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International Journal of Molecular... May 2024In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes and , a...
In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes and , a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.
Topics: Animals; Aedes; RNA Interference; RNA, Small Interfering; Mosquito Vectors; Larva; RNA, Double-Stranded; Gene Silencing; Gene Knockdown Techniques
PubMed: 38791257
DOI: 10.3390/ijms25105218 -
Antioxidants (Basel, Switzerland) Apr 2024Research on ozonated sunflower oil (OSO) is mostly restricted to its topical application, whereas the functional and toxicological assessment of oral OSO consumption is...
Oral Supplementation of Ozonated Sunflower Oil Augments Plasma Antioxidant and Anti-Inflammatory Abilities with Enhancement of High-Density Lipoproteins Functionality in Rats.
Research on ozonated sunflower oil (OSO) is mostly restricted to its topical application, whereas the functional and toxicological assessment of oral OSO consumption is yet to be solved. Herein, OSO was orally supplemented in rats to assess the impact on plasma antioxidant status, low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Also, the functionality of HDL from the OSO-supplemented rats (OSO-HDL) was tested against carboxymethyllysine (CML)- induced hyperinflammation in embryo and adult zebrafish. The results revealed that four weeks of OSO supplementation (3 g/kg BW/day) had no adverse effect on rats' hematological and blood biochemical profiles. Nonetheless, decreased interleukin (IL)-6, and LDL-C levels, along with enhanced ferric ion reduction ability (FRA) and sulfhydryl content, were observed in the plasma of OSO-supplemented rats compared to the control and sunflower oil (SO) supplemented group. In addition, OSO supplementation stabilized apoA-I/HDL and augmented HDL-allied paraoxonase (PON)-1 activity. The microinjection of OSO-HDL (10 nL, 2 mg/mL) efficiently prevented the CML (500 ng)-induced zebrafish embryo mortality and developmental deformities. Similarly, OSO-HDL thwarted CML-posed neurotoxicity and demonstrated a significant hepatoprotective effect against CML-induced fatty liver changes, hepatic inflammation, oxidative stress, and apoptosis, as well as exhibiting a noticeable influence to revert CML-induced dyslipidemia. Conclusively, OSO supplementation demonstrated no toxic effects on rats, ameliorated plasma antioxidant status, and positively influenced HDL stability and functionality, leading to a protective effect against CML-induced toxicity in zebrafish.
PubMed: 38790634
DOI: 10.3390/antiox13050529 -
Cells May 2024Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle...
Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus-both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.
Topics: Animals; Mice; Coculture Techniques; Gastrointestinal Tract; Intestine, Small; Myenteric Plexus; Neurons; Organoids; Submucous Plexus
PubMed: 38786037
DOI: 10.3390/cells13100815 -
Biosensors May 2024Intracellular delivery, the process of transporting substances into cells, is crucial for various applications, such as drug delivery, gene therapy, cell imaging, and... (Review)
Review
Intracellular delivery, the process of transporting substances into cells, is crucial for various applications, such as drug delivery, gene therapy, cell imaging, and regenerative medicine. Among the different approaches of intracellular delivery, mechanoporation stands out by utilizing mechanical forces to create temporary pores on cell membranes, enabling the entry of substances into cells. This method is promising due to its minimal contamination and is especially vital for stem cells intended for clinical therapy. In this review, we explore various mechanoporation technologies, including microinjection, micro-nano needle arrays, cell squeezing through physical confinement, and cell squeezing using hydrodynamic forces. Additionally, we highlight recent research efforts utilizing mechanoporation for stem cell studies. Furthermore, we discuss the integration of mechanoporation techniques into microfluidic platforms for high-throughput intracellular delivery with enhanced transfection efficiency. This advancement holds potential in addressing the challenge of low transfection efficiency, benefiting both basic research and clinical applications of stem cells. Ultimately, the combination of microfluidics and mechanoporation presents new opportunities for creating comprehensive systems for stem cell processing.
Topics: Stem Cells; Humans; Microfluidics; Animals; Drug Delivery Systems
PubMed: 38785730
DOI: 10.3390/bios14050256 -
Addiction Biology May 2024Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions...
Addictive properties of propofol have been demonstrated in both humans and animals. The nucleus accumbens (NAc) shell (NAsh) in the brain, along with the interactions between N-methyl-D-aspartate receptor (NMDAR) and the dopamine D1 receptor (D1R), as well as their downstream ERK/CREB signalling pathway in the NAc, are integral in regulating reward-seeking behaviour. Nevertheless, it remains unclear whether NMDARs and the NMDAR-D1R/ERK/CREB signalling pathway in the NAsh are involved in mediating propofol addiction. To investigate it, we conducted experiments with adult male Sprague-Dawley rats to establish a model of propofol self-administration behaviour. Subsequently, we microinjected D-AP5 (a competitive antagonist of NMDARs, 1.0-4.0 μg/0.3 μL/site) or vehicle into bilateral NAsh in rats that had previously self-administered propofol to examine the impact of NMDARs within the NAsh on propofol self-administration behaviour. Additionally, we examined the protein expressions of NR2A and NR2B subunits, and the D1R/ERK/CREB signalling pathways within the NAc. The results revealed that propofol administration behaviour was enhanced by D-AP5 pretreatment in NAsh, accompanied by elevated expressions of phosphorylation of NR2A (Tyr1246) and NR2B (Tyr1472) subunits. There were statistically significant increases in the expressions of D1Rs, as well as in the phosphorylated ERK1/2 (p-ERK1/2) and CREB (p-CREB). This evidence substantiates a pivotal role of NMDARs in the NAsh, with a particular emphasis on the NR2A and NR2B subunits, in mediating propofol self-administration behaviour. Furthermore, it suggests that this central reward processing mechanism may operate through the NMDAR-D1R/ERK/CREB signal transduction pathway.
Topics: Animals; Nucleus Accumbens; Propofol; Receptors, N-Methyl-D-Aspartate; Male; Rats, Sprague-Dawley; Receptors, Dopamine D1; Self Administration; Rats; Signal Transduction; Cyclic AMP Response Element-Binding Protein; MAP Kinase Signaling System
PubMed: 38782631
DOI: 10.1111/adb.13401 -
The Journal of Clinical and Aesthetic... May 2024Nose reshaping with hyaluronic acid (HA) fillers, also known as medical rhinoplasty, is an increasingly popular, minimally invasive aesthetic procedure. As the demand...
INTRODUCTION
Nose reshaping with hyaluronic acid (HA) fillers, also known as medical rhinoplasty, is an increasingly popular, minimally invasive aesthetic procedure. As the demand for nasal reshaping continues to rise, it is essential to develop safe and efficient injection techniques and assess satisfaction to ensure optimal outcomes and patient-centered care.
OBJECTIVE
This study aims to evaluate patient and physician satisfaction with hyaluronic acid filler applications using microinjection technique for nasal reshaping.
METHODS
The study included healthy adult patients who underwent medical rhinoplasty with the same HA filler using the microinjection technique. Patient satisfaction levels were evaluated at one and six months after the last injection using the Global Patient Satisfaction Scale (GPSS). Additionally, an independent dermatologist conducted a clinical evaluation for each patient by comparing before and after clinical pictures, using the Aesthetic Improvement Scale (AIS). Any side effects were recorded during each session and follow-up period for six months.
RESULTS
A total of 40 patients (37 women and 3 men) participated in the study. The most frequently targeted anatomical areas for filler injections were the nasal tip (100%), columella (100%), nasal prominence (100%), nasal dorsum (85%), and nasal root (82.5%). Injections distal to the nasolabial fold (NFL) were performed in 2.5 percent of patients. Patients expressed high satisfaction with the results at both one and six months after the procedure (mean GPSS, respectively; 4.65 and 4.47). Similarly, clinicians reported satisfaction with outcomes at the same time points (mean AIS, respectively; 1.7 and 1.4). Apart from mild pain during the procedure and transient erythema afterward, no side effects were recorded.
CONCLUSION
Medical rhinoplasty with HA fillers using the microinjection technique is an effective and reliable procedure. This technique provides safe and aesthetically pleasing results from both patient and dermatologist perspectives, making it a favorable option for nasal reshaping with HA dermal fillers.
PubMed: 38779374
DOI: No ID Found -
Heliyon May 2024To construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function.
OBJECTIVE
To construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function.
METHODS
Using CRISPR/Cas9, the expression vector -Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8 T cell proportions in the spleen were evaluated.
RESULTS
Two F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous -engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8 T cells decreased.
CONCLUSION
HPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.
PubMed: 38765051
DOI: 10.1016/j.heliyon.2024.e29881 -
Frontiers in Cell and Developmental... 2024Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However,...
Prohibitin (PHB) is an essential scaffold protein that modulates signaling pathways controlling cell survival, metabolism, inflammation, and bone formation. However, its specific role in periodontium development remains less understood. This study aims to elucidate the expression pattern and function of PHB in periodontium development and its involvement in alveolar bone formation. Immunolocalization of PHB in the periodontium of postnatal (PN) mice were examined. morpholino was micro-injected into the right-side mandible at PN5, corresponding to the position where the alveolar bone process forms in relation to the lower first molar. The micro-injection with a scramble control (PF-127) and the left-side mandibles were used as control groups. Five days post-micro-injection, immunohistochemical analysis and micro-CT evaluation were conducted to assess bone mass and morphological changes. Additionally, expression patterns of signaling molecules were examined following downregulation using 24-h cultivation of developing dental mesenchyme at E14.5. The immunostaining of PHB showed its localization in the periodontium at PN5, PN8, and PN10. The cultivation of dental mesenchyme resulted in alterations in Bmps, Runx2, and Wnt signalings after knock-down. At 5 days post-micro-injection, knocking down showed weak immunolocalizations of runt-related transcription factor (RUNX2) and osteocalcin (OCN). However, knocking down led to histological alterations characterized by decreased bone mass and stronger localizations of Ki67 and PERIOSTIN in the periodontium compared 1 to control groups. The micro-CT evaluation showed decreased bone volume and increased PDL space in the knock-down specimens, suggesting its regulatory role in bone formation. The region-specific localization of PHB in the margin where alveolar bone forms suggests its involvement in alveolar bone formation and the differentiation of the periodontal ligament. Overall, our findings suggest that plays a modulatory role in alveolar bone formation by harmoniously regulating bone-forming-related signaling molecules during periodontium development.
PubMed: 38756696
DOI: 10.3389/fcell.2024.1369634 -
ELife May 2024During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes...
During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.
Topics: Animals; Endoplasmic Reticulum; Meiosis; Oocytes; Formins; Mitochondria; Mice; Actins; Swine; Female; Spindle Apparatus
PubMed: 38747713
DOI: 10.7554/eLife.92732 -
BioRxiv : the Preprint Server For... May 2024Despite the profound behavioral effects of the striatal dopamine (DA) activity and the inwardly rectifying potassium channel ( ) being a key determinant of striatal...
Despite the profound behavioral effects of the striatal dopamine (DA) activity and the inwardly rectifying potassium channel ( ) being a key determinant of striatal medium spiny neuron (MSN) activity that also profoundly affects behavior, previously reported DA regulations of Kir are conflicting and incompatible with MSN function in behavior. Here we show that in normal mice with an intact striatal DA system, the predominant effect of DA activation of D1Rs in D1-MSNs is to cause a modest depolarization and increase in input resistance by inhibiting Kir, thus moderately increasing the spike outputs from behavior-promoting D1-MSNs. In parkinsonian (DA-depleted) striatum, DA increases D1-MSN intrinsic excitability more strongly than in normal striatum, consequently strongly increasing D1-MSN spike firing that is behavior-promoting; this DA excitation of D1-MSNs is stronger when the DA depletion is more severe. The DA inhibition of Kir is occluded by the Kir blocker barium chloride (BaCl ). In behaving parkinsonian mice, BaCl microinjection into the dorsal striatum stimulates movement but occludes the motor stimulation of D1R agonism. Taken together, our results resolve the long-standing question about what D1R agonism does to D1-MSN excitability in normal and parkinsonian striatum and strongly indicate that D1R inhibition of Kir is a key ion channel mechanism that mediates D1R agonistic behavioral stimulation in normal and parkinsonian animals.
PubMed: 38746264
DOI: 10.1101/2024.04.29.590632