In-vivo Conditions Research Articles

Impact of the external knee flexion moment on patello-femoral loading derived from in vivo loads and kinematics

IntroductionAnterior knee pain and other patello-femoral (PF) complications frequently limit the success of total knee arthroplasty as the final treatment of end stage osteoarthritis. However, knowledge about the in-vivo loading conditions at the PF joint remains limited, as no direct measurements are available. We hypothesised that the external knee flexion moment (EFM) is highly predictive of the PF contact forces during activities with substantial flexion of the loaded knee.Materials and methodsSix patients (65–80 years, 67–101 kg) with total knee arthroplasty (TKA) performed two activities of daily living: sit-stand-sit and squat. Tibio-femoral (TF) contact forces were measured in vivo using instrumented tibial components, while synchronously internal TF and PF kinematics were captured with mobile fluoroscopy. The measurements were used to compute PF contact forces using patient specific musculoskeletal models. The relationship between the EFM and the PF contact force was quantified using linear regression.ResultsMean peak TF contact forces of 1.97–3.24 times body weight (BW) were found while peak PF forces reached 1.75 to 3.29 times body weight (BW). The peak EFM ranged from 3.2 to 5.9 %BW times body height, and was a good predictor of the PF contact force (R2 = 0.95 and 0.88 for sit-stand-sit and squat, respectively).DiscussionThe novel combination of in vivo TF contact forces and internal patellar kinematics enabled a reliable assessment of PF contact forces. The results of the regression analysis suggest that PF forces can be estimated based solely on the EFM from quantitative gait analysis. Our study also demonstrates the relevance of PF contact forces, which reach magnitudes similar to TF forces during activities of daily living.

Vitrification and rapid rewarming of precision-cut liver slices for pharmacological and biomedical research.

High-throughput in vitro pharmacological toxicity testing is essential for drug discovery. Precision-cut liver slices (PCLS) provide a robust system for screening that is more representative of the complex 3D structure of the whole liver than isolated hepatocytes. However, PCLS are not available as off-the-shelf products, significantly limiting their translational potential. Cryopreservation could solve this bottleneck by effectively preserving PCLS indefinitely until their time of use. Conventional cryopreservation (slow cooling in DMSO-forming ice) results in poor PCLS viability and function and, therefore, has proven unsuitable. Here, we explore an "ice-free" cryopreservation approach called vitrification and focus on culturing and assessing PCLS for 3 days post-vitrification and rewarming, given that most acute drug toxicity tests are conducted over 24h. Rat liver slices were diffusively loaded with a cryoprotective agent (CPA) cocktail consisting of EG and Sucrose. The CPA-loaded PCLS were placed on a polymer cryomesh, vitrified in liquid nitrogen (LN2), and rapidly rewarmed in CPA. The vitrified and rewarmed PCLS were subsequently cultured in a controlled volume of serum-free, chemically defined media for 3 days. The cryopreserved PCLS maintained high viability, morphology, function, enzymatic activity, and drug toxicity response. Results show that the vitrified PCLS perform comparably to untreated controls and significantly outperform conventionally cryopreserved PCLS in all assessments ( p < 0.05). Rapid vitrification and rewarming of PCLS using cryomesh enabled successful preservation and culture. This approach maintained high viability, function, enzymatic activity, and drug response for 3 days in culture, similar to controls. The implications of using vitrification to store PCLS are extensive. This technology provides an exciting opportunity for the development of an "off-the-shelf" cold supply chain of human PCLS from organs declined for transplant, which are sliced, cryopreserved, and stored in a repository and available for on-demand shipping for industrial and academic biomedical research. This would also allow PCLS to become a scalable, reproducible, wide-ranging, and population-representative source of tissue that can accurately mimic in-vivo conditions of the human liver. These transformative technologies could revolutionize our practice in studying not just the metabolism of drugs but also increase our capacity to study the zonal progression of many liver diseases and conduct other exciting biomedical research.

The Utility of Total Thrombus-Formation Analysis System (T-TAS) in the Thrombosis and Hemostasis Field: A Scoping Review.

A wide variety of laboratory hemostasis tests is available, but the majority is plasma-based, static and unable to assess platelet function and fibrin formation simultaneously. The Total Thrombus-Formation Analysis System (T-TAS) is a microchip-based flow chamber system that simulates invivo conditions for evaluating whole blood thrombogenicity. A comprehensive overview of its applicability in different thrombosis and hemostasis related clinical situations is lacking and therefore this scoping review was performed. A literature search was done using the electronic databases PubMed, Scopus and Embase on January 7, 2024. Original studies assessing the usefulness of the T-TAS in thrombosis and hemostasis related clinical situations were eligible for this scoping review. A total of 28 studies were included; six studies investigating the role of the T-TAS in congenital bleeding disorders, five studies using the T-TAS to assess 1-year bleeding risk in patients on antiplatelet or anticoagulant medications, four studies investigating the effects of thrombocytopenia and hemodialysis on thrombus formation as measured by the T-TAS, 11 studies testing the applicability of the T-TAS in the monitoring of anticoagulant and antiplatelet therapies and eventually two studies on the ability of the T-TAS to assess the thrombogenicity in different disease entities. The T-TAS method is an interesting technology that mimics the complex biological coagulation process using shear forces, creating a "blood vessel component on a chip". More research is needed, but it could eventually function as a screening test for platelet function and coagulation. Moreover, it could be used to detect the presence of anticoagulant and/or antiplatelet medication.

A Critical Look at the Evolution of Flow Analysis

The inception of flow analysis (FA) is closely related to the need for a better understanding of chemical reactions. At the beginning of the last century, these reactions were intensively investigated, and the main involved parameters were the reactant amounts and the available reaction time.¹ It was soon realized that solution mixing and detection were often time-dependent, so they were better accomplished on a fluidic basis.² This stimulated the development of simple flow-based instruments³ that allowed chemical reactions to be more efficiently investigated. The availability of commercial colorimetric detectors in the 1930s4 increased the demand for chemical analysis and, thus, the laboratory workload. This motivated the proposal of the AutoAnalyzer®, the first commercial air-segmented flow analyzer.5 It was intensively used for large-scale assays, persisted for several decades, and involved only a few manufacturing companies. As the implementation of official, recommended, or tentative methods was preferred, the AutoAnalyzer underwent a relatively slow evolution yet an amazing worldwide acceptance. Later on, flow analyzers without segmentation were proposed.6 An example is the flow injection analyzer, which involves precise sample insertion, controlled dispersion, and reproducible timing. Exploitation of these features improved the simplicity, versatility, and flexibility of the analyzer. Further evolution led to the emergence of the sequential injection analyzer and derived ones, which occasionally involved segmented and unsegmented streams. Nowadays, flow analysis is approaching maturity, as evidenced by the number of applications, books, book chapters, tutorials, thesis, academic courses, workshops, scientific articles, events, seminars, commercially available instruments (some of them specially designed for educational purposes), patents, etc. Intensive development of expert flow systems,7 usually exploiting manifold programming,8 is underway. Micro-flow analyzers are also being proposed, especially for large-scale assays under laboratory, in-situ and in-vivo conditions. Compliance with the principles of Green9 and White10 Analytical Chemistry has always been a positive factor for FA development. For a FA healthy evolution, inertia or setback should be avoided, and synergy rather than divergence should be ensured. To this end, flashback and consensus are relevant. Flashback permits the evaluation a particular situation in the past, whereas consensus is more related to future developments.

The White Matter Fiber Tract Deforms Most in the Perpendicular Direction During In Vivo Volunteer Impacts.

White matter (WM) tract-related strains are increasingly used to quantify brain mechanical responses, but their dynamics in live human brains during invivo impact conditions remain largely unknown. Existing research primarily looked into the normal strain along the WM fiber tracts (i.e., tract-oriented normal strain), but it is rarely the case that the fiber tract only endures tract-oriented normal strain during impacts. In this study, we aim to extend the invivo measurement of WM fiber deformation by quantifying the normal strain perpendicular to the fiber tract (i.e., tract-perpendicular normal strain) and the shear strain along and perpendicular to the fiber tract (i.e., tract-oriented shear strain and tract-perpendicular shear strain, respectively). To achieve this, we combine the three-dimensional strain tensor from the tagged magnetic resonance imaging with the diffusion tensor imaging (DTI) from an open-access dataset, including 44 volunteer impacts under two head loading modes, i.e., neck rotations (N = 30) and neck extensions (N = 14). The strain tensor is rotated to the coordinate system with one axis aligned with DTI-revealed fiber orientation, and then four tract-related strain measures are calculated. The results show that tract-perpendicular normal strain peaks are the largest among the four strain types (p < 0.05, Friedman's test). The distribution of tract-related strains is affected by the head loading mode, of which laterally symmetric patterns with respect to the midsagittal plane are noted under neck extensions, but not under neck rotations. Our study presents a comprehensive invivo strain quantification toward a multifaceted understanding of WM dynamics. We find that the WM fiber tract deforms most in the perpendicular direction, illuminating new fundamentals of brain mechanics. The reported strain images can be used to evaluate the fidelity of computational head models, especially those intended to predict fiber deformation under noninjurious conditions.

Withaferin-A induced vimentin S56 phosphorylation dissociates NEDD9 signaling loop to regress progressive metastatic melanoma into lung adenocarcinoma

Metastasis is complex and insidious type of disease involves multiple signaling nexus, which have implications in understanding disease pathogenesis. Treatment failure for metastatic cancer is frequently high due to aggressive adaptation of cancerous cells to invade to neighboring organs. Cytoskeleton intermediate filamentous protein Vimentin and scaffolding protein Neural precursor cell expressed Developmentally Down-regulated protein 9 (NEDD9) play a key role in metastatic events by regulating multiple metastatic events. Interaction between these proteins is necessary to promote metastatic progression. Withaferin A (WFA), a natural pharamacophore, known to target Vimentin to induce antitumor potential. However exact molecular mechanism still yet to be elucidated. We hypothesize, Vimentin-NEDD9 signaling nexus is necessary for metastatic progression and targeting this interwoven signaling loop with effective pharamacophore WFA halts metastatic progression of melanoma into lung. To elucidate the same, we carried out gene expression measurement through quantitative Reverses Transcription Polymerase Chain Reaction (qRT-PCR), Immunoblot and Immunohistochemistry. Assessment of interactive signaling by Co-immunoprecipitation, Immunofluorescence, Co-localization and Proximity ligation assay. Phosphorylation studies through transfection of phospho specific mutant constructs generated through site directed mutagenesis. WFA induced cellular behavioral changes by migration, invasion assays and Immunoblot analysis. The B16F10 induced mouse metastatic melanoma model to asses NEDD9-Vimentin expression and anti-metastasis induced by WFA. The results postulates, elevated levels and interaction between NEDD9-Vimentin proteins, have positive correlation in metastatic progression of melanoma into lung in both in-vitro and in-vivo condition, establishing it as therapeutic target. Pharmacologically, WFA targets this complex by extending its activity by not only inducing specific Serine 56 phosphorylation of Vimentin, also dissociates NEDD9 signaling loop to halt Epithelial-mesenchymal transition (EMT) and subsequent metastatic events. Eventually, modulation of the relevant metastatic genes E-Cadherin, N-Cadherin, SNAIL, MMP-2 & MMP-9 resulted in regression of metastatic melanoma progression to lung. The study validates WFA induced S56 phosphorylation is necessary to abrupt the NEDD9-Vimentin metastatic signaling complex to regress aggressive metastatic melanoma. The investigation emphasized more mechanistic approach of WFA. Understanding and targeting such integrative mechanical input in the tumor microenvironment will be a better therapeutic strategy to combat metastasis.

Enhancing the wound healing potential using earthworm clitellum factors and elucidating its molecular mechanism in an in-vitro and earthworm model

Earthworm, Eudrilus eugeniae cannot survive and regenerate without clitellum segments. In regenerating worms, the clitellum’s epithelial and circular muscular layers are reduced to one-third, and longitudinal cell layers to half. In C2C12 cells, Clitellum Factors (CF − 5, 25 and 50%) and Regenerative Clitellum Factors (RCF − 5, 25, 50, 75%) ameliorate the cell viability up to 20–28% and 30–38% respectively than the control. In contrast, extracts from body segments negatively influence cell viability up to 80%. In a scratch-wound assay, 25% RCF and 5% CF achieved 99.86% and 81.54% wound closure in 24 h, respectively, compared to 40% in controls. RCF and CF also possess enhanced anti-microbial activity against gram + ve bacteria. Western Blotting reveals that Wnt3a, HoxD3 and VEGF were remarkably upregulated in RCF and CF treated samples and their upregulated stemness property is effectively regulated by p53, TCTP, H2AX, Cleaved Caspase-3 proteins. Immunofluorescence data clearly states that Wnt3a and Caspase-3 signals are more profoundly observed in nuclear over cytoplasm in RCF treated samples and H2AX shows less nuclear signals than CF. In in-vivo earthworm model conditions, RCF remarkably promotes the survivability and wound healing ability by promoting the Wnt3a and VEGF expression together with downregulation of Cox2.

Effects of electric fields on the release and content of extracellular vesicles.

Extracellular vesicles (EVs) are small membrane-bound structures that originate from various cell types and carry molecular cargos to influence the behaviour of recipient cells. The use of EVs as biomarkers for diagnosis and as delivery vehicles for treatment in a wide range of human disease is a rapidly growing field in research and clinical practice. We hypothesized that electric fields (EFs) could influence the release and content of EVs. To examine this hypothesis, we developed a specialized bioreactor enabling cells to thrive in a three-dimensional setting, replicating in-vivo conditions amidst programmable EF environments. We established a three-step EV purification protocol to achieve high-density production of EVs. We also performed mass spectrometry-based proteomics analysis on EV-carrying proteins and used high-resolution nanoparticle flowcytometry for single-vesicle analysis. Findings from this report suggest that electrical stimulation, employing physiologically relevant amplitudes typical in therapeutic deep brain stimulation, influences the release of EVs and their cargo content in a frequency-dependent fashion. This conclusion could carry significant implications for both fundamental biological understanding and medical advancements. First, it raises an intriguing question about how the endogenous electrical activity of neuronal and other cellular assemblies influence the production and composition of EVs. Second, it reveals a novel underlying mechanism of how therapeutic electrical stimulations can modulate EVs and treat human brain disorders. Third, it provides a novel approach to utilize electrical stimulation for generating desired EV cargos in a programmable setting.

TRPV4 modulation affects mitochondrial parameters in adipocytes and its inhibition upregulates lipid accumulation

Enhanced lipid-droplet formation by adipocytes is a complex process and relevant for obesity. Using knock-out animals, involvement of TRPV4, a thermosensitive ion channel in the obesity has been proposed. However, exact role/s of TRPV4 in adipogenesis and obesity remain unclear and contradictory. Here we used in vitro culture of 3T3L-1 preadipocytes and primary murine-mesenchymal stem cells as model systems, and a series of live-cell-imaging to analyse the direct involvement of TRPV4 exclusively at the adipocytes that are free from other complex signalling as expected in in-vivo condition. Functional TRPV4 is endogenously expressed in pre- and in mature-adipocytes. Pharmacological inhibition of TRPV4 enhances differentiation of preadipocytes to mature adipocytes, increases expression of adipogenic and lipogenic genes, enhances cholesterol, promotes bigger lipid-droplet formation and reduces the lipid droplet temperature. On the other hand, TRPV4 activation enhanced the browning of adipocytes with increased UCP-1 levels. TRPV4 regulates mitochondrial-temperature, Ca2+-load, ATP, superoxides, cardiolipin, membrane potential (ΔΨm), and lipid-mitochondrial contact sites. TRPV4 also regulates the extent of actin fibres, affecting the cells mechanosensing ability. These findings link TRPV4-mediated mitochondrial changes in the context of lipid-droplet formation involved in adipogenesis and confirm the direct involvement of TRPV4 in adipogenesis. These findings may have broad implication in treating adipogenesis and obesity in future.

Promising Drug Delivery System for Cervical Cancer – A Future Approach

There is evidence that peptide targeting can be applied in cervical cancer therapy and for diagnosis involving the use of molecular imaging, hence developing peptide probes for this disease. Cervical cancer, which also has a high possibility of being prevented, is among the common tumors affecting women globally. Despite the occurrence being reduced greatly by screening, the illness remains fatal and its claims many lives annually, especially in the developing nations. In exercising such a perspective, specific public health efforts known to encourage physical activity, proper diet, and early detection of cervical cancer must begin to be taken seriously. When combined, doctors, health officers, and lawmakers could speed up the global deployment of such programs. California’s American Cancer Society, the present review enlists a host of pharmaceutical goods available in the market. This perspective of present-day research and manufacturing is devoted to the development of various types of nanocarriers for possible utilization in medication transport. Thus, overall cancer mortality appears to be not substantially different, though cancer incidence rates for both sexes are 50% lower in impoverished countries when compared to affluent nations. At the moment, there are new peptide probes that are developed solely for cervical cancer-perfectly suitable for targeting peptides applicable in therapeutic and diagnostic imaging. M13KE phage dodecapeptide (12-mer) peptide library was screened for recognition of human immunodeficiency virus type 1 (HIV-1) by immunofluorescence and flow cytometry; the S7 clone was identified as most suitable. A disease that affects many women is gynecologic health problems, and cervical cancer is the leading presenting complaint experienced regularly. Among these studies, QSAR, drug-likeness, PCA, dynamic cross-correlation matrix, molecular docking, molecular dynamics and quantum calculation properties can be listed. In the first literature survey, after identifying the potent antibacterial and anticancer activity of the molecule apigenin, some other promising derivatives were identified and more research has been carried out on them, focusing on their synthesized derivatives as inhibitors of DNA polymerase theta and cervical cancer caused by human papillomavirus. The results were streamlined by the use of in-silico molecular docking, which showed that all Apigenin derivatives and the targeted proteins had potential energy-binding relationships. Further validation is essential because this study used in-silico computational methodologies and produced excellent results. Applying in-silico molecular docking made the data more comprehensible, which showed the possible energy bindings of the targeted proteins and all the Apigenin variants. Due to the fact that this work utilized in-silico computational methods and obtained significant results, further validation is required. Hence, the present wet-lab experiments coupled with both in-vitro and in-vivo conditions, additional validation of the results.

Synthesis, Preclinical Toxicity, and Biodistribution of [18F]AVT-011 to Assess the P-Glycoprotein Function.

Introduction: Many studies have reported the role of P-glycoprotein (Pgp) in chemoresistance in various pathological conditions such as cancer and neurodegenerative diseases, such as Alzheimer's. In this study, we are reporting the high-performance liquid chromatography (HPLC)-based purification of fluorine-18 [18F]AVT-011 and its preclinical evaluation. Methods: AVT-011 was labeled with 18F using the nucleophilic substitution method by heating the reaction mixture at 110°C for 10 min, followed by purification using preparative HPLC and C18ec cartridge. The in vitro cell uptake study was carried out in U87 cells with and without an inhibitor. The preclinical toxicity was carried out in CD1 mice in three groups, including control, AVT-011 treated, and [18F]AVT-011 treated. The biodistribution study was done in CD1 mice (n = 12) after intravenous injection of 4-6 MBq [18F]AVT-011, and mice were sacrificed at various time intervals. A dose of 3.7 ± 0.7 MBq of [18F]AVT-011 was injected intravenously in the healthy Swiss albino mice, and the whole-body micro-positron emission tomography was acquired at 0-, 30-, 60-, and 120-min postinjection. Results: The radiochemical purity of [18F]AVT-011 was 97 ± 1.5% as evaluated by radio-HPLC with a yield of 14 ± 2% and was stable up to 95% under in vitro conditions in blood and invivo conditions up to 4 h. The in vitro cell uptake study showed a significant difference in control (27.4 ± 2.1%) and blocked U987 cells (73.2 ± 3.2%) after incubation of 120 min. The tissue distribution in mice showed the highest uptake in the liver (17.3 ± 2.4%), kidneys (16.6 ± 3.1%), lungs (10.4 ± 2.9%), and spleen (5.6 ± 0.8%) at 15 min, and the activity was washed out with time. The radioactivity cleared through the hepatorenal pathway. The animal imaging study also demonstrates a similar biodistribution pattern. Conclusions: [18F]AVT-011 showed higher specific activity than the cartridge-based method but showed similar biological activity.

An investigation of PLLA hybrid stent design to overcome thick strut problems

Biodegradable polymer-based stents simultaneously provide scaffolding, drug release, and biodegradation to eliminate chronic inflammation. The most important factors hindering the wide use of these stents are thick struts, low radial strength, and large footprints formed on the inner wall of the artery as a result of stent expansion. Negative Poisson’s Ratio (NPR), also known as the Auxetic design, has shown great potential to provide radial strength with less strut thickness. However, a detailed mechanical evaluation proving improvement in stent performance parameters is not available in the literature. In this study, the performance parameters of two stent designs based on the Auxetic geometry with PLLA were analyzed under in-vivo conditions using an in-silico model consisting of the artery, crimper, and expander FE model. For this purpose, one design utilizes Auxetic unit cell, which is already available in the literature, while the other uses a newly proposed Hybrid design combining Auxetic and Chevron type geometries. Additionally, a specially heated coaxial balloon-catheter system was considered as a deployment tool between glass transition and body temperature, and carried out for thin-strut stent simulations. The Hybrid design is shown to resolve the foreshortening problem of Auxetic design and collapse pressure of commercial PLLA stents. In this present study validates the potential of Hybrid design to overcome problems for polymer-based biodegradable stents.

Design of biphasic Fe and Zn doped hydroxyapatite: Novel strategy for combating osteomyelitis infections

Hydroxyapatite (HA) bioceramics are widely used materials for orthopaedic applications, offering enhanced cellular adhesion and differentiation. However, the lack of antibacterial features makes the HA biomaterials prone to bacterial infections in in-vivo conditions. The osteomyelitis-like conditions are often treated with prolonged doses of antibiotics and may require surgical removal. Such problems can be tackled by inducing the antibacterial characteristics in the biomaterials which prevent such infections. Various research studies have been conducted to improve the mechanical and antibacterial characteristics of biomaterials like HA through doping. The present study focuses on the synthesis, characterization, and antibacterial study of Zn and Fe doped HA (ZFHAp) for application in the treatment of osteomyelitis. Response Surface Methodology (RSM) based Central Composite Design (CCD) approach was used to optimize the process parameters using the Design Expert software. The analyses indicated the presence of Calcium Iron Hydrogen Phosphate (CIHP) and Parascholzite (PS) as major phases. The optimized experimental conditions were estimated as 0.09 M Zn and 0.04 M Fe at 65 °C, corresponding to 71.29 % and 28.71 % of the CIHP and PS phases, respectively. The synthesized nanopowder showed mixed morphology comprising spherical and rod-shaped particles due to biphasic composition. The ZFHAp sample showed a broad-spectrum antibacterial activity, with IC50 values of 4.02 mg/ml and 4.79 mg/ml against E. coli and S. aureus, respectively. The optimized sample showed excellent biocompatibility with the osteoblast-like cells (MG-63) with improved biomineralization properties. The antibacterial and osteoinductive characteristics of the synthesized biphasic Fe and Zn doped HA suggest its potential application against osteomyelitis-related conditions.

Calcium ions do not influence the Aβ(25–35) triggered morphological changes of lipid membranes

We have studied the effect of calcium ions (Ca2+) at various concentrations on the structure of lipid vesicles in the presence of amyloid-beta peptide Aβ(25–35). In particular, we have investigated the influence of calcium ions on the formation of recently documented bicelle-like structures (BLSs) emerged as a result of Aβ(25–35) triggered membrane disintegration. First, we have shown by using small-angle X-ray and neutron scattering that peptide molecules rigidify the lipid bilayer of gel phase DPPC unilamellar vesicles (ULVs), while addition of the calcium ions to the system hinders this effect of Aβ(25–35). Secondly, the Aβ(25–35) demonstrates a critical peptide concentration at which the BLSs reorganize from ULVs due to heating and cooling the samples through the lipid main phase transition temperature (Tm). However, addition of calcium ions does not affect noticeably the Aβ-induced formation of BLSs and their structural parameters, though the changes in peptide's secondary structure, e.g. the increased α-helix fraction, has been registered by circular dichroism spectroscopy. Finally, according to 31P nuclear magnetic resonance (NMR) measurements, calcium ions do not affect the lipid-peptide arrangement in BLSs and their ability to align in the magnetic field of NMR spectrometer. The influences of various concentrations of calcium ions on the lipid-peptide interactions may prove biologically important because their local concentrations vary widely in in-vivo conditions. In the present work, calcium ions were investigated as a possible tool aimed at regulating the lipid-peptide interactions that demonstrated the disruptive effect of Aβ(25–35) on lipid membranes.

Radiosensitizing Effects of Metformin on Ionizing Irradiation: An Overview of Animal and Clinical Studies

Background: Radiation therapy is the most common modality to treat various types of cancers. The use of radiosensitizers as an adjuvant therapy to increase the sensitivity of tumors against radiation and to improve the efficiency of radiotherapy has risen over the past years. Metformin, the first-line drug in type two diabetes has attracted the researcher’s attention because of its anti-cancer and anti-proliferative properties and many studies have been conducted to investigate the radiosensitization effects of metformin in different types of malignancies. Objective: This review aims to gather the existing evidence of radiosensitizing effects of metformin in animal and clinical studies. Method: Various databases like PubMed, Scopus, and Google Scholar were searched over a period of 35 years for this review. Results: The results include 54 studies on the radiosensitization effect of metformin in in-vivo and clinical conditions including breast, colorectal, prostate, lung, liver, and other cancers. Conclusion: This review shows that metformin in animal and clinical studies appears to be a potential radiosensitizer for many types of cancers. However, further investigations are needed to determine whether metformin can be used as a radiosensitizer in adjuvant radiotherapy.

P-609 Impact of low versus air oxygen tension on the oocyte maturation in PCOS women undergoing capacitation IVM: a sibling oocyte pilot study

Abstract Study question Does low or air oxygen tension improve the oocyte maturation rate in biphasic capacitation IVM (CAPA-IVM)? Summary answer Low oxygen tension resulted in a lower maturation rate and two-pronuclei fertilization rate compared with air oxygen tension in the CAPA-IVM culture. What is known already CAPA-IVM, with a pre-maturation step, improves the competence of oocytes matured in-vitro. The current CAPA-IVM culture condition is under air oxygen concentration. However, the impact of oxygen tension on in-vitro oocyte maturation needs to be better understood, and it remains uncertain whether the maturation process aligns with in-vivo conditions. Study design, size, duration A sibling oocyte pilot study of 416 cumulus-oocyte complexes (COCs) recruited from fifteen polycystic ovary syndrome (PCOS) women was performed at IVFMD, My Duc Phu Nhuan Hospital, Ho Chi Minh City, Vietnam from November 2023 to January 2024. The study was approved by the Institutional Ethics Committee (22/23/DD-BVMD, dated October 12th, 2023) and conducted according to Good Clinical Practice and Declaration of Helsinki 2002 principles. All participants provided written informed consent. Participants/materials, setting, methods Women (18–37 years) diagnosed with PCOS undergoing CAPA-IVM were recruited. At collection, COCs from each woman were divided into two groups based on the oxygen tension used in the two steps of CAPA-IVM culture, low oxygen tension (5% O2, 6% CO2 at 37 °C) and air oxygen tension (20% O2, 6% CO2 at 37 °C). Embryos were cultured to blastocyst stage. Primary outcome was the oocyte maturation rate. Secondary outcomes were embryology outcomes. Main results and the role of chance A total of 416 COCs from fifteen eligible women were assigned to the low oxygen (207 COCs) and air oxygen tension (209 COCs) groups. Mean age and body max index were 29.0±2.5 years and 22.6±2.6, respectively. The maturation rate was significantly lower in the low oxygen group compared to the air oxygen group (54.1% vs. 66.5%, p = 0.01, respectively). The two-pronuclei fertilization rate per COC was significantly decreased in the low oxygen group compared to the air oxygen group (35.7% vs. 49.3%, p = 0.005, respectively). Rates of day-3 embryos, blastocyst formation rate, and good quality blastocyst rate were also lower but not significant in the low oxygen tension group as compared to the air oxygen tension group. Limitations, reasons for caution The study’s limitations include a relatively small sample size and a single ethnicity group. There was a lack of data on clinical outcomes after transferring embryos from the study. Wider implications of the findings Oocyte maturation rate and two-pronuclei fertilization rate were significantly lower when oxygen tension was reduced in the process of CAPA-IVM culture. Therefore, air oxygen tension should be considered as a standard for oocyte culture in the pre-maturation step of CAPA-IVM. Trial registration number NCT06087354

Quality Assessment by Bile Composition in Normothermic Machine Perfusion of Rat Livers.

Background: The persistent challenge of organ scarcity in liver transplantation leads to an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) is used for improved preservation. Due to the mimicked invivo conditions during normothermic machine perfusion, the liver is metabolically active, which allows quality assessment during perfusion. Bile seems to be of rising interest in clinical studies, as it is easily collectible for analysis. As there are currently no data on biliary bile acids during NMP, the primary objective of this study was to use our experimental rodent NMP model to assess changes in bile composition through organ damage during perfusion to inform clinical evaluation of donor organs during NMP. Methods: Thirty livers from male Sprague-Dawley rats in five groups underwent 6 h of NMP using either erythrocyte-supplemented DMEM or Steen solution, with or without 30 min of warm ischemia time (WIT). We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at 3-hour intervals. Bile samples were analyzed for biliary pH, LDH, and gamma glutamyltransferase, as well as biliary bile acids via mass spectrometry and UHPLC. Results: Compared with regular livers, liver injury parameters were significantly higher in our donation after circulatory death (DCD) model. Bile production was significantly reduced in livers exposed to WIT, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers experiencing WIT. However, regular livers produced a higher total amount of biliary bile acids during perfusion. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced during perfusion due to the missing enterohepatic circulation. Conclusions: WIT-induced liver injury affects bile composition within our small-animal NMP model. We hypothesize this phenomenon to be attributed to the energy-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile. Our results may inform clinical studies, in which biliary bile acids might have a potential as a quantifiable viability marker in human NMP liver transplantation studies.

Host Range Studies of Alternaria Species on Sunflower and Efficacy Studies of Fungicides Against A. Alternata Under Both in-Vitro and in-Vivo Conditions

Alternaria blight is the major profound disease of sunflowers induced by divergent species of Alternaria. Among numerous species of phytopathogenic fungi the genus Alternaria Nees ex Fr. is distributed extensively throughout the world. It is extremely virulent in infection in all economically important plants, including grains, pulses, oilseeds, spices, vegetables, and ornamentals. Current studies aim to explore the widened host range of Alternaria species, influenced by strain variability and environmental factors, leading to yield losses and compromised crop quality. Additionally, these studies conducted to understand expanded host range of sunflower Alternaria species on various crops and weeds within the Asteraceae family. The host range study of Alternaria spp. recovered from sunflowers belonged to six Alternaria species collected from different commercial sunflower growing regions of Karnataka. A. alternata, A. tenuissima, and A. helianthi exhibit a broad host range, infecting various plants of the Asteraceae family, including African marigold, safflower, China aster, and Parthenium. Conversely, A. gossypina, A. burnsii, and A. solani specifically infect sunflowers and do not affect other host species. The results of the host range test indicate that Alternaria spp. infecting sunflower can demonstrate pathogenicity towards other hosts as well, especially under favorable conditions. In vitro testing of novel fungicide compounds against the pathogen revealed that Fluopyram 17.7% + Tebuconazole 17.7% 400 SC, applied at concentrations of 100, 250, 500, 750, and 1000 ppm, effectively prohibited the mycelial growth of the pathogen. Conversely, the most favorable outcomes in the field were observed with seed treatment using Fluxapyroxad FS at 1.5 g/kg seed, resulting in the minimal disease severity (7.36% PDI) and highest seed yield (2153 Kg/ha). This was followed by a foliar spray application of Fluopyram 17.7% + Tebuconazole 17.7% 400 SC at 1 ml/L upon disease onset.

A cyclic di-GMP-binding adaptor protein interacts with aN5-glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both invitro and invivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

Suppressing the HIFU interference in ultrasound guiding images with a diffusion-based deep learning model

Background and objectivesIn ultrasound guided high-intensity focused ultrasound (HIFU) surgery, it is necessary to transmit sound waves at different frequencies simultaneously using two transducers: one for the HIFU therapy and another for the ultrasound imaging guidance. In this specific setting, real-time monitoring of non-invasive surgery is challenging due to severe contamination of the ultrasound guiding images by strong acoustic interference from the HIFU sonication. MethodsThis paper proposed the use of a deep learning (DL) solution, specifically a diffusion implicit model, to suppress the HIFU interference. We considered the images contaminated with HIFU interference as low-resolution images, and those free from interference as high-resolution. While suppressing HIFU interference using the diffusion implicit (HIFU-Diff) model, the task was transformed into generating a high-resolution image through a series of forward diffusion steps and reverse sampling. A series of ex-vivo and in-vivo experiments, conducted under various parameters, were designed to validate the performance of the proposed network. ResultsQuantitative evaluation and statistical analysis demonstrated that the HIFU-Diff network achieved superior performance in reconstructing interference-free images under a variety of ex-vivo and in-vivo conditions, compared to the most commonly used notch filtering and the recent 1D FUS-Net deep learning network. The HIFU-Diff maintains high performance with ‘unseen’ datasets from separate experiments, and its superiority is more pronounced under strong HIFU interferences and in complex in-vivo situations. Furthermore, the reconstructed interference-free images can also be used for quantitative attenuation imaging, indicating that the network preserves acoustic characteristics of the ultrasound images. ConclusionsWith the proposed technique, HIFU therapy and the ultrasound imaging can be conducted simultaneously, allowing for real-time monitoring of the treatment process. This capability could significantly enhance the safety and efficacy of the non-invasive treatment across various clinical applications. To the best of our knowledge, this is the first diffusion-based model developed for HIFU interference suppression.