormone Metabolites in Naked Mole-Rats.

Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat (Heterocephalus glaber, NMR) using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3ß,11ß,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3ß-11ß-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5ß-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.

Broad Electronic Modulation of Two-Dimensional Metal-Organic Frameworks over Four Distinct Redox States.

Two-dimensional (2D) inorganic materials have emerged as exciting platforms for (opto)electronic, thermoelectric, magnetic, and energy storage applications. However, electronic redox tuning of these materials can be difficult. Instead, 2D metal-organic frameworks (MOFs) offer the possibility of electronic tuning through stoichiometric redox changes, with several examples featuring one to two redox events per formula unit. Here, we demonstrate that this principle can be extended over a far greater span with the isolation of four discrete redox states in the 2D MOFs LixFe3(THT)2 (x = 0-3, THT = triphenylenehexathiol). This redox modulation results in 10,000-fold greater conductivity, p- to n-type carrier switching, and modulation of antiferromagnetic coupling. Physical characterization suggests that changes in carrier density drive these trends with relatively constant charge transport activation energies and mobilities. This series illustrates that 2D MOFs are uniquely redox flexible, making them an ideal materials platform for tunable and switchable applications.

Unraveling two distinct polymorph transition mechanisms in one n-type single crystal for dynamic electronics.

Cooperativity is used by living systems to circumvent energetic and entropic barriers to yield highly efficient molecular processes. Cooperative structural transitions involve the concerted displacement of molecules in a crystalline material, as opposed to typical molecule-by-molecule nucleation and growth mechanisms which often break single crystallinity. Cooperative transitions have acquired much attention for low transition barriers, ultrafast kinetics, and structural reversibility. However, cooperative transitions are rare in molecular crystals and their origin is poorly understood. Crystals of 2-dimensional quinoidal terthiophene (2DQTT-o-B), a high-performance n-type organic semiconductor, demonstrate two distinct thermally activated phase transitions following these mechanisms. Here we show reorientation of the alkyl side chains triggers cooperative behavior, tilting the molecules like dominos. Whereas, nucleation and growth transition is coincident with increasing alkyl chain disorder and driven by forming a biradical state. We establish alkyl chain engineering as integral to rationally controlling these polymorphic behaviors for novel electronic applications.

Highly socially vulnerable communities exhibit disproportionately increased viral loads as measured in community wastewater.

Wastewater surveillance has proven to be a useful tool for evidence-based epidemiology in the fight against the SARS-CoV-2 virus. It is particularly useful at the population level where acquisition of individual test samples may be time or cost-prohibitive. Wastewater surveillance for SARS-CoV-2 has typically been performed at wastewater treatment plants; however, this study was designed to sample on a local level to monitor the spread of the virus among three communities with distinct social vulnerability indices in Shreveport, Louisiana, located in a socially vulnerable region of the United States. Twice-monthly grab samples were collected from September 30, 2020, to March 23, 2021, during the Beta wave of the pandemic. The goals of the study were to examine whether: 1) concentrations of SARS-CoV-2 RNA in wastewater varied with social vulnerability indices and, 2) the time lag of spikes differed during wastewater monitoring in the distinct communities. The size of the population contributing to each sample was assessed via the quantification of the pepper mild mottle virus (PMMoV), which was significantly higher in the less socially vulnerable community. We found that the communities with higher social vulnerability exhibited greater viral loads as assessed by wastewater when normalized with PMMoV (Kruskal-Wallis, p < 0.05). The timing of the spread of the virus through the three communities appeared to be similar. These results suggest that interconnected communities within a municipality experienced the spread of the SARS-CoV-2 virus at similar times, but areas of high social vulnerability experienced more intense wastewater viral loads.

Aggression, Boldness, and Exploration Personality Traits in the Subterranean Naked Mole-Rat (Heterocephalus glaber) Disperser Morphs.

Animal personality traits (consistent behavioral differences between individuals in their behavior across time and/or situation) affect individual fitness through facets, such as dispersal. In eusocial naked mole-rat (Heterocephalus glaber) colonies, a disperser morph may arise with distinct morphological, behavioral, and physiological characteristics. This study aimed to quantify the personality traits of a cohort of disperser morphs of naked mole-rat (NMR). Behavioral tests were performed on twelve disperser morphs (six males and six females) in an observation tunnel system that was novel and unfamiliar. Novel stimuli (fresh snakeskin, tissue paper, and conspecific of the same sex) were introduced for fifteen minutes, and the behavioral acts of the individual were recorded. A total of 30 behaviors were noted during the behavioral tests of which eight were used to quantify aggression, boldness, and exploration. The NMR disperser morphs showed consistent individual differences in boldness, and exploration across time and test, indicating a distinct personality. In addition, new naked mole-rat responses including disturbance behaviors; confront, barricade, and stay-away, were recorded. Further investigations into the relationships between animal personality traits and social hierarchy position in entire colonies are needed for more informative results as we further investigate the role of personality in cooperatively breeding societies.

The Operating Room Black Box: Understanding Adherence to Surgical Checklists.

OBJECTIVE: We report for the first time the use of the Operating Room Black Box (ORBB) to track checklist compliance, engagement, and quality. BACKGROUND: Implementation of operative checklists is associated with improved outcomes. Compliance is difficult to monitor. Most studies report either no assessment of checklist compliance or deployed in-person short-term assessment. The ORBB a novel artificially intelligence-driven data analytic platform affords the opportunity to assess checklist compliance without disrupting surgical workflow. METHODS: This was a retrospective review of prospectively collected ORBB data. Operative cases included elective surgery at a quaternary referral center. Cases were analyzed as prepolicy change (first 9 months) or as a postpolicy change (last 9 months). Measures of checklist compliance, engagement, and quality were assessed. RESULTS: There were 3879 cases that were performed and monitored for checklist compliance between August 15, 2020, and February 20, 2022. The overall scores for compliance, engagement, and quality were 81%, 84%, and 67% respectively. When broken down by phase, the scores for time-out were compliance 100%, engagement 98%, and quality 61%. Scores for the debrief phase were 81% for compliance, 98% for engagement, and 66% for quality. After a hospital policy change, the debrief scores improved significantly (85%; P <0.001 for compliance, 88%; P <0.001 for engagement and 71%; P <0.001 for quality). CONCLUSIONS: ORBB provides the unprecedented ability to assess not only compliance with surgical safety checklists but also engagement and quality. Utilization of this technology allows the assessment of compliance in near real time and to accurately address safety threats that may arise from noncompliance.

An inverse planned oil release validation method for estimating oil slick thickness from thermal contrast remote sensing by in-scene calibration.

This study demonstrates a method to estimate floating oil slick thickness based on remote sensing of thermal infrared contrast. The approach was demonstrated for thick oil slicks from natural seeps in the Coal Oil Point seep field, offshore southern California. Airborne thermal infrared and visible spectrum remote sensing imagery were acquired along with position and orientation data by the SeaSpires™ science package. Remote sensing data were acquired in the cross-slick direction of oil slick segments that were targeted for collection, termed "collects." A collect consisted of booming, skimming, and offloading the oil slick segment into buckets for analysis at the laboratory. Each collect provided an in-scene calibration point of oil thickness versus brightness temperature contrast, ΔTB , where TB is the sensor-reported temperature based on the emitted thermal radiation and differs from the true temperature due to the oil's emissivity. ΔTB is the TB difference between the oil and oil-free sea surface. Thus, this study is a reverse planned oil-release experiment that demonstrates the value of natural seeps for oil spill science. • Novel approach to quantify floating oil thickness • Custom modified weir skimmer used with added floor and structural strengthening.

Defining the link between oxidative stress, behavioural reproductive suppression and heterothermy in the Natal mole-rat (Cryptomys hottentotus natalensis).

Sub-lethal effects, such as oxidative stress, can be linked to various breeding and thermophysiological strategies, which themselves can be linked to seasonal variability in abiotic factors. In this study, we investigated the subterranean, social living Natal mole-rat (Cryptomys hottentotus natalensis), which, unlike other social mole-rat species, implements heterothermy seasonally in an attempt to avoid exercise-induced hyperthermia and relies solely on behavioural reproductive suppression to maintain reproductive skew in colonies. Subsequently, we investigated how oxidative stress varied between season, sex and breeding status in Natal mole-rats. Oxidative markers included total oxidant status (TOS measure of total peroxides present), total antioxidant capacity (TAC), OSI (oxidative stress index) and malondialdehyde (MDA) to measure oxidative stress. Breeding and non-breeding mole-rats of both sexes were captured during the summer (wet season) and winter (dry season). Seasonal environmental variables (air temperature, soil temperature and soil moisture) had a significant effect on TOS, OSI and MDA, where season affected each sex differently. Unlike other social mole-rat species that use both physiological and behavioural means of reproductive suppression, no oxidative costs to reproduction were present in the Natal mole-rats. Males had significantly higher MDA than females, which was most apparent in summer (wet season). We conclude that the significant oxidative damage in males is a consequence of exercise-induced oxidative stress, exacerbated by increased burrow humidities and poorer heat dissipation abilities as a function of body mass. This study highlights the importance of both breeding and thermophysiological strategies in affecting oxidative stress.

Ovarian Cancer Ascites Inhibits Transcriptional Activation of NK Cells Partly through CA125.

Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy.

Common synaptic phenotypes arising from diverse mutations in the human NMDA receptor subunit GluN2A.

Dominant mutations in the human gene GRIN2A, encoding NMDA receptor (NMDAR) subunit GluN2A, make a significant and growing contribution to the catalogue of published single-gene epilepsies. Understanding the disease mechanism in these epilepsy patients is complicated by the surprising diversity of effects that the mutations have on NMDARs. Here we have examined the cell-autonomous effect of five GluN2A mutations, 3 loss-of-function and 2 gain-of-function, on evoked NMDAR-mediated synaptic currents (NMDA-EPSCs) in CA1 pyramidal neurons in cultured hippocampal slices. Despite the mutants differing in their functional incorporation at synapses, prolonged NMDA-EPSC current decays (with only marginal changes in charge transfer) were a common effect for both gain- and loss-of-function mutants. Modelling NMDA-EPSCs with mutant properties in a CA1 neuron revealed that the effect of GRIN2A mutations can lead to abnormal temporal integration and spine calcium dynamics during trains of concerted synaptic activity. Investigations beyond establishing the molecular defects of GluN2A mutants are much needed to understand their impact on synaptic transmission.

The Cost of Reproduction in a Cooperatively Breeding Mammal: Consequences of Seasonal Variation in Rainfall, Reproduction, and Reproductive Suppression.

Biological investments, such as reproduction, are influenced by both biotic and abiotic factors and their interactions. The trade-off between reproduction and survival has been well established. Seasonally breeding species, therefore, may exhibit variations in these trade-offs, but there is a dearth of knowledge concerning this. This study investigated the physiological cost of reproduction (measured through oxidative stress) across seasons in the cooperatively breeding highveld mole-rat (Cryptomys hottentotus pretoriae), one of the few seasonal breeding mole-rats. Oxidative stress indicates elevated reactive oxygen species (ROS) levels, which can overwhelm antioxidant defences resulting in damaged proteins, lipids and DNA, which overall can reduce longevity and compromise reproduction. Oxidative markers such as total oxidant status (TOS-measure of total peroxides present), total antioxidant capacity (TAC), oxidative stress index (OSI), and malondialdehyde (MDA) are utilised to measure oxidative stress. In this study, breeding and non-breeding male (NBM) and female mole-rats were captured during the dry season (breeding period) and wet season (non-breeding period). There was an apparent cost of reproduction in the highveld mole-rat; however, the seasonality pattern to the cost of reproduction varied between the sexes. Breeding females (BFs) had significantly higher MDA during the breeding period/dry season in comparison to the non-breeding period/wet season; this is possibly a consequence of bearing and nursing offspring. Contrastingly, breeding males (BMs) showed increased oxidative damage in the non-breeding/wet season compared to the breeding/dry season, possibly due to increased activities of protecting their mating rights for the next breeding/dry season, but this was not significant. Interestingly, during the non-breeding period/wet season, non-breeding females (NBFs) are released from their reproductive suppression, which resulted in increases in TOS and OSI, which again indicated that just the mere ability to be able to breed results in a cost (oxidative stress). Therefore we can speculate that highveld mole-rats exhibited seasonal variation in redox balance brought about by variation in abiotic variables (e.g., rainfall), physiology and behaviour. We conclude that physiological changes associated with reproduction are sufficient to induce significant acute oxidative stress in the plasma of female highveld mole-rats, which become alleviated following transition to the non-breeding season/wet period suggesting a possible hormetic effect.

Plasma oxidative stress in reproduction of two eusocial African mole-rat species, the naked mole-rat and the Damaraland mole-rat.

One of the most prominent life-history trade-offs involves the cost of reproduction. Oxidative stress has been proposed to be involved in this trade-off and has been associated with reduced life span. There is currently an unclear relationship between oxidative cost and the reproduction-longevity trade-off. The current study, using a non-lethal and minimally invasive (only a single blood sample and no euthanasia) method, investigated whether an oxidative cost (oxidative stress) to reproduction would be apparent in two long-lived eusocial mole-rats, the naked mole-rat (NMR), Heterocephalus glaber, and the Damaraland mole-rat (DMR), Fukomys damarensis, where breeding colony members live longer than non-breeder conspecifics. We measured the direct redox balance in plasma by measuring the oxidative stress index (OSI) based on the ratio of total oxidant status and total antioxidant activity in breeders and non-breeders of both sexes, in the two species. NMR had significantly higher OSI between breeders and non-breeders of each sex, whereas DMR showed no significant differences except for total antioxidant capacity (TAC). The mode of reproductive suppression and the degree of reproductive investment in NMR may explain to some degree the redox balance difference between breeders and non-breeders. DMR show minimal physiological changes between breeders and non-breeders except for the mode of reproduction, which may explain some variations in TAC and TOS values, but similar OSI between breeders and non-breeders.

Near Complete Pathologic Response to PD-1 Inhibitor and Radiotherapy in a Patient with Locally Advanced Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains deadly despite advances in systemic therapies and surgical techniques. While there is increasing utilization of immune therapies across diverse cancer types, PDAC remains generally resistant to these treatments. We report a case of locally advanced PDAC treated with preoperative radiation and anti-PD-1 immunotherapy guided by preoperative PD-L1 tumor analysis. After 4 months of preoperative therapy, the patient was submitted to resection, demonstrating a near-complete pathologic response on final tumor analysis. We will discuss the relevant literature and current state of immunotherapeutics for PDAC.

Open and Closed Endotracheal Suction Systems Divergently Affect Pulmonary Function in Mechanically Ventilated Subjects.

BACKGROUND: In mechanically ventilated subjects, intra-tracheal secretions can be aspirated with either open suction systems (OSS) or closed suction systems (CSS). In contrast to CSS, conventional OSS require temporarily disconnecting the patient from the ventilator, which briefly diminishes PEEP and oxygen supply. On the other hand, CSS are more expensive and less effective at aspirating secretions. Thus, it was hypothesized that the 2 procedures differentially affect pulmonary and cardiovascular parameters after suction. METHODS: Subjects in the ICU (N = 66) were quasi-randomized for initial treatment with OSS or CSS in a crossover design. To compare the potential for these suction systems to compromise cardiorespiratory stability, changes in cardiopulmonary physiology were assessed from before to just after use of each suction system (three 10-s aspirations). RESULTS: For most pulmonary and cardiovascular parameters (ie, peak inspiratory pressure, airway resistance, pressure plateau, heart rate, and arterial pressures), the effects of aspiration inversely correlated with baseline values for that parameter, with a similar regression slope between suction systems. However, when controlling for baseline values, OSS caused significantly greater increases in airway resistance and peak inspiratory pressure (P < .001 and < .01 vs CSS, respectively). CONCLUSIONS: Elevated airway resistance prior to endotracheal suction may justify use of a CSS and contraindicate a conventional OSS in mechanically ventilated subjects. Adoption of this approach into clinical guidelines may prevent suction-induced pulmonary injury in subjects, especially for those with underlying diseases involving increased airway resistance or increased alveolar pressure. (ClinicalTrials.gov registration: NCT03256214.).

Brain-derived Neurotropic Factor val66met is a Strong Predictor of Decision Making and Attention Performance on the CONVIRT Virtual Reality Cognitive Battery.

The val66met polymorphism of the brain-derived neurotrophic factor gene has been associated with changes in components of executive functioning such as decision making; however, this relationship remains unclear. Val66met-related changes in attention and visual processing speed may explain potential changes in decision making. Furthermore, chronic stress disrupts executive functions and alters autonomic activity. Because the relationship between val66met and cognition has not been investigated in the context of chronic stress or stress-related autonomic changes, in this study 55 healthy university students completed self-report measures of chronic stress and mental health. Participants then completed a virtual reality cognitive test battery (CONVIRT) measuring decision making, attention, and visual processing reaction times. To measure autonomic activity, saliva alpha amylase and heart rate variability (HRV) were assessed at baseline and after CONVIRT testing. Saliva samples were used to identify val66met genotype. Regression analyses demonstrated that val66met was the strongest predictor of decision making and attention, but not visual processing, where valine/methionine (Val/met) participants had faster reaction times than Val/val participants. Val/met participants also had higher perceived chronic stress and heightened increases in sympathetic activity, but not parasympathetic activity. Neither stress nor autonomic activity moderated the effect of val66met on decision making or attention. This study is the first to investigate the role of val66met in decision making, attention, and visual processing while taking into account chronic stress and autonomic activity. This multifactorial approach revealed that carriers of the Val/met genotype may have better decision making and attention than Val/val carriers.