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ELife Apr 2024We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail...
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism. SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
Topics: Animals; Cell Movement; Fibroblast Growth Factors; High-Temperature Requirement A Serine Peptidase 1; Neural Crest; Serpin E2; Signal Transduction; Xenopus laevis; Xenopus Proteins
PubMed: 38634469
DOI: 10.7554/eLife.91864 -
STAR Protocols Jun 2024Precise integration of DNA constructs greater than 3 kb into mouse zygotes is difficult. Here, we present a protocol for large DNA transgenesis in mice using the...
Precise integration of DNA constructs greater than 3 kb into mouse zygotes is difficult. Here, we present a protocol for large DNA transgenesis in mice using the Cas9+Bxb1 toolbox. We describe steps for choosing mouse strains with preplaced attachment sites. We then detail procedures for microinjecting mouse zygotes with the plasmid donor DNA construct to generate transgenic mice by recombination-mediated cassette exchange. This protocol has the potential for application in exploring the functional implications of large structural variations in cancer. For complete details on the use and execution of this protocol, please refer to Low et al. and Hosur et al..
Topics: Animals; Mice; Mice, Transgenic; Gene Transfer Techniques; DNA; CRISPR-Cas Systems; Zygote; Microinjections; Plasmids; Female
PubMed: 38625797
DOI: 10.1016/j.xpro.2024.103022 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Jan 2024Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN,...
OBJECTIVES
Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and in TG with a TN model, evaluate whether has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN.
METHODS
A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+ agomir group (=6), the sham rats were microinjected with agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+ antagonist group (=6), TN rats were microinjected with antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and and whether directly affected the 3'-UTR terminal of . The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress . The inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of . The behavioral changes of rats before and after administration were observed, and the expression changes of and Kv1.1 in TG of rats after intervention were detected.
RESULTS
Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of mRNA and protein in TG of the TN group was down-regulated (both <0.05), and the expression of was up-regulated (<0.05). The results of dual luciferase report showed that the luciferase activity of rno- mimics and WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type WT, respectively (<0.001). Compared with low dose rno- mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno- mimics (20 nmol/L) cotransfection group was significantly decreased (<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of in the TN group, the mechanical pain threshold and the expression of mRNA and protein in TG were increased. After overexpression of in the sham group, the mechanical pain threshold and the expression of mRNA and protein in TG were decreased.
CONCLUSIONS
Both Kv1.1 and are involved in TN and can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.
Topics: Animals; Rats; Antagomirs; Down-Regulation; Luciferases; MicroRNAs; Neuralgia; Rats, Sprague-Dawley; RNA, Messenger; Trigeminal Neuralgia; Kv1.1 Potassium Channel
PubMed: 38615163
DOI: 10.11817/j.issn.1672-7347.2024.230273 -
Sensors (Basel, Switzerland) Mar 2024Microinjection is usually applied to the treatment of some retinal disorders, such as retinal vein cannulation and displaced submacular hemorrhage. Currently, the...
Microinjection is usually applied to the treatment of some retinal disorders, such as retinal vein cannulation and displaced submacular hemorrhage. Currently, the microinjection procedure is usually performed by using the viscous fluid control of a standard vitrectomy system, which applies a fixed air pressure through foot pedal activation. The injection process with the fixed pressure is uncontrollable and lacks feedback, the high flow rate of the injected drug may cause damage to the fundus tissue. In this paper, a liquid-driven microinjection system with a flow sensor is designed and developed specifically for fundus injection. In addition, a PID sliding mode control (SMC) method is proposed to achieve precise injection in the injection system. The experimental results of fundus simulation injection demonstrate that the microinjection system meets the requirements of fundus injection and reduces the impact of the injection process on the fundus tissue.
Topics: Animals; Microinjections; Abomasum; Computer Simulation; Fundus Oculi; Retinal Vein
PubMed: 38610350
DOI: 10.3390/s24072140 -
Frontiers in Genetics 2024RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK...
RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Wild-type (WT) and mutant genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway. Recurrent and novel variants that we reported showed increased or RAS signaling pathway activity because of gain-of-function variants. Clinical features are similar to those previously reported, suggesting that gain-of-function variants cause this disease in patients.
PubMed: 38601074
DOI: 10.3389/fgene.2024.1383176 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Mar 2024To compare the anti-inflammatory, antitumor and anti-bacterial effects of the single extract (in granules) and the prepared drug in pieces of Forsythia Suspense (, a...
OBJECTIVE
To compare the anti-inflammatory, antitumor and anti-bacterial effects of the single extract (in granules) and the prepared drug in pieces of Forsythia Suspense (, a traditional Chinese herbal medicine).
METHODS
In zebrafish embryo models of CuSO exposure, tail transection and LPS microinjection-induced inflammation, the anti-inflammatory effects of 10 μg/mL DEX, single extract of Forsythia Suspense, and the water extract of the prepared drug (400, 600, and 800 μg/mL) were evaluated by observing neutrophil counts, RT- qPCR, HE staining and survival analysis. Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining. The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs.
RESULTS
In the zebrafish embryo models of inflammation, both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation, reduced the mRNA expressions of TNF-α, IL-6, P38, Jnk, Erk and P65, and increased the survival rate of zebrafish. They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells (HCT116), pancreas adenocarcinoma cells (PANC-1), lung cancer cells (A549), liver cancer cells (Hep3B) and cervical carcinoma cells (Hela) in zebrafish embryos, and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL.
CONCLUSION
The two dosage forms of Forsythia Suspense have similar anti-inflammatory, antitumor and antibacterial effects, but their effects for inhibiting IL-6, P65, and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.
Topics: Animals; Humans; Forsythia; Zebrafish; Interleukin-6; Anti-Inflammatory Agents; Inflammation; Anti-Bacterial Agents; RNA, Messenger; Drugs, Chinese Herbal
PubMed: 38597452
DOI: 10.12122/j.issn.1673-4254.2024.03.22 -
IScience Apr 2024Microinjecting yeast cells has been challenging for decades with no significant breakthrough due to the ultra-tough cell wall and low stiffness of the traditional...
Microinjecting yeast cells has been challenging for decades with no significant breakthrough due to the ultra-tough cell wall and low stiffness of the traditional injector tip at the micro-scale. Penetrating this protection wall is the key step for artificially bringing foreign substance into the yeast. In this paper, a yeast cell model was built by using finite element analysis (FEA) method to analyze the penetrating process. The key parameters of the yeast cell wall in the model (the Young's modulus, the shear modulus, and the Lame constant) were calibrated according to a general nanoindentation experiment. Then by employing the calibrated model, the injection parameters were optimized to minimize the cell damage (the maximum cell deformation at the critical stress of the cell wall). Key guidelines were suggested for penetrating the cell wall during microinjection.
PubMed: 38591007
DOI: 10.1016/j.isci.2024.109503 -
Advanced Science (Weinheim,... Jun 2024Bacterial skin infections are highly prevalent and pose a significant public health threat. Current strategies are primarily focused on the inhibition of bacterial...
Bacterial skin infections are highly prevalent and pose a significant public health threat. Current strategies are primarily focused on the inhibition of bacterial activation while disregarding the excessive inflammation induced by dead bacteria remaining in the body and the effect of the acidic microenvironment during therapy. In this study, a novel dual-functional MgB microparticles integrated microneedle (MgB MN) patch is presented to kill bacteria and eliminate dead bacteria for skin infection management. The MgB microparticles not only can produce a local alkaline microenvironment to promote the proliferation and migration of fibroblasts and keratinocytes, but also achieve >5 log bacterial inactivation. Besides, the MgB microparticles effectively mitigate dead bacteria-induced inflammation through interaction with lipopolysaccharide (LPS). With the incorporation of these MgB microparticles, the resultant MgB MN patches effectively kill bacteria and capture dead bacteria, thereby mitigating these bacteria-induced inflammation. Therefore, the MgB MN patches show good therapeutic efficacy in managing animal bacterial skin infections, including abscesses and wounds. These results indicate that reactive metal borides-integrated microneedle patches hold great promise for the treatment of clinical skin infections.
Topics: Animals; Needles; Anti-Bacterial Agents; Mice; Skin Diseases, Bacterial; Disease Models, Animal; Humans; Transdermal Patch; Microinjections
PubMed: 38582511
DOI: 10.1002/advs.202309622 -
Brain Structure & Function Jun 2024In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate...
In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.
Topics: Animals; Hypercapnia; Male; Galanin; Neurons; Carbon Dioxide; Proto-Oncogene Proteins c-fos; Neural Pathways; Respiratory Center; Rats; Chemoreceptor Cells; Rats, Sprague-Dawley; Brain Stem
PubMed: 38578351
DOI: 10.1007/s00429-024-02782-8 -
Journal of Pharmacy & Pharmaceutical... 2024Microneedle (MN)-assisted drug delivery technology has gained increasing attention over the past two decades. Its advantages of self-management and being minimally... (Review)
Review
Microneedle (MN)-assisted drug delivery technology has gained increasing attention over the past two decades. Its advantages of self-management and being minimally invasive could allow this technology to be an alternative to hypodermic needles. MNs can penetrate the stratum corneum and deliver active ingredients to the body through the dermal tissue in a controlled and sustained release. Long-acting polymeric MNs can reduce administration frequency to improve patient compliance and therapeutic outcomes, especially in the management of chronic diseases. In addition, long-acting MNs could avoid gastrointestinal reactions and reduce side effects, which has potential value for clinical application. In this paper, advances in design strategies and applications of long-acting polymeric MNs are reviewed. We also discuss the challenges in scale manufacture and regulations of polymeric MN systems. These two aspects will accelerate the effective clinical translation of MN products.
Topics: Humans; Skin; Microinjections; Administration, Cutaneous; Pharmaceutical Preparations; Drug Delivery Systems; Polymers
PubMed: 38571937
DOI: 10.3389/jpps.2024.12434