RESUMO
The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.
RESUMO
The foundation of dielectrophoresis (DEP) as a tool for biological investigation is the use of the Clausius-Mossotti (C-M) factor to model the observed behaviour of cells experiencing DEP across a frequency range. Nevertheless, it is also the case that at lower frequencies, the DEP spectrum deviates from predictions; there exists a rise in DEP polarisability, which varies in frequency and magnitude with different cell types and medium conductivities. In order to evaluate the origin of this effect, we have studied DEP spectra from five cell types (erythrocytes, platelets, neurons, HeLa cancer cells and monocytes) in several conditions including medium conductivity and cell treatment. Our results suggest the effect manifests as a low-pass dispersion whose cut-off frequency varies with membrane conductance and capacitance as determined using the DEP spectrum; the effect also varies as a logarithm of medium conductivity and Debye length. These together suggest that the values of membrane capacitance and conductance depend not only on the impedance of the membrane itself, but also of the surrounding double layer. The amplitude of the effect in different cell types compared to the C-M factor was found to correlate with the depolarisation factors for the cells' shapes, suggesting that this ratio may be useful as an indicator of cell shape for DEP modelling.
Assuntos
Condutividade Elétrica , Eletroforese , Eletroforese/métodos , Humanos , Células HeLa , Eritrócitos/citologia , Eritrócitos/química , Neurônios/fisiologia , Plaquetas/citologia , Plaquetas/química , Animais , Monócitos/citologiaRESUMO
BACKGROUND: Although experimental research supports that resistance training (RT), especially with greater dietary protein intake, improves muscle mass and strength in older adults, comparable research on tendons is needed. OBJECTIVES: We assessed the effects of a protein-rich diet emphasizing lean beef, compared with 2 control diets, on RT-induced changes in skeletal muscle and tendon size and strength in older women. METHODS: We randomly assigned women [age: 66 ± 1 y, body mass index (BMI): 28 ± 1] to groups that consumed 1) 0.8 g total protein/kg body weight/day from mixed food sources (normal protein control, n = 16); 2) 1.4 g/kg/d protein from mixed food sources (high protein control, n = 17); or 3) 1.4 g/kg/d protein emphasizing unprocessed lean beef (high protein experimental group, n = 16). Participants were provided with all foods and performed RT 3 times/wk, 70% of 1-repetition maximum for 12 wk. We measured quadriceps muscle volume via magnetic resonance imaging (MRI). We estimated patellar tendon biomechanical properties and cross-sectional area (CSA) using ultrasound and MRI. RESULTS: Dietary intake did not influence RT-induced increases in quadriceps strength (P < 0.0001) or muscle volume (P < 0.05). We noted a trend for an RT effect on mean tendon CSA (P = 0.07), with no differences among diets (P > 0.05). Proximal tendon CSA increased with RT (P < 0.05) with no difference between dietary groups (P > 0.05). Among all participants, midtendon CSA increased with RT (P ≤ 0.05). We found a decrease in distal CSA in the 0.8 g group (P < 0.05) but no change in the 1.4 g group (P > 0.05). Patellar tendon MRI signal or biomechanical properties were unchanged. CONCLUSIONS: Our findings indicated that greater daily protein intake, emphasizing beef, did not influence RT-induced changes in quadriceps muscle strength or muscle volume of older women. Although we noted trends in tendon CSA, we did not find a statistically significant impact of greater daily protein intake from beef on tendon outcomes. This trial was registered at clinicaltrials.gov as NCT04347447.
Assuntos
Proteínas Alimentares , Músculo Esquelético , Treinamento Resistido , Idoso , Bovinos , Feminino , Humanos , Pessoa de Meia-Idade , Adaptação Fisiológica , Dieta , Proteínas Alimentares/administração & dosagem , Proteínas Alimentares/farmacologia , Imageamento por Ressonância Magnética , Força Muscular , Músculo Esquelético/fisiologia , Carne Vermelha , Tendões/fisiologiaRESUMO
Primates are adept at dealing with fluctuating availability of resources and display a range of responses to minimize the effects of food scarcity. An important component of primate conservation is to understand how primates adapt their foraging and ranging patterns in response to fluctuating food resources. Animals optimize resource acquisition within the home range through the selection of resource-bearing patches and choose between contrasting foraging strategies (resource-maximizing vs. area-minimizing). Our study aimed to characterize the foraging strategy of a folivorous primate, Verreaux's sifaka (Propithecus verreauxi), by evaluating whether group home range size varied between peak and lean leaf seasons within a seasonally dry tropical forest in Madagascar. We hypothesized that Verreaux's sifaka used the resource maximization strategy to select high-value resource patches so that during periods of resource depression, the home range area did not significantly change in size. We characterized resource availability (i.e., primary productivity) by season at Kirindy Mitea National Park using remotely-sensed Enhanced Vegetation Index data. We calculated group home ranges using 10 years of focal animal sampling data collected on eight groups using both 95% and 50% kernel density estimation. We used area accumulation curves to ensure each group had an adequate number of locations to reach seasonal home range asymptotes. Neither 95% home ranges nor 50% core areas differed across peak and lean leaf resource seasons, supporting the hypothesis that Verreaux's sifaka use a resource maximization strategy. With a better understanding of animal space use strategies, managers can model anticipated changes under environmental and/or anthropogenic resource depression scenarios. These findings demonstrate the value of long-term data for characterizing and understanding foraging and ranging patterns. We also illustrate the benefits of using satellite data for characterizing food resources for folivorous primates.
Assuntos
Comportamento de Retorno ao Território Vital , Estações do Ano , Strepsirhini , Animais , Strepsirhini/fisiologia , Madagáscar , Florestas , Comportamento Alimentar , EcossistemaRESUMO
The study of how animals adapt their behaviors depending on weather variables has gained particular significance in the context of climate change. This exploration offers insights into endangered species' potential threats and provides information on the direction to take in conservation activities. In this context, noninvasive, cost-effective, and potentially long-term monitoring systems, such as Passive Acoustic Monitoring (PAM), become particularly appropriate. Our study investigates the relationship between weather variables and the vocal behavior of Indri indri, the sole singing lemur species, within Madagascar's Maromizaha New Protected Area. Using PAM, we explore the factors shaping the vocalization patterns of this primate species in response to some environmental factors in their natural habitat. Analysis of an extensive audio data set collected across different years revealed the differential influence of temperature and precipitation on Indri indri vocal activity. We found that rainfall negatively influenced the emission of the vocalizations while warmer temperatures correlated with a greater emission of songs. The various environmental factors we considered also affected the timing of vocal emissions, showing the same pattern. Furthermore, our study confirms, once again, the strength of PAM as a valuable tool for studying vocal animal communication quickly, giving us information about long-term behavioral patterns that would be difficult to get in other ways. This research gives us further valuable information about how indris use vocalizations in their environment and how they adjust to environmental changes.
RESUMO
While substantial progress has been made in improving water and sanitation services in low- and middle-income countries, aligned basic services such as greywater, stormwater, and solid waste management have progressed little in recent decades. Data was collected in Khulna city, Bangladesh via a household survey (n = 192) of low-income areas exploring domestic water use and greywater volumes, characteristics, and disposal practices. Most households (71%) use a piped water supply for domestic purposes, supplemented by seasonal rainwater harvesting (26%) and greywater use (13%). Of the total water used by households (mean: 594 L/household/day and equivalent to 116 L/person/day), approximately 58% becomes greywater through bathing, dishwashing, religious practices, handwashing, laundry, and mopping. Greywater produced ranges from 61-1274 L/household/day, with a mean of 345 L/household/day and equivalent to 78.4 L/person/day. Greywater characteristics vary depending on the activity, individual behaviours and any products used during cooking, bathing, or cleaning. After generation, households dispose greywater to open drains (67%), nearby waterbodies (17%) directly to the ground (9%), or decentralised wastewater treatment system (7%). Without services for greywater management, greywater disposal may have considerable public and environmental health implications, necessitating careful attention and oversight from service-providers and stakeholders beyond the household-level.
Assuntos
Conservação dos Recursos Hídricos , Características da Família , Águas Residuárias , Abastecimento de Água , Humanos , Bangladesh , Cidades , Conservação dos Recursos Hídricos/métodosRESUMO
Introduction: The venous thromboembolism (VTE) risk screening forms were developed to allow for recording identified risk factors for VTE including thrombophilia, history of VTE, postpartum hemorrhage, and cesarean delivery, and documentation of specific actions taken to mitigate these risks and reduce complications due to VTE. Methods: Compliance with hospital guidelines in assessing VTE risk and appropriate prescribing of thromboprophylaxis was evaluated prior to the introduction of VTE risk screening forms (March 2022). Efficacy of the new VTE risk screening forms was also assessed (April 2023). Patient discharge summaries and patient medical records including medication charts were used to review the documentation of VTE risk assessments and details of thromboprophylaxis prescribing. Results: Of 74 postnatal patients, 37.8% had VTE risk assessment documented prior to the introduction of VTE risk screening forms. Of 37 patients identified to be at moderate to high risk of VTE requiring pharmacological prophylaxis, 70.3% (n = 26) were appropriately prescribed pharmacological prophylaxis. After the risk screening forms were introduced, a total of 67 antenatal, postnatal, and gynecologic patients were studied. Of these, 32.8% (n = 22) of patients had all required fields completed appropriately. When using the forms, 26.9% (n = 7) of postnatal and 88% (n = 22) of gynecological patients were rated as medium or high risk, and all received medical review within 24 hours. Pharmacological prophylaxis was indicated in 88% (n = 22) of gynecological, 43.8% (n = 7) of antenatal, and 38.5% (n = 10) of postnatal patients, and all were appropriately prescribed. Conclusion: The guideline review and introduction of VTE risk screening forms was valuable to provide guidance in the risk assessment for VTE and to identify patients requiring prophylaxis.
RESUMO
Small, isolated populations are often characterised by low levels of genetic diversity. This can result in inbreeding depression and reduced capacity to adapt to changes in the environment, and therefore higher risk of extinction. However, sometimes these populations can be rescued if allowed to increase in size or if migrants enter, bringing in new allelic variation and thus increasing genetic diversity. This study uses experimental manipulation of population size and migration to quantify their effects on fitness in a challenging environment to better understand genetic rescue. Using small, replicated populations of Tribolium castaneum experimentally evolved to different temperature regimes we tested genetic and demographic rescue, by performing large-scale manipulations of population size and migration and examining fitness consequences over multiple generations. We measured fitness in high temperature (38°C) thermal lines maintained at their usual 'small' population size of N = 100 individuals, and with 'large' scaled up duplicates containing N≈10,000 individuals. We compared these large lines with and without migration (m = 0.1) for 10 generations. Additionally, we assessed the effects of outcrossing at an individual level, by comparing fitness of hybrid (thermal line × stock) offspring with within-line crosses. We found that, at the population level, a rapid increase in the number of individuals in the population resulted in reduced fitness (represented by reproductive output and survival through heatwave conditions), regardless of migration. However, at an individual level, the hybrid offspring of migrants with native individuals generally demonstrated increased longevity in high temperature conditions compared with individuals from thermal selection lines. Overall, these populations showed no evidence that demographic manipulations led to genetic or evolutionary rescue. Following the effects of migration in individuals over several generations may be the next step in unravelling these conflicting results. We discuss these findings in the context of conservation intervention.
RESUMO
Introduction: The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions. Materials and methods: We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function. Results: Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from -40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 µM) and CPA (1 µM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased. Conclusion: We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.
RESUMO
Hippocampal pyramidal neuronal activity has been previously studied using conventional patch clamp in isolated cells and brain slices. We here introduce the loose patch clamping study of voltage-activated currents from in situ pyramidal neurons in murine cornus ammonis 1 hippocampal coronal slices. Depolarizing pulses of 15-ms duration elicited early transient inward, followed by transient and prolonged outward currents in the readily identifiable junctional region between the stratum pyramidalis (SP) and oriens (SO) containing pyramidal cell somas and initial segments. These resembled pyramidal cell currents previously recorded using conventional patch clamp. Shortening the depolarizing pulses to >1-2 ms continued to evoke transient currents; hyperpolarizing pulses to varying voltages evoked decays whose time constants could be shortened to <1 ms, clarifying the speed of clamping in this experimental system. The inward and outward currents had distinct pharmacological characteristics and voltage-dependent inactivation and recovery from inactivation. Comparative recordings from the SP, known to contain pyramidal cell somas, demonstrated similar current properties. Recordings from the SO and stratum radiatum demonstrated smaller inward and outward current magnitudes and reduced transient outward currents, consistent with previous conventional patch clamp results from their different interneuron types. The loose patch clamp method is thus useful for in situ studies of neurons in hippocampal brain slices.
Assuntos
Técnicas de Patch-Clamp , Células Piramidais , Animais , Técnicas de Patch-Clamp/métodos , Camundongos , Células Piramidais/fisiologia , Potenciais da Membrana/fisiologia , Hipocampo/fisiologia , Hipocampo/citologia , Neurônios/fisiologia , Região CA1 Hipocampal/fisiologia , Região CA1 Hipocampal/citologia , Camundongos Endogâmicos C57BL , MasculinoRESUMO
BACKGROUND AND PURPOSE: Radiotherapy trial quality assurance (RT QA) is crucial for ensuring the safe and reliable delivery of radiotherapy trials, and minimizing inter-institutional variations. While previous studies focused on outlining and planning quality assurance (QA), this work explores the process of Image-Guided Radiotherapy (IGRT), and adaptive radiotherapy. This study presents findings from during-accrual QA in the RAIDER trial, evaluating concordance between online and offline plan selections for bladder cancer participants undergoing adaptive radiotherapy. RAIDER had two seamless stages; stage 1 assessed adherence to dose constraints of dose escalated radiotherapy (DART) and stage 2 assessed safety. The RT QA programme was updated from stage 1 to stage 2. MATERIALS AND METHODS: Data from all participants in the adaptive arms (standard dose adaptive radiotherapy (SART) and DART) of the trial was requested (33 centres across the UK, Australia and New Zealand). Data collection spanned September 2015 to December 2022 and included the plans selected online, on Cone-Beam Computed Tomography (CBCT) data. Concordance with the plans selected offline by the independent RT QA central reviewer was evaluated. RESULTS: Analysable data was received for 72 participants, giving a total of 884 CBCTs. The overall concordance rate was 83% (723/884). From stage 1 to stage 2 the concordance in the plans selected improved from 75% (369/495) to 91% (354/389). CONCLUSION: During-accrual IGRT QA positively influenced plan selection concordance, highlighting the need for ongoing support when introducing a new technique. Overall, it contributes to advancing the understanding and implementation of QA measures in adaptive radiotherapy trials.
RESUMO
Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.POUT was a phase III, randomized, open-label trial, including 261 patients with muscle-invasive or lymph node-positive, nonmetastatic upper tract urothelial cancer (UTUC) randomly assigned after radical nephroureterectomy to platinum-based chemotherapy (132) or surveillance (129). Primary outcome analysis demonstrated that chemotherapy improved disease-free survival (DFS). At that time, the planned secondary outcome analysis of overall survival (OS) was immature. By February 2022, 50 and 67 DFS events had occurred in the chemotherapy and surveillance groups, respectively, at a median follow-up of 65 months. The 5-year DFS was 62% versus 45%, univariable hazard ratio (HR), 0.55 (95% CI, 0.38 to 0.80, P = .001). The restricted mean survival time (RMST) was 18 months longer (95% CI, 6 to 30) in the chemotherapy arm. There were 46 and 60 deaths in the chemotherapy and control arms, respectively. The 5-year OS was 66% versus 57%, with univariable HR, 0.68 (95% CI, 0.46 to 1.00, P = .049) and RMST difference 11 months (95% CI, 1 to 21). Treatment effects were consistent across chemotherapy regimens (carboplatin or cisplatin) and disease stage. Toxicities were similar to those previously reported, and there were no clinically relevant differences in quality of life between arms. In summary, although OS was not the primary outcome measure, the updated results add further support for the use of adjuvant chemotherapy in patients with UTUC, suggesting long-term benefits.
Assuntos
Nefroureterectomia , Humanos , Nefroureterectomia/métodos , Quimioterapia Adjuvante , Feminino , Intervalo Livre de Doença , Masculino , Idoso , Pessoa de Meia-Idade , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/cirurgia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/mortalidade , Neoplasias Urológicas/cirurgia , Neoplasias Urológicas/patologia , Neoplasias Ureterais/tratamento farmacológico , Neoplasias Ureterais/mortalidade , Neoplasias Ureterais/cirurgia , Neoplasias Ureterais/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/mortalidade , Neoplasias Renais/cirurgia , Neoplasias Renais/patologiaRESUMO
Measurement of cellular resting membrane potential (RMP) is important in understanding ion channels and their role in regulation of cell function across a wide range of cell types. However, methods available for the measurement of RMP (including patch clamp, microelectrodes, and potential-sensitive fluorophores) are expensive, slow, open to operator bias, and often result in cell destruction. We present non-contact, label-free membrane potential estimation which uses dielectrophoresis to determine the cytoplasm conductivity slope as a function of medium conductivity. By comparing this to patch clamp data available in the literature, we have demonstratet the accuracy of this approach using seven different cell types, including primary suspension cells (red blood cells, platelets), cultured suspension cells (THP-1), primary adherent cells (chondrocytes, human umbilical mesenchymal stem cells), and adherent (HeLa) and suspension (Jurkat) cancer cell lines. Analysis of the effect of ion channel inhibitors suggests the effects of pharmaceutical agents (TEA on HeLa; DMSO and neuraminidase on red blood cells) can also be measured. Comparison with published values of membrane potential suggest that the differences between our estimates and values recorded by patch clamp are accurate to within published margins of error. The method is low-cost, non-destructive, operator-independent and label-free, and has previously been shown to allow cells to be recovered after measurement.
Assuntos
Eletroforese , Potenciais da Membrana , Humanos , Potenciais da Membrana/fisiologia , Eletroforese/métodos , Células HeLa , Células Jurkat , Técnicas de Patch-Clamp/métodos , Eritrócitos/citologia , Eritrócitos/metabolismoRESUMO
BACKGROUND: Recurrence of nonmuscle-invasive bladder cancer (NMIBC) is common after transurethral resection of bladder tumor (TURBT). Photodynamic diagnosis (PDD) provides better diagnostic accuracy and more complete tumor resection and may reduce recurrence. However, there is limited evidence on the longer-term clinical effectiveness and cost-effectiveness of PDD-guided resection. METHODS: In this pragmatic, open-label, parallel-group randomized trial conducted in 22 U.K. National Health Service hospitals, we recruited participants with a suspected first diagnosis of NMIBC at intermediate or high risk for recurrence on the basis of routine visual assessment before being listed for TURBT. Participants were assigned (1:1) to PDD-guided TURBT or to standard white light (WL)guided TURBT. The primary clinical outcome was time to recurrence at 3 years of follow-up, analyzed by modified intention to treat. RESULTS: A total of 538 participants were enrolled (269 in each group), and 112 participants without histologic confirmation of NMIBC or who had had cystectomy were excluded. After 44 months' median follow-up, 86 of 209 in the PDD group and 84 of 217 in the WL group had recurrences. The hazard ratio for recurrence was 0.94 (95% confidence interval [CI], 0.69 to 1.28; P=0.70). Three-year recurrence-free rates were 57.8% (95% CI, 50.7 to 64.2) and 61.6% (95% CI, 54.7 to 67.8) in the PDD and WL groups, respectively, with an absolute difference of −3.8 percentage points (95% CI, −13.37 to 5.59) favoring PDD. Adverse events occurred in less than 2% of participants, and rates were similar in both groups, as was health-related quality of life. PDD-guided TURBT was £876 (95% CI, −766 to 2518; P=0.591) more costly than WL-guided TURBT over a 3-year follow-up, with no evidence of a difference in quality-adjusted life years (−0.007; 95% CI, −0.133 to 0.119; P=0.444). CONCLUSIONS: PDD-guided TURBT did not reduce recurrence rates, nor was it cost-effective compared with WL at 3 years. (Funded by the National Institute for Health and Care Research Health Technology Assessment program; ISRCTN number, ISRCTN84013636.)