Fluorescence Microscopy Research Articles

Nanofat Improves Vascularization and Tissue Integration of Dermal Substitutes without Affecting Their Biocompatibility.

Dermal substitutes require sufficient tissue integration and vascularization to be successfully covered with split-thickness skin grafts. To rapidly achieve this, we provide the proof of principle for a novel vascularization strategy with high translational potential. Nanofat was generated from subcutaneous adipose tissue of green fluorescence protein (GFP)+ C57BL/6J donor mice and seeded onto small samples (4 mm in diameter) of the clinically approved dermal substitute Integra®. These samples and non-seeded controls were then implanted into full-thickness skin defects in the dorsal skinfold chamber of C57BL/6J wild-type mice and analyzed by intravital fluorescence microscopy, histology and immunohistochemistry over a 14-day period. Nanofat-seeded dermal substitutes exhibited an accelerated vascularization, as indicated by a significantly higher functional microvessel density on days 10 and 14 when compared to controls. This was primarily caused by the reassembly of GFP+ microvascular fragments inside the nanofat into microvascular networks. The improved vascularization promoted integration of the implants into the surrounding host tissue, which finally exhibited an increased formation of a collagen-rich granulation tissue. There were no marked differences in the inflammatory host tissue reaction to nanofat-seeded and control implants. These findings demonstrate that nanofat significantly improves the in vivo performance of dermal substitutes without affecting their biocompatibility.

Optogenetic estimation of synaptic connections in brain slices

BackgroundDetection of synaptic connections is essential for understanding neural circuits. By using optogenetics, current injection, and glutamate uncaging to activate presynaptic cells and simultaneously recording the subsequent response of postsynaptic cells, the presence of synaptic connections can be confirmed. However, these methods present throughput challenges, such as the need for simultaneous multicellular patch-clamp recording and two-photon microscopy. These challenges lead to a trade-off between sacrificing resolution and experimental throughput. New methodWe adopted the laser, typically used for local field ablation, and combined this with post hoc analysis. We successfully approximated the synaptic connection probabilities using only an epi-fluorescence microscope and single-cell recordings. ResultsWe sequentially stimulated the channelrhodopsin 2-expressing cells surrounding the recorded cell and approximated the synaptic connection probabilities. This probability value was comparable to that obtained from simultaneous multi-cell patch-clamp recordings, which included more than 600 pairs. Comparison with existing methodsOur setup allows us to estimate connection probabilities within 100 s, outperforming existing methods. We successfully estimated synaptic connection probabilities using only the optical path typically used by an epi-fluorescence microscope and single-cell recordings. It may also be suitable for dendritic ablation experiments. ConclusionsThe proposed method simplifies the estimation of connection probabilities, which is expected to advance the study of neural circuits in conditions such as autism and schizophrenia where connection probabilities vary. Furthermore, this approach is applicable not only to local circuits but also to long-range connections, thus increasing experimental throughput.

The relationship between serum interleukin-1β and circulating CD16+ neutrophils for assessing progression-free time in advanced ovarian cancer

BACKGROUND: Ovarian cancer is the most aggressive gynecological tumor with high mortality. The ambivalent, both helper and suppressive effects of neutrophils on the cells of innate and adaptive immunity determines their important immunoregulatory role in the development of malignant tumors. AIM: Evaluation of the effect of serum interleukin-1β and circulating neutrophils CD16+ on progression-free time in advanced ovarian cancer. MATERIAL AND METHODS: In 42 primary patients with serous ovarian adenocarcinoma (median age 54 years) with ascites and 15 patients with benign ovarian tumors (median age 60 years) admitted for examination and treatment to the Regional Clinical Oncology Dispensary before receiving neoadjuvant antitumor treatment (2015–2020), the level of interleukin-1β in the blood serum was determined using enzyme immunoassay, and the number of CD16+ neutrophils was determined by fluorescence microscopy using monoclonal antibodies. Statistical processing was performed using Statistica 13 and Jamovi 2.4.14 programs. Progression-free time analysis of patients was performed using the Cox and Kaplan–Meier regression methods. RESULTS: It was found that the number of circulating CD16+ neutrophils in advanced ovarian cancer with ascites was lower than in benign ovarian tumors [48.0; 95% confidence interval (CI) 46.59–48.12 versus 50.0; 95% CI 48.51–49.76; p=0.017]. At the same time, the level of interleukin-1β in the blood serum in advanced ovarian cancer with ascites was higher than in benign ovarian tumors [2.52 (95% CI 2.50–3.37) versus 1.26 (95% CI 0.97–1.55) (p=0.0001)]. In multivariate Cox analysis in patients with ascites (odds ratio 4.334; 95% CI 1.83–10.23; p=0.001), both the CD16+ neutrophil count (odds ratio 0.73; 95% CI 0.62–0.86; p=0.0001) and serum interleukin-1β levels (odds ratio 1.90; 95% CI 1.41–2.57; p=0.0001) had prognostic significance for assessing progression-free time. CONCLUSION: The number of circulating CD16+ neutrophils and serum interleukin-1β levels simultaneously affect progression-free time in advanced ovarian cancer.

Non-aqueous Pickering emulsions stabilized by natroglaucocerinite-like layered double hydroxides particles

This study explores non-aqueous Pickering emulsions stabilized by natroglaucocerinite-like layered double hydroxides (Zn/Al LDH), featuring two immiscible phases: glycerin (Gly) and various oils (silicone, castor, soybean, or vaseline) over a span of 15 days. The stability of the emulsions was assessed through macroscopic observations of flocculation, creaming, sedimentation, and coalescence. The wettability of the solid particles at the phase interface was analyzed using tensiometry and contact angle measurements, while fluorescence microscopy was used to determine the type of emulsion. Emulsification effectiveness was found to be influenced by the glycerin volume fraction, oil type, and LDH particle concentration. Oils like silicone and vaseline required both a higher LDH concentration and glycerin fraction for complete oil emulsification, whereas castor and soybean oils showed better emulsification at higher glycerin fractions. These findings underscore the potential of LDH as a versatile and eco-friendly solution for applications that demand the reduction or control of specific solvents.

A-169 Artificial Intelligence based Complete Blood Count

Abstract Background The OLO device (Sight diagnostics, Israel) is an artificial intelligence based blood counter. OLO contains a fluorescent microscope and a camera embedded inside it. The pictures are processed by an image processing algorithm based on “machine-vision technology” to identify and quantify cells. The measurement analysis is accompanied by messages (flags) that are used to warn of deviations in the counted parameters, which help the operator refer the sample for microscopic analysis. In this study, we compared the results of 19 complete blood count (CBC) parameters obtained from OLO to the CBC parameters obtained from the ADVIA 2120 device (Siemens, USA). In addition we examined the reliability of OLO alerts (flags) by comparing it to the microscopic analysis findings. Methods 99 blood samples from patients aged >3 months were sampled by the ADVIA and OLO devices. Regression analysis was performed in comparing the results of 19 parameters between the devices. Specificity and sensitivity values ​​of the OLO device alerts were calculated. Results A good correlation was found for CBC parameters between OLO and ADVIA with Pearson correlation (R) of 0.992 (WBC), 0.985 (RBC), 0.991 (HB) and 0.983 (PLT). A fundamental difference in measurement and classification technologies between OLO and ADVIA lead to low correlation measured for monocytes. By comparing OLO alerts against blood smears findings, a total of 14 samples were alerted by OLO but only slight left shift/single pathological cell were found in the smears which is below threshold defined by the manufacturer. These 14 samples were transferred to the true positive category, therefore OLO device alerts showed sensitivity and specificity of 81.8% and 90% (respectively) in identifying pathological cells. Conclusions The comparison findings present a technology based on image processing and artificial intelligence that have a good correlation to the ADVIA reference device. In addition, OLO has the ability to warn of pathological findings even below the threshold defined by the manufacturer. The artificial learning is expected to be improved so that this device can integrate CBC and microscopic differentiation simultaneously, a great advantage to the hematology laboratories workflow.

Mitochondrial Structure, Dynamics, and Physiology: Light Microscopy to Disentangle the Network.

Mitochondria serve as energetic and signaling hubs of the cell: This function results from the complex interplay between their structure, function, dynamics, interactions, and molecular organization. The ability to observe and quantify these properties often represents the puzzle piece critical for deciphering the mechanisms behind mitochondrial function and dysfunction. Fluorescence microscopy addresses this critical need and has become increasingly powerful with the advent of superresolution methods and context-sensitive fluorescent probes. In this review, we delve into advanced light microscopy methods and analyses for studying mitochondrial ultrastructure, dynamics, and physiology, and highlight notable discoveries they enabled.

Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections.

Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)-Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host-parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae.

Xiao-Er-Kang-Du capsules regulate autophagy against the influenza B virus (Victoria strain) through the mTOR/ULK1/Beclin1/VPS34 pathway

Ethnopharmacological relevanceXiao-er-kang-du (XEKD) capsule is a Chinese herbal formula used for treatment of upper respiratory tract infection caused by various viruses in pediatric patients in China. XEKD is used clinically for the treatment of influenza-like symptoms, including fever, chills, cough, stuffy and runny nose, headache, and sore throat, with remarkable efficacy. However, the pharmacologic mechanism of XEKD against influenza B virus (IBV) infection is unclear. Aim of the studyThe main purpose of the present work is to explore the curative effect as well as possible mechanisms of XEKD against influenza B virus (IBV) (Victoria strain). Materials and methodsBoth in vivo and in vitro experiments were performed to confirm the antiviral properties of XEKD. High-performance liquid chromatography was used to analyze the active components and assess the stability of XEKD. In addition, the mechanism of action of XEKD against IBV (Victoria) was investigated by western blot, immunofluorescence, and immunohistochemical analyses, in addition to confocal fluorescence microscopy. ResultsThe findings revealed that XEKD demonstrated antiviral effects against IBV infection in both in vivo and in vitro via the mTOR/ULK1/Beclin1/VPS34 pathway and promote cellular autophagy to mitigate IBV-induced lung tissue damage. The results of this work are expected to lead to a deeper understanding of the mechanism underlying the effect of the XEKD capsule against IBV infections. ConclusionsIBV infection was found to inhibit autophagy, which exacerbated inflammatory damage. XEKD regulates autophagy through the mTOR/ULK1/Beclin1/VPS34 pathway and exerts antiviral effects, thereby laying a foundation for further development of XEKD as a potential therapeutic against IBV infection.

Acne-related UVA-induced facial fluorescence: An exploratory study from physiological properties to tissue structure information

UVA-induced facial fluorescence (UVAF) is recognized as an objective measurement technique to quantify the severity of acne. However, notable inconsistencies in quantitative outcomes have been observed in various studies, possibly due to the fact that different colors of fluorescence represent different pathophysiological implications. This study investigated the pathophysiological importance of UVAF color differences and improved its reliability in assessing acne severity. MIDOO Smart Skin Imager was used to capture UVAF and analyze the correlation between fluorescence colors and acne lesions. Techniques such as two-photon excited fluorescence microscopy, scanning electron microscopy, western blot, and high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) were used to examine the biochemical composition and structure of comedonal plugs and follicular casts associated with different fluorescence colors. We found that green fluorescence correlates with non-inflammatory acne lesions (comedones), while orange-red fluorescence shows no correlation with either type of lesion. Green fluorescence is associated with higher levels of keratin, indicating keratinization, while orange-red fluorescence is associated with porphyrin from S. epidermidis. UVAF color differences - orange-red are from porphyrins and green from keratin. This distinction helps to understand the structural and physiological bases of facial fluorescence, with potential implications for clinical evaluations of acne.

Evaluation of Multiplex loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis complex from clinically suspected cases of pulmonary tuberculosis

Tuberculosis (TB) is the second leading cause of death from a single infectious agent worldwide. Bangladesh ranks 7th among the 30 high TB burdened countries in the world. Accurate detection of Mycobacterium tuberculosis complex (MTBC) is challenging for developing countries as most of the resource poor settings are not suitable to perform molecular techniques.The purpose of the study was to compare the multiplex TB-LAMP assay with MTB/NTM qPCR, culture, Z-N staining, and fluorescence microscopy in order to assess the effectiveness of the LAMP assay for detecting cases of pulmonary tuberculosis.This research work was done from March 2022 to February 2023. Fulfilling the inclusion criteria 130 sputum samples were collected. TB-LAMP assay, qPCR, culture in L-J media, Z-N staining and fluorescence microscopy were performed.Out of 130 samples qPCR detected MTBC in 56.92% cases and TB-LAMP detected 53.85%. MTBC was detected by culture 46.15%, by Fluorescence microscopy 40.77% and Z-N staining 36.92%. TB-LAMP detected 16.93% more cases than Z-N staining and 13.08% more cases than fluorescence microscopy. The sensitivity, specificity, positive, and negative predictive values of multiplex-LAMP assay were 95%, 81.4%, 81.4% and 95% respectively considering culture as a gold-standard. MTBC negative culture samples (18.57%) showed positivity by LAMP assay as well as by qPCR. This study detected 7.69% non-tuberculous mycobacteria (NTM) by qPCR. All NTM positive samples were negative by TB-LAMP.TB-LAMP is an easy to perform, cost-effective, reliable assay with high sensitivity and specificity. World Health Organization recommended TB-LAMP as a rapid molecular test for rapid detection of tuberculosis and as replacement of microscopy in resource poor settings/hard to reach areas. Bangladesh being a high TB burden country it is essential to implement TB-LAMP to achieve End TB Strategy by 2035.

Enhanced spinal cord repair using bioengineered induced pluripotent stem cell-derived exosomes loaded with miRNA

BackgroundA spinal cord injury (SCI) can result in severe impairment and fatality as well as significant motor and sensory abnormalities. Exosomes produced from IPSCs have demonstrated therapeutic promise for accelerating spinal cord injury recovery, according to a recent study.ObjectiveThis study aims to develop engineered IPSCs-derived exosomes (iPSCs-Exo) capable of targeting and supporting neurons, and to assess their therapeutic potential in accelerating recovery from spinal cord injury (SCI).MethodsiPSCs-Exo were characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blot. To enhance neuronal targeting, iPSCs-Exo were bioengineered, and their uptake by neurons was visualized using PKH26 labeling and fluorescence microscopy. In vitro, the anti-inflammatory effects of miRNA-loaded engineered iPSCs-Exo were evaluated by exposing neurons to LPS and IFN-γ. In vivo, biodistribution of engineered iPSC-Exo was monitored using a vivo imaging system. The therapeutic efficacy of miRNA-loaded engineered iPSC-Exo in a SCI mouse model was assessed by Basso Mouse Scale (BMS) scores, H&E, and Luxol Fast Blue (LFB) staining.ResultsThe results showed that engineered iPSC-Exo loaded with miRNA promoted the spinal cord injure recovery. Thorough safety assessments using H&E staining on major organs revealed no evidence of systemic toxicity, with normal organ histology and biochemistry profiles following engineered iPSC-Exo administration.ConclusionThese results suggest that modified iPSC-derived exosomes loaded with miRNA have great potential as a cutting-edge therapeutic approach to improve spinal cord injury recovery. The observed negligible systemic toxicity further underscores their potential safety and efficacy in clinical applications.

Development of erythromycin loaded PLGA nanoparticles for improved drug efficacy and sustained release against bacterial infections and biofilm formation

Bacterial infections are a common cause of sepsis, often leading to high patient mortality. Such infections are challenging to treat due to bacterial resistance to many existing drugs. Erythromycin (Ery) is a macrolide antibiotic used against bacterial infections with reported resistance. Recently, synthetic poly-lactide co-glycolic acid (PLGA) polymer nanoparticles (NPs) have displayed improved drug delivery characteristics and biocompatibility. In this study, PLGA-Ery NPs were synthesized by the o/w emulsion diffusion method, having a particle size of 159 ± 23 nm and displayed 71.89 % of encapsulation efficiency. The PLGA-Ery NPs showed 1.5, 2.1 and 1.5-fold improved MIC and antibacterial efficacy against E. coli, S. aureus, and P. aeruginosa, respectively than the pure drug. As illustrated by scanning electron microscopy, PLGA-Ery NPs caused damage to the bacterial cell walls. Furthermore, a surface coating with PLGA-Ery NPs on a glass surface showed efficient inhibition (>90 %) of the biofilm formation by P. aeruginosa, as determined by fluorescence microscopy and MTT assay. This study demonstrates that PLGA-Ery NPs can increase the efficiency of erythromycin and can suppress the growth and biofilm formation of P. aeruginosa. Such polymeric nanoparticles drug nanoformulations have potential as an antimicrobial and as a surface coating for medical devices.

Hydrogen sulfide donor NaHS inhibits formaldehyde-induced epithelial-mesenchymal transition in human lung epithelial cells via activating TGF-β1/Smad2/3 and MAPKs signaling pathways

Formaldehyde (FA) long term exposure leads to abnormal pulmonary function and small airway obstruction of the patients. Hydrogen sulfide (H2S) is one of the recognized gaseous transmitters involved in a wide range of cellular functions. It is unknown the involvement of H2S in FA-induced lung injury. The purpose of this study is to investigate the therapeutic potential and mechanism of H2S on FA-induced epithelial-mesenchymal transition (EMT) of human lung epithelial cells. The cell viability of Beas2B and A549 cells after FA treatment were assessed using MTT assay. The endogenous H2S was visualized by fluorescence microscopy using of the 7-azido-4-methylcoumarin (AzMC). Cell morphology was observed under phase contrast microscope. The mRNAs and proteins level were evaluated by reverse transcription-polymerase chain reaction and western blotting assays. FA treatment downregulated the endogenous H2S levels and the mRNAs and proteins level of H2S synthesizing enzymes, such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in Beas2B and A549 cells. FA treatment changed the cell morphology of Beas2B cells from cuboid to a spindle-shape, while declined the protein level of E-cadherin and increased the protein level of Vimentin. Moreover, FA treatment increased the proteins level of transforming growth factor-β1 (TGF-β1), phosphorylated-Smad2 (p-Smad2), phosphorylated-Smad3 (p-Smad3), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and phosphorylated-P38 (p-P38). Furthermore, the inhibitors of TGF-β receptor type 1 (TGFβRI) and mitogen-activated protein kinases (MAPKs) signaling pathways reversed FA-induced decrease in E-cadherin expression and increase in Vimentin expression in Beas2B cells. Sodium hydrogen sulfide (NaHS) increased the level of H2S, while reversed FA-induced the low expression of E-cadherin and the high expression of Vimentin, TGF-β1, p-Smad2, p-Smad3, p-ERK, p-JNK, and p-P38. These findings indicates FA treatment downregulating the endogenous H2S in human lung epithelial cells. NaHS may inhibit FA-induced EMT in human lung epithelial cells via modulating TGF-β1/Smad2/3 and MAPKs signaling pathways. Therefore, we demonstrated that supplementation of exogenous H2S may inhibit FA-induced lung injury.

Colonic targeting insulin-loaded trimethyl chitosan nanoparticles coated pectin for oral delivery: In vitro and In vivo studies

Colon-targeted delivery offers several benefits for oral protein delivery, such as low proteolytic enzyme activity, natural pH, and extended residence time, which improve the bioavailability of the encapsulated protein. Therefore, we hypothesize that developing a novel colonic nanocarrier system based on modified chitosan, which enhances solubility at natural pH and is coated with a colon-degradable polymer, will provide an effective delivery system for oral insulin. This study aims to synthesize insulin-loaded pectin-trimethyl chitosan nanoparticles (Ins-P-TMC-NPs) as an oral insulin delivery system and evaluate their efficacy both in vitro and in streptozotocin-induced diabetic male rats. N-trimethyl chitosan (TMC), synthesized via a methylation method, was used to prepare insulin-TMC nanoparticles coated with pectin via the ionic gelation method. The nanoparticles were characterized for their physicochemical properties, cumulative release profile, and surface morphology. In vitro biological cytotoxicity and cellular uptake of the nanoparticles were evaluated against HT-29 cells using an MTT assay and fluorescent microscopy, respectively. The in vivo blood glucose-lowering effect was assessed in diabetic male Sprague–Dawley rats, with in vivo toxicity evaluated in the liver, spleen, duodenum, pancreas, and kidney through histological studies (H&E staining). The results showed that Ins-P-TMC-NPs were spherical, with an average size of 379.40 ± 40.26 nm, a polydispersity index of 24.10 ± 1.03 %, a zeta potential of +17.20 ± 0.52 mV, and a loading efficiency of 83.21 ± 1.23 %. Compared to uncoated TMC nanoparticles, Ins-P-TMC-NPs reduced insulin loss in simulated gastrointestinal fluid by approximately 67.23 ± 0.97 % and provided controlled insulin release in simulated colonic fluid. In vitro bioactivity studies revealed that Ins-P-TMC-NPs were non-toxic, with cell viability of 91.12 ± 0.91 %, and exhibited high cellular uptake in the HT-29 cell line with a fluorescence intensity of 37.80 ± 2.40. Furthermore, in vivo studies demonstrated a sustained reduction in blood glucose levels after oral administration, peaking after 8 h with a glucose reduction of 87 ± 1.03 %. Histological sections showed no signs of toxicity. Overall, the developed colon-targeted oral insulin delivery system shows great potential as a candidate for oral insulin administration.

Overexpression of sialyl Lewisa carrying mucin-type glycoprotein in prostate cancer cell line contributes to aggressiveness and metastasis

Metastasis-promoting Lewis and sialyl Lewis antigens expressed on glycoproteins such as mucins are frequently displayed on the surface of prostate cancer cells and could thus be ideal candidates as measures of prostate cancer aggressiveness. The current study describes the altered expression of sialyl Lewisa (sLea) antigen attached to glycoproteins and key glycosyltransferases between normal prostate (RWPE-1) and cancerous cell lines (LNCaP and DU145). Our results suggest that the expression of sLea on different glycoproteins correlates with the aggressiveness of prostate cancer cells, as determined by flow cytometry and fluorescence microscopy. Blotting studies revealed that sLea-bearing glycoproteins, similar to mucins, are predominantly expressed in the more aggressive DU145 cells, followed by LNCaP cells. Immunohistochemistry technique showed a gradient of sLea expression, with low levels in low-grade prostate cancer (stage II/III) and increasing levels in high-grade cancer (stage IV), indicating its potential as a prognostic marker. Additionally, in qRT-PCR analysis significant upregulation of the glycosyltransferases GALNT5 and ST3GAL6 was observed, correlating with the increased sLea expression in LNCaP (3.2- and 14.5-fold) and DU145 (3.3- and 23.75-fold) cells. Our data indicates a correlation between sLea selectin ligand expression and prostate cancer aggressiveness. Furthermore, GALNT5 and ST3GAL6 could serve as benchmarks in PCa malignancy.

Chitosan/siRNA nanoparticles targeting PARP-1 attenuate Neuroinflammation and apoptosis in hyperglycemia-induced oxidative stress in Neuro2a cells

Hyperglycemia induces an excessive production of superoxide by the mitochondria's electron-transport chain triggers several pathways of injury contributing to the development of diabetic complications. This increase in oxidative and nitrosative stress triggers the activation of PARP-1, a nuclear enzyme, through mechanisms such as DNA damage. siRNA-chitosan nanoparticles were formed based on electrostatic interaction, their particle size, zeta potential, STEM, and cellular uptake were characterized. Neuro2a cells were treated with low glucose (LG) and high glucose (HG) for 24 and 48 h. Neuro2a cells were pre-treated with negative siRNA, naked siRNA, siRNA-Lipofectamine™300, and ChNPs-5. qRT-PCR was used to analyze the expression of regulatory, inflammatory, and apoptotic biomarkers. The siRNA-chitosan complex at the weight ratio 1:3000 were approximately uniform spheres with particle size 150.5 nm and a positive zeta potential of about +41.5 mV. The uptake of FITC-labeled nanoparticles into Neuro2a cells was visualized using fluorescence microscopy with no significant cytotoxicity compared to the control cells. High glucose stimulation of Neuro2a cells increased PARP1 expression, and with siRNA-ChNP (1:3000) treatment, significant inhibition of PARP1 expression is observed that consequently reversed the expression of regulatory genes like SIRT1, FOXO1, FOXO3, and p53. PARP-1 inhibition reduced HG-induced inflammatory response, including NF-kB, IL6, IL1β, TNFα, iNOS, and TGF-β expression, and HG-induced apoptosis response, such as Cas-3, Cas-9, BAK, BAX, and AIF expression. This study highlights the crucial role of siRNA delivery via ChNPs and PARP-1 inhibition in hyperglycemia-induced oxidative stress in Neuro2a cells and PARP-1 inhibition may be a feasible strategy for the treatment of hyperglycemia-induced oxidative stress.

Research on the Rheological Properties and Modification Mechanism of Epoxy Resin/SBS Composite-Modified Asphalt

Both epoxy resin (ER) and SBS are considered effective pavement materials for avoiding ruts. However, epoxy resin asphalt suffers from poor low-temperate performance and a high material cost in practical applications. Aiming to tackle these issues, a new type of composite asphalt modifier (ER-SBS) has been fabricated by combining epoxy resin with SBS. This work prepared modified asphalt with different doping amounts using the above composite asphalt modifier (ER-SBS), intending to explore the properties of composite-modified asphalt and the modification mechanism of the modifier. Furthermore, the effects of the composite modifier at different doping amounts on the viscoelastic property of asphalt were explored through rheological tests, and then the prepared composite-modified asphalt was compared with matrix asphalt and SBS-modified asphalt. In addition, the modification mechanism of the composite modifier was investigated by fluorescence microscopy and infrared spectroscopy. The difference in pavement performance between the composite-modified asphalt and SBS-modified asphalt was compared by a rut test and dynamic modulus test. The research results showed that the composite modifier improved the high- and low-temperature performances and viscoelastic property of matrix asphalt. When the doping amount was raised to 9%, the composite-modified asphalt exhibited better a modification effect than SBS-modified asphalt. The rut test results indicated that composite-modified asphalt demonstrated a stronger deformation resistance than SBS-modified asphalt. The dynamic modulus test showed that the composite-modified asphalt has better viscoelastic properties and temperature sensitivity. Fluorescence microscopy suggested that the crosslinking between the composite modifier and asphalt forms a mesh structure which greatly improves its resistance to deformation. From infrared spectroscopy, the composite modifier clearly functions as a physical modifier.

Inducing Multicolour emission in MEH-PPV/TiO2 nanocomposites

Fluorescence-based polymers have a wide variety of applications such as light-emitting diodes, optoelectronics, and biosensors. The present study endeavors towards the effect of TiO2 nanoparticles (calcined at various temperature) on the emission of Poly [2-methoxy-5-(2-ethylhexyloxy)-1, 4-phenylenevinylene] (MEH-PPV) and to induce multicolor emission. The TiO2 nanoparticles synthesized by sol-gel method were calcined at 400°C and 600°C. In situ polymerization was adopted to synthesize MEH-PPV / TiO2 nanocomposites and the structural characteristics of the nanocomposites were studied using Fourier transformed Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). The photo-physical characteristics of the nanocomposites were investigated using UV-Visible spectroscopy, Photoluminescence spectroscopy, and Fluorescence microscopy. TiO2 nanoparticles calcined at 400°C demonstrates anatase phase, whereas the particles calcined at 700°C exhibits mixed phase of rutile and anatase. The study reveal that calcination temperature has a strong impact on the morphology of the synthesized nanoparticles. The photoluminescence spectra reveal that the incorporation of TiO2 nanoparticles enhances the red orange emission intensity of MEH-PPV, additionally exhibits emission at multiple wavelengths. Fluorescence microscopy evidences multiple colour emission from MEH-PPV/TiO2 nanocomposites. The multiple emission in the polymer nanocomposite is arised from the oxygen vacancies present in anatase and rutile phases of TiO2 nanoparticle.

Coloring Multilayer Zirconia May Affect Its Optical and Mechanical Properties

The coloring process of monolithic dental zirconia caused considerable debate on the possible effects of different coloring methods. The main objective of this study was to investigate the influence of pigments in 3 multilayer 5-mol% yttria partially stabilized zirconia (5Y-PSZ) disks (Lava Esthetic A2 [Zr-AGG_A2] and Bleach [Zr-AGG_BL], both 3M Oral Care, and Katana STML A2 [Zr-NoAGG], Kuraray Noritake). The influence of pigment addition on the translucency parameter (TP00), fracture toughness, Vickers hardness, biaxial strength, and hydrothermal stability was assessed and correlated with the microstructure and phase composition. The pigment composition and distribution were evaluated by light and fluorescence microscopy, electron probe microanalysis, and nano–scanning electron microscopy. The chemical and phase composition and aging behavior were assessed using X-ray fluorescence and X-ray diffraction, respectively, while the aging sensitivity of the pigments was evaluated using micro-Raman spectroscopy. In contrast to Zr-NoAGG, possessing a typical 5Y-PSZ microstructure, the pigment additions in both Zr-AGG_A2/BL zirconia resulted in large yellow and blue fluorescent Er-, Hf-, and Al-containing agglomerates composed of small grains (0.57 µm and 0.38 µm, respectively, vs. 0.92 µm for the surrounding grains) with lower Y2O3 content. Zr-AGG_A2 had the lowest aging resistance, with transformation degradation occurring exclusively within the pigment agglomerates. All zirconia grades had a high Y2O3 content (4.2%–5.7 mol%) tetragonal ZrO2 phase and a high (42%–55 wt%) cubic ZrO2 phase content. Although no statistical differences were measured for hardness and toughness, Zr-NoAGG had a significantly higher TP00, higher flexural strength, and lower mechanical reliability compared to both Zr-AGG_A2/BL zirconia. The rare-earth oxide-containing zirconia agglomerates that were added as pigments to the multilayered monolithic Zr-AGG_A2/BL zirconia are the cause for their lower optical and mechanical properties and reduced aging resistance.

Dynamical stiffening of dsDNA confined and stretched in a nanochannel

The internal dynamics of double-stranded DNA (dsDNA) confined within channels of diameters ranging from the persistence length to twice that length were investigated using fluorescence microscopy. By analysing spatiotemporal intensity fluctuations, we derived the intermediate dynamic structure factor. The structure factor consistently exhibited behaviour characteristic of Rouse dynamics, regardless of the channel diameter. Notably, as the channel diameter decreased, the DNA molecules became increasingly elongated along the channel, leading to shorter relaxation times. These findings indicate a dynamic stiffening effect, where the effective spring constant of a stretched polymer chain increases. We propose that this effect has significant implications for controlling DNA motion in biological and biotechnological applications.