The European intercomparison of in-vivo monitoring laboratories: the EIVIC-2020 project.

The EIVIC project was launched in 2020, and the main goal was the organisation of a European intercomparison of in-vivo monitoring laboratories dealing with direct measurements of gamma-emitting radionuclides incorporated into the body of exposed workers. This project was organised jointly by members of EURADOS Working Group 7 on internal dosimetry (WG7), the Federal Office for Radiation Protection (BfS, Germany) and the Radioprotection and Nuclear Safety Institute (IRSN, France). The objective was to assess the implementation of individual-monitoring requirements in EU Member States on the basis of in-vivo measurements and to gain insight into the performance of in-vivo measurements using whole-body counters. In this context, a total of 41 in-vivo monitoring laboratories from 21 countries, together with JRC (EC) and IAEA participated. The results were submitted in terms of activity (Bq) of the radionuclides identified inside phantoms that were circulated to all participants. The measured data were compared with reference activity values to evaluate the corresponding bias according to the standards ISO 28218 and ISO 13528. In general, the results of the different exercises are good, and most facilities are in conformity with the criteria for the bias and z-scores in the ISO standards. Furthermore, information about technical and organisational characteristics of the participating laboratories was collected to test if they had a significant influence on the reported results.

Design of Bio-Optical Transceiver for In Vivo Biomedical Sensor Applications.

This paper presents an enhanced version of our previously developed bio-optical transceiver, presenting a significant advancement in nanosensor technology. Using self-assembled polymers, this nanodevice is capable of electron detection while maintaining biocompatibility, an essential feature for in vivo medical biosensors. This enhancement finds significance in the field of infectious disease control, particularly in the early detection of respiratory viruses, including high-threat pathogens such as SARS-CoV-2. The proposed system harnesses bioluminescence by converting electric signaling to visible blue light, effectively opening the path of linking nano-sized mechanisms to larger-scale systems, thereby pushing the boundaries of in vivo biomedical sensing. The performance evaluation of our technology is analytical and is based on the use of Markov chains, through which we assess the bit error probability. The calculated improvements indicate that this technology qualifies as a forerunner in terms of supporting the communication needs of smaller, safer, and more efficient manufactured sensor technologies for in vivo medical applications.

Use of the mCherry fluorescent protein to optimize the expression of class I lanthipeptides in Escherichia coli.

BACKGROUND: Lanthipeptides are a rapidly expanding family of ribosomally synthesized and post-translationally modified natural compounds with diverse biological functions. Lanthipeptide structural and biosynthetic genes can readily be identified in genomic datasets, which provides a substantial repository for unique peptides with a wide range of potentially novel bioactivities. To realize this potential efficiently optimized heterologous production systems are required. However, only a few class I lanthipeptides have been successfully expressed using Escherichia coli as heterologous producer. This may be attributed to difficulties experienced in the co-expression of structural genes and multiple processing genes as well as complex optimization experiments. RESULTS: Here, an optimized modular plasmid system is presented for the complete biosynthesis for each of the class I lanthipeptides nisin and clausin, in E. coli. Genes encoding precursor lanthipeptides were fused to the gene encoding the mCherry red fluorescent protein and co-expressed along with the required synthetases from the respective operons. Antimicrobially active nisin and clausin were proteolytically liberated from the expressed mCherry fusions. The mCherry-NisA expression system combined with in vivo fluorescence monitoring was used to elucidate the effect of culture media composition, promoter arrangement, and culture conditions including choice of growth media and inducer agents on the heterologous expression of the class I lanthipeptides. To evaluate the promiscuity of the clausin biosynthetic enzymes, the optimized clausin expression system was used for the heterologous expression of epidermin. CONCLUSION: We succeeded in developing novel mCherry-fusion based plug and play heterologous expression systems to produce two different subgroups of class I lanthipeptides. Fully modified Pre-NisA, Pre-ClausA and Pre-EpiA fused to the mCherry fluorescence gene was purified from the Gram-negative host E. coli BL21 (DE3). Our study demonstrates the potential of using in vivo fluorescence as a platform to evaluate the expression of mCherry-fused lanthipeptides in E. coli. This allowed a substantial reduction in optimization time, since expression could be monitored in real-time, without the need for extensive and laborious purification steps or the use of in vitro activity assays. The optimized heterologous expression systems developed in this study may be employed in future studies for the scalable expression of novel NisA derivatives, or novel genome mined derivatives of ClausA and other class I lanthipeptides in E. coli.

Ultrasonographic monitoring of fetal eye growth parameters throughout gestation in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is considered an ideal species for developing genetically modified nonhuman primates (NHP) models of human disease, particularly eye disease. They have been proposed as a suitable bridge between rodents and other NHP models due to their similar ophthalmological features to humans. Prenatal ultrasonography is an accurate and reliable diagnostic tool for monitoring fetal development and congenital malformation. We monitored fetal eye growth and development using noninvasive ultrasonography in 40 heads of clinically normal fetuses during pregnancy to establish the criteria for studying congenital eye anomalies in marmosets. The coronal, sagittal, and transverse planes were useful to identify the facial structures for any associated abnormalities. For orbital measurements, biorbital distance (BOD), ocular diameter (OD), interorbital distance (IOD), and total axial length (TAL) were measured in the transverse plane and carefully identified for intraorbital structures. As a result, high correlations were observed between delivery-based gestational age (GA) and biparietal diameter (BPD), BOD, OD, and TAL. The correlation assessments based on BOD provide more reliable results for monitoring eye growth and development in normal marmosets than any other parameters since BOD has the highest correlation coefficient according to both delivery-based GA and BPD among ocular measurements. In conclusion, orbital measurements by prenatal ultrasonography provide reliable indicators of marmoset eye growth, and it could offer early diagnostic criteria to facilitate the development of eye disease models and novel therapies such as genome editing technologies in marmosets.

Near-Infrared Spectroscopy for the In Vivo Monitoring of Biodegradable Implants in Rats.

Magnesium (Mg) alloys possess unique properties that make them ideal for use as biodegradable implants in clinical applications. However, reports on the in vivo assessment of these alloys are insufficient. Thus, monitoring the degradation of Mg and its alloys in vivo is challenging due to the dynamic process of implant degradation and tissue regeneration. Most current works focus on structural remodeling, but functional assessment is crucial in providing information about physiological changes in tissues, which can be used as an early indicator of healing. Here, we report continuous wave near-infrared spectroscopy (CW NIRS), a non-invasive technique that is potentially helpful in assessing the implant-tissue dynamic interface in a rodent model. The purpose of this study was to investigate the effects on hemoglobin changes and tissue oxygen saturation (StO2) after the implantation of Mg-alloy (WE43) and titanium (Ti) implants in rats' femurs using a multiwavelength optical probe. Additionally, the effect of changes in the skin on these parameters was evaluated. Lastly, combining NIRS with photoacoustic (PA) imaging provides a more reliable assessment of tissue parameters, which is further correlated with principal component analysis.

Age-specific calibration for in vivo monitoring of thyroid: is it necessary?

Usually, an age-specific calibration of detectors used for in vivo monitoring of 131I thyroid radioactivity is not performed in practice. This study aimed to investigate the reduction in uncertainty that one can expect if an age-specific calibration is performed. For this, voxel and stylized computational phantoms of the thyroid, corresponding to children at different age groups, were used to simulate the calibration process of 131I detectors used for thyroid monitoring. SCK•CEN physical phantoms were also used for this purpose. Both analytical and Monte Carlo methods (MCNPX version 2.6.0) were used to estimate the counting efficiencies of the considered detectors. The results show that the uncertainties in the assessment of thyroid activity at a distance of 20 cm would be reduced from a range of +8% to +30%, to a range from - 6% to +15% when age-specific calibration was performed. Using a calibration based on thyroids of adults would result in an overestimation of the thyroid activity for children by up to 30% at a detector-neck distance of about 20 cm; a larger overestimation may be expected at closer distances. It is concluded that age-specific calibration of in vivo monitoring systems for the thyroid is important and has to be taken into consideration to improve the reliability of thyroid dose assessment for children.

Upconversion nanoparticle platform for efficient dendritic cell antigen delivery and simultaneous tracking.

Upconversion nanoparticles (UCNPs) represent a group of NPs that can convert near-infrared (NIR) light into ultraviolet and visible light, thus possess deep tissue penetration power with less background fluorescence noise interference, and do not induce damage to biological tissues. Due to their unique optical properties and possibility for surface modification, UCNPs can be exploited for concomitant antigen delivery into dendritic cells (DCs) and monitoring by molecular imaging. In this study, we focus on the development of a nano-delivery platform targeting DCs for immunotherapy and simultaneous imaging. OVA 254-267 (OVA24) peptide antigen, harboring a CD8 T cell epitope, and Pam3CysSerLys4 (Pam3CSK4) adjuvant were chemically linked to the surface of UCNPs by amide condensation to stimulate DC maturation and antigen presentation. The OVA24-Pam3CSK4-UCNPs were thoroughly characterized and showed a homogeneous morphology and surface electronegativity, which promoted a good dispersion of the NPs. In vitro experiments demonstrated that OVA24-Pam3CSK4-UCNPs induced a strong immune response, including DC maturation, T cell activation, and proliferation, as well as interferon gamma (IFN-γ) production. In vivo, highly sensitive upconversion luminescence (UCL) imaging of OVA24-Pam3CSK4-UCNPs allowed tracking of UCNPs from the periphery to lymph nodes. In summary, OVA24-Pam3CSK4-UCNPs represent an effective tool for DC-based immunotherapy.

Design and Analysis of a Sample-and-Hold CMOS Electrochemical Sensor for Aptamer-based Therapeutic Drug Monitoring.

In this paper, we present the design and the analysis of an electrochemical circuit for measuring the concentrations of therapeutic drugs using structure-switching aptamers. Aptamers are single-stranded nucleic acids, whose sequence is selected to exhibit high affinity and specificity toward a molecular target, and change its conformation upon binding. This property, when coupled with a redox reporter and electrochemical detection, enables reagent-free biosensing with a sub-minute temporal resolution for in vivo therapeutic drug monitoring. Specifically, we design a chronoamperometry-based electrochemical circuit that measures the direct changes in the electron transfer (ET) kinetics of a methylene blue reporter conjugated at the distal-end of the aptamer. To overcome the high-frequency noise amplification issue when interfacing with a large-size (> 0.25 mm2) implantable electrode, we present a sample-and-hold (S/H) circuit technique in which the desired electrode potentials are held onto noiseless capacitors during the recording of the redox currents. This allows disconnecting the feedback amplifiers to avoid its noise injection while reducing the total power consumption. A prototype circuit implemented in 65-nm CMOS demonstrates a cell-capacitance-insensitive input-referred noise (IRN) current of 15.2 pArms at a 2.5-kHz filtering bandwidth. We tested our system in human whole blood samples and measured the changes in the ET kinetics from the redox-labeled aptamers at different kanamycin concentrations. By employing principal component analysis (PCA) to compensate for the sampling errors, we report a molecular noise floor (at SNR = 1) of 3.1 µM with sub 1-sec acquisition time at 0.22-mW power consumption.

New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors.

The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model.IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

3D-printed CuO nanoparticle-functionalized flow reactor enables online fluorometric monitoring of glucose.

An online flow reactor was fabricated by using a fused deposition modeling three-dimensional printing (3DP) technology along with thermoplastic poly(lactic acid) filaments incorporating copper oxide nanoparticles (CuO NPs). In the presence of glucose, the flow reactor displays multi-catalytic activities because accelerates the oxidation of 2',7'-dichlorodihydrofluorescein to form fluorescein which displays green fluorescence under 480 nm excitation (emission wavelength: 530 nm). The CuO NPs exert two functions to mediate electron transfer at a basic reaction condition, viz. direct oxidation of glucose to generate reactive oxygen species (ROS), and prompting the ROS to oxidize 2',7'-dichlorofluorescin diacetate. The flow reactor coupled to a microdialysis sampler and a fluorometer was applied for online fluorometric monitoring of brain extracellular glucose levels in living rats based on scanning of time-resolved fluorescence intensities. After optimization of (a) the manufacture of the flow reactor, (b) the reaction conditions (pH 10; 50 °C), and (c) the online analytical system, the detection limit of the method (when using 10-µL samples of microdialysate) is as low as 6.1 µM (linear range: 0.05-5 mM) with a sampling frequency of 7.5 h-1. To illustrate the method's applicability, analyses of spiked off-line-collected rat brain microdialysates were conducted. In addition, rat brain extracellular glucose levels were monitored in-vivo and online upon neuronal depolarization triggered by perfusing a high-K+ medium. The results demonstrate that functionalizing raw 3DP materials with appropriate nanomaterials can simplify the manufacturing of analytical devices and related analytical procedures. This will extend the diversity and adaptability of current 3DP-enabling analytical strategies. Graphical abstract Schematic presentation of an online flow reactor fabricated using a fused deposition modeling 3D printer along with poly(lactic acid) (PLA) filaments incorporating CuO NPs. The manufactured flow reactor displays multi-catalytic activities and simplifies online fluorometric monitoring of living rat brain extracellular glucose.

Impaired cerebral microcirculation induced by ammonium chloride in rats is due to cortical adenosine release.

BACKGROUND & AIMS: Liver failure results in hyperammonaemia, impaired regulation of cerebral microcirculation, encephalopathy, and death. However, the key mediator that alters cerebral microcirculation remains unidentified. In this study we show that topically applied ammonium significantly increases periarteriolar adenosine tone on the brain surface of healthy rats and is associated with a disturbed microcirculation. METHODS: Cranial windows were prepared in anaesthetized Wistar rats. The flow velocities were measured by speckle contrast imaging and compared before and after 30 min of exposure to 10 mM ammonium chloride applied on the brain surface. These flow velocities were compared with those for control groups exposed to artificial cerebrospinal fluid or ammonium plus an adenosine receptor antagonist. A flow preservation curve was obtained by analysis of flow responses to a haemorrhagic hypotensive challenge and during stepwise exsanguination. The periarteriolar adenosine concentration was measured with enzymatic biosensors inserted in the cortex. RESULTS: After ammonium exposure the arteriolar flow velocity increased by a median (interquartile range) of 21.7% (23.4%) vs. 7.2% (10.2%) in controls (n = 10 and n = 6, respectively, p <0.05), and the arteriolar surface area increased. There was a profound rise in the periarteriolar adenosine concentration. During the hypotensive challenge the flow decreased by 27.8% (14.9%) vs. 9.2% (14.9%) in controls (p <0.05). The lower limit of flow preservation remained unaffected, 27.7 (3.9) mmHg vs. 27.6 (6.4) mmHg, whereas the autoregulatory index increased, 0.29 (0.33) flow units per millimetre of mercury vs. 0.03 (0.21) flow units per millimetre of mercury (p <0.05). When ammonium exposure was combined with topical application of an adenosine receptor antagonist, the autoregulatory index was normalized. CONCLUSIONS: Vasodilation of the cerebral microcirculation during exposure to ammonium chloride is associated with an increase in the adenosine tone. Application of a specific adenosine receptor antagonist restores the regulation of the microcirculation. This indicates that adenosine could be a key mediator of the brain dysfunction seen during hyperammonaemia and is a potential therapeutic target. LAY SUMMARY: In patients with liver failure, disturbances in brain function are caused in part by ammonium toxicity. In our project we studied how ammonia, through adenosine release, affects the blood flow in the brain of rats. In our experimental model we demonstrated that the detrimental effect of ammonia on blood flow regulation was counteracted by blocking the adenosine receptors in the brain. With this observation we identified a novel potential treatment target. If we can confirm our findings in a future clinical study, this might help patients with liver failure and the severe condition called hepatic encephalopathy.

A novel homologous model for noninvasive monitoring of endometriosis progression.

To date, several groups have generated homologous models of endometriosis through the implantation of endometrial tissue fluorescently labeled by green fluorescent protein (GFP) or tissue from luciferase-expressing transgenic mice into recipient animals, enabling noninvasive monitoring of lesion signal. These models present an advantage over endpoint models, but some limitations persist; use of transgenic mice is laborious and expensive, and GFP presents poor tissue penetration due to the relatively short emission wavelength. For this reason, a homologous mouse model of endometriosis that allows in vivo monitoring of generated lesions over time and mimics human lesions in recipient mice would be most desirable. In this regard, using C57BL/6 and B6N-Tyrc-Brd/BrdCrCrl mice, we optimized a decidualization protocol to obtain large volumes of decidual endometrium and mimic human lesions. Subsequently, to obtain a more robust and reliable noninvasive monitoring of lesions, we used the fluorescent reporter mCherry, which presents deeper tissue penetration and higher photostability, showing that endometrial tissue was properly labeled with 1 × 108 PFU/mL mCherry adenoviral vectors. mCherry-labeled endometriotic tissue was implanted in recipient mice, generating lesions that displayed characteristics typical of human endometriotic lesions, such as epithelial cells forming glands, local inflammation, collagen deposits, and new vessel formation. In vivo monitoring demonstrated that subcutaneous implantation on ventral abdomen of recipient mice provided the most intense and reliable signal for noninvasive lesion monitoring over a period of at least 20 days. This homologous model improves upon previously reported models of endometriosis and provides opportunities to study mechanism underlying endometriotic lesion growth and progression. We created a cost-effective but accurate homologous mouse model of endometriosis that allows the study of growth and progression of endometriotic lesions over early time points in lesion development through noninvasive monitoring.

Chlorophyll fluorescence imaging as a tool to monitor the progress of a root pathogen in a perennial plant.

MAIN CONCLUSION: The chlorophyll fluorescence parameter ΦNO is an excellent metric for the non-destructive monitoring of disease progression, measured over a broad range of light intensities. The suitability of the slow induction chlorophyll fluorescence parameters ΦPSII, ΦNPQ, and ΦNO to monitor in vivo disease progression in a host-root pathogen pathosystem was evaluated and compared to the established method of monitoring disease by measuring Fv/Fm. Using the infection of ginseng plants (Panax quinquefolius L.) with Pythium irregulare Buisman as a model, light response curves were used to establish the optimal irradiance for the resolution of differences between fluorescence parameters ΦPSII, ΦNPQ and ΦNO. As infection progressed only changes in ΦNO remained consistent with increased irradiance, and increased as infection progressed. Furthermore, ΦNO showed a high sensitivity for distinguishing increased disease load. In contrast, the magnitude in change of ΦPSII and ΦNPQ were sensitive to irradiance levels. The magnitude of increase in ΦNO per unit disease score was equivalent to the corresponding decline in Fv/Fm values. Thus ΦNO is as sensitive as Fv/Fm in monitoring biotic stress. The ability to measure ΦNO under a wide range of light intensities, including natural light, potentially without the need for dark adaptation, means that it can be used in the development of a general protocol for non-invasive, in vivo monitoring of plant health, from the laboratory to the field scale.

In Vivo Monitoring of the Growth of Fertilized Eggs of Medaka Fish (Oryzias latipes) by Near-Infrared Spectroscopy and Near-Infrared Imaging-A Marked Change in the Relative Content of Weakly Hydrogen-Bonded Water in Egg Yolk Just before Hatching.

The present study develops further our previous study of in vivo monitoring at the molecular level of the embryonic development in Japanese medaka fish (Oryzias latipes) using near-infrared (NIR) spectroscopy and NIR imaging. NIR spectra were measured nondestructively for three major parts of fertilized medaka eggs (the embryonic body, oil droplets, and egg yolk) from the first day after fertilization to the day just before hatching (JBH). Changes in the contents of chemical components such as proteins, water, and lipids were monitored in situ during embryonic development. A marked change in the relative content of weakly hydrogen-bonded water was observed in the egg yolk JBH. Principal component analysis (PCA) was carried out using the NIR spectra data of the egg yolk and embryo on the fifth day after fertilization. The PCA clearly separates the egg yolk data from the embryo body parts. Principal component PC1 and PC2 loading plots suggest that the hydrogen bonding structure of water in the egg yolk is considerably different to those of the other parts and the fraction of weakly hydrogen-bonded water in the egg yolk is smaller than that in the embryonic body. NIR images developed from the intensities of peaks of second derivative spectra owing to water and proteins show their different distribution patterns. Images of the ratio of strongly and weakly hydrogen-bonded water confirmed that oil droplets and embryonic body parts have higher and lower ratios, respectively, of strongly hydrogen-bonded water than do the other parts. The images developed from the intensity of the peaks at 4864 and 4616 cm(-1) related to the proteins indicated that the egg yolk contains a higher concentration of protein than do the other parts. The peaks at 5756 and 4530 cm(-1) caused by the protein secondary structures of α-helix and ß-sheet showed the configuration of the egg cell membrane. The present study might lead to new understanding at the molecular level regarding the growth of fertilized eggs and provides a new tool to visualize egg development in a nondestructive manner.

Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey regulatory T cells.

We expanded flow-sorted Foxp3(+) cynomolgus monkey regulatory T cells (Treg) >1000-fold after three rounds of stimulation with anti-CD3 mAb-loaded artificial antigen-presenting cells, rapamycin (first round only) and IL-2. The expanded Treg maintained their expression of Treg signature markers, CD25, CD27, CD39, Foxp3, Helios, and CTLA-4, as well as CXCR3, which plays an important role in T cell migration to sites of inflammation. In contrast to expanded effector T cells (Teff), expanded Treg produced minimal IFN-γ and IL-17 and no IL-2 and potently suppressed Teff proliferation. Following cryopreservation, thawed Treg were less viable than their freshly-expanded counterparts, although no significant changes in phenotype or suppressive ability were observed. Additional rounds of stimulation/expansion restored maximal viability. Furthermore, adoptively-transferred autologous Treg expanded from cryopreserved second round stocks and labeled with CFSE or VPD450 were detected in blood and secondary lymphoid tissues of normal or immunosuppressed recipients at least two months after their systemic infusion.

PET imaging of neovascularization with (68)Ga-3PRGD2 for assessing tumor early response to Endostar antiangiogenic therapy.

Antiangiogenic therapy is an effective strategy to inhibit tumor growth. Endostar, as an approved antiangiogenesis agent, inhibits the newborn vascular endothelial cells, causing the decrease of integrin αvß3 expression. Radiolabeled 3PRGD2, a novel PEGlayted RGD dimer probe (PEG4-E[PEG4-c(RGDfK)]2) showed highly specific targeting capability to integrin αvß3, which could be used for monitoring the efficacy of Endostar antiangiogenic therapy. In this study, (68)Ga-3PRGD2 PET imaging was performed in Endostar treated/untreated Lewis Lung Carcinoma (LLC) mice on days 3, 7, 14, and 21 post-treatment for monitoring the tumor response to Endostar treatment, with the (18)F-FDG imaging as control. As a result, (68)Ga-3PRGD2 PET reflected the tumor response to Endostar antiangiogenic therapy much earlier (day 3 post-treatment vs day 14 post-treatment) and more accurately than that of (18)F-FDG metabolic imaging, which provides new opportunities to develop individualized therapeutic approaches, establish optimized dosages and dose intervals for effective treatment that improve the survival rate of patients.

Needle-in-needle electrochemical sensor for in-vivo monitoring of anticancer drug etoposide.

Therapeutic drug monitoring (TDM) serves as a potent tool for adjusting drug concentration within a reasonable range. However, continuous monitoring of anticancer drugs in-vivo presents a significant challenge. Herein, we propose a needle-in-needle electrochemical sensor based on an acupuncture needle electrode, capable of monitoring the anticancer drug etoposide in the peritoneal cavity of living rats. The acupuncture needle was modified with Au nanoparticles and etoposide-templated molecularly imprinted polymer (MIP), resulting in high sensitivity and selectivity in the electrochemical detection of etoposide. The modified acupuncture needle (0.16 mm diameter) was anchored inside a syringe needle (1.40 mm diameter), allowing the outer syringe needle to protect the modified materials of the inner acupuncture needle during skin piercing. Due to the unique needle-in-needle design, high stability was obtained during in-vivo etoposide monitoring. Connecting to a smartphone-controlled portable electrochemical workstation, the needle-in-needle sensor offers great convenience in point-of-care TDM. Moreover, the electrode materials on the acupuncture needle were carefully characterized and optimized. Under the optimized conditions, low detection limits and wide linear range were achieved. This work provides new insights into acupuncture needle electrochemical sensors and further expands the feasibility for real-time and in-vivo detection.

In Vivo Monitoring of Glycerolipid Metabolism in Animal Nutrition Biomodel-Fed Smart-Farm Eggs.

Although many studies have examined the biochemical metabolic pathways by which an egg (egg yolk) lowers blood lipid levels, data on the molecular biological mechanisms that regulate and induce the partitioning of hepatic glycerolipids are missing. The aim of this study was to investigate in vivo monitoring in four study groups using an animal nutrition biomodel fitted with a jugular-vein cannula after egg yolk intake: CON (control group, oral administration of 1.0 g of saline), T1 (oral administration of 1.0 g of pork belly fat), T2 (oral administration of 1.0 g of smart-farm egg yolk), and T3 (oral administration of T1 and T2 alternately every week). The eggs induced significant and reciprocal changes in incorporating 14C lipids into the total glycerolipids and releasing 14CO2, thereby regulating esterification and accelerating oxidation in vivo. The eggs increased phospholipid secretion from the liver into the blood and decreased triacylglycerol secretion by regulating the multiple cleavage of fatty acyl-CoA moieties' fluxes. In conclusion, the results of the current study reveal the novel fact that eggs can lower blood lipids by lowering triacylglycerol secretion in the biochemical metabolic pathway of hepatic glycerolipid partitioning while simultaneously increasing phospholipid secretion and 14CO2 emission.

Luciferase transduction and selection protocol for reliable in vivo bioluminescent measurements in cancer research.

Bioluminescence imaging has become an essential non-invasive tool in cancer research for monitoring various cellular processes and tumor progression in vivo. In this article, we aimed to propose a transduction and selection protocol for reliable in vivo bioluminescent measurements in immunocompetent mouse models. Using two different heterogenous luciferase-expressing cell models, we underlined factors influencing transduction. The protocol was tested through an in vitro luciferase activity assay as well as using in vivo longitudinal monitoring of metastases formation (In Vivo Imaging System®). The data were cross validated with histological assessment. Our results demonstrated stable and proportional in vitro and in vivo bioluminescent signals correlating with actual metastatic burden. Furthermore, ex vivo analysis confirmed the accuracy of bioluminescent imaging in quantifying metastatic surface area. This protocol should ensure reliable and reproducible measurements in cancer research utilizing luciferase-positive cell lines, confirming the validity and accuracy of preclinical studies in immunocompetent models.