Material modeling and recent findings in transcatheter aortic valve implantation simulations.

BACKGROUND AND OBJECTIVE: Transcatheter aortic valve implantation (TAVI) has significantly transformed the management of aortic valve (AV) diseases, presenting a minimally invasive option compared to traditional surgical valve replacement. Computational simulations of TAVI become more popular and offer a detailed investigation by employing patient-specific models. On the other hand, employing accurate material modeling procedures and applying basic modeling steps are crucial to determining reliable numerical results. Therefore, this review aims to outline the basic modeling approaches for TAVI, focusing on material modeling and geometry extraction, as well as summarizing the important findings from recent computational studies to guide future research in the field. METHODS: This paper explains the basic steps and important points in setting up and running TAVI simulations. The material properties of the leaflets, valves, stents, and tissues utilized in TAVI simulations are provided, along with a comprehensive explanation of the geometric extraction methods employed. The differences between the finite element analysis, computational fluid dynamics, and fluid-structure interaction approaches are pointed out and the important aspects of TAVI modeling are described by elucidating the recent computational studies. RESULTS: The results of the recent findings on TAVI simulations are summarized to demonstrate its powerful potential. It is observed that the material properties of aortic tissues and components of implanted valves should be modeled realistically to determine accurate results. For patient-specific AV geometries, incorporating calcific deposits on the leaflets is essential for ensuring the accuracy of computational findings. The results of numerical TAVI simulations indicate the significance of the selection of optimal valves and precise deployment within the appropriate anatomical position. These factors collectively contribute to the effective functionality of the implanted valve. CONCLUSIONS: Recent studies in the literature have revealed the critical importance of patient-specific modeling, the selection of accurate material models, and bio-prosthetic valve diameters. Additionally, these studies emphasize the necessity of precise positioning of bio-prosthetic valves to achieve optimal performance in TAVI, characterized by an increased effective orifice area and minimal paravalvular leakage.

Inhibition of cytochrome P450 epoxygenase promotes endothelium-to-mesenchymal transition and exacerbates doxorubicin-induced cardiovascular toxicity.

BACKGROUND: Doxorubicin (DOX) is a potent chemotherapy widely used in treating various neoplastic diseases. However, the clinical use of DOX is limited due to its potential toxic effect on the cardiovascular system. Thus, identifying the pathway involved in this toxicity may help minimize chemotherapy risk and improve cancer patients' quality of life. Recent studies suggest that Endothelial-to-Mesenchymal transition (EndMT) and endothelial toxicity contribute to the pathogenesis of DOX-induced cardiovascular toxicity. However, the molecular mechanism is yet unknown. Given that arachidonic acid and associated cytochrome P450 (CYP) epoxygenase have been involved in endothelial and cardiovascular function, we aimed to examine the effect of suppressing CYP epoxygenases on DOX-induced EndMT and cardiovascular toxicity in vitro and in vivo. METHODS AND RESULTS: To test this, human endothelial cells were treated with DOX, with or without CYP epoxygenase inhibitor, MSPPOH. We also investigated the effect of MSPPOH on the cardiovascular system in our zebrafish model of DOX-induced cardiotoxicity. Our results showed that MSPPOH exacerbated DOX-induced EndMT, inflammation, oxidative stress, and apoptosis in our endothelial cells. Furthermore, we also show that MSPPOH increased cardiac edema, lowered vascular blood flow velocity, and worsened the expression of EndMT and cardiac injury markers in our zebrafish model of DOX-induced cardiotoxicity. CONCLUSION: Our data indicate that a selective CYP epoxygenase inhibitor, MSPPOH, induces EndMT and endothelial toxicity to contribute to DOX-induced cardiovascular toxicity.

Fabrication Strategies for Bioceramic Scaffolds in Bone Tissue Engineering with Generative Design Applications.

The aim of this study is to provide an overview of the current state-of-the-art in the fabrication of bioceramic scaffolds for bone tissue engineering, with an emphasis on the use of three-dimensional (3D) technologies coupled with generative design principles. The field of modern medicine has witnessed remarkable advancements and continuous innovation in recent decades, driven by a relentless desire to improve patient outcomes and quality of life. Central to this progress is the field of tissue engineering, which holds immense promise for regenerative medicine applications. Scaffolds are integral to tissue engineering and serve as 3D frameworks that support cell attachment, proliferation, and differentiation. A wide array of materials has been explored for the fabrication of scaffolds, including bioceramics (i.e., hydroxyapatite, beta-tricalcium phosphate, bioglasses) and bioceramic-polymer composites, each offering unique properties and functionalities tailored to specific applications. Several fabrication methods, such as thermal-induced phase separation, electrospinning, freeze-drying, gas foaming, particle leaching/solvent casting, fused deposition modeling, 3D printing, stereolithography and selective laser sintering, will be introduced and thoroughly analyzed and discussed from the point of view of their unique characteristics, which have proven invaluable for obtaining bioceramic scaffolds. Moreover, by highlighting the important role of generative design in scaffold optimization, this review seeks to pave the way for the development of innovative strategies and personalized solutions to address significant gaps in the current literature, mainly related to complex bone defects in bone tissue engineering.

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

An optimized CT-dense agent perfusion and micro-CT imaging protocol for chick embryo developmental stages.

Compared to classical techniques of morphological analysis, micro-CT (µ-CT) has become an effective approach allowing rapid screening of morphological changes. In the present work, we aimed to provide an optimized micro-CT dense agent perfusion protocol and µ-CT guidelines for different stages of chick embryo cardiogenesis. Our study was conducted over a period of 10 embryonic days (Hamburger-Hamilton HH36) in chick embryo hearts. During the perfusion of the micro-CT dense agent at different developmental stages (HH19, HH24, HH27, HH29, HH31, HH34, HH35, and HH36), we demonstrated that durations and volumes of the injected contrast agent gradually increased with the heart developmental stages contrary to the flow rate that was unchanged during the whole experiment. Analysis of the CT imaging confirmed the efficiency of the optimized parameters of the heart perfusion.

In vivo testing of novel nitric oxide-releasing nanoparticles for alleviating heart failure using the zebrafish embryo model.

Heart failure (HF) is a multifactorial, heterogeneous systemic disease that is considered one of the leading causes of death and morbidity worldwide. It is well-known that endothelial dysfunction (ED) plays an important role in cardiac disease etiology. A reduction in the bioavailability of nitric oxide (NO) in the bloodstream leads to vasoconstriction and ED. Many studies indicated diminishment of peripheral arteries vasodilation that is mediated by the endothelium in the of patients with chronic HF. With the advancement of nanomedicine, nanotechnology can provide adequate solutions for delivering exogenous NO with the aid of nanoparticles (NPs) to treat ED. The properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable both passive and active delivery of drugs. This prompted us to investigate the efficacy of our newly-developed hydrogel nanoparticles (NO-RPs) for the delivery and sustained release of NO gas to alleviate cardiac failure and inflammation in the heart failure zebrafish model. The hydrogel NO-RPs incorporate SPIONS and NO precursor. The sustainend release of NO in the NO-RPs (4200 s), overcomes the problem of the short half life of NO in vivo which is expected to ameliorate the reduced NO bioavailabilty, and its consequences in endothelial and cardiac dysfunction. Zebrafish embryos were used as the animal model in this study to determine the effect of SPIONs-loaded NO-RPs on the cardiovascular system. Cardiac failure was induced in 24hpf embryos by exposure to aristolochic acid (AA)(0.25, 0.5 µM) for 8 h, followed by the SPIONs-loaded NO-RPs (0.25, 0.5 mg/ml) for 48 h, experimental groups included: control group which is healthy non treated zebrafish embryos, AA injured zebrafish embryos (HF) model,and NO-RP treated HF zebrafish embryos. Survival rate was assessed at 72hpf. Cardiac function was also evaluated by analyzing cardiac parameters including heartbeat, major blood vessels primordial cardinal vein and dorsal aorta (PCV &DA) diameter, blood flow velocity in PCV & DA vessels, cardiac output, and PCV & DA shear stresses. All cardiac parameters were analyzed with the aid of MicroZebraLab blood flow analysis software from Viewpoint. In addition, we studied the molecular effects of the developed NO-RPs on the mRNA expression of selected pro-inflammatory markers: IL-6, and Cox-2. Our findings demonstrated that the NO-RPs improved the survival rate in the heart failure zebrafish model and reversed heart failure by enhancing blood flow perfusion in Zebrafish embryos, significantly. In addition, RT-PCR results showed that the NO-RPs significantly reduced the expression of pro-inflammatory markers (lL-6&COX-2) in the heart failure zebrafish model. Our study confirmed that the developed SPIONs-loaded NO-RPs are effective tool to alleviate cardiac failure and inflammation in the HF zebrafish model.

Trilayer composite scaffold for urethral reconstruction:in vitroevaluation of mechanical, biological, and angiogenic properties.

Regeneration of damaged urethral tissue remains a major challenge in the field of lower urinary tract reconstruction. To address this issue, various synthetic and natural biodegradable biomaterials are currently being explored for the fabrication of scaffolds that promote urethral regeneration and healing. In this study, we present an approach to fabricate a trilayer hybrid scaffold comprising a central layer of poly(lactic acid) (PLA) between two layers of chitosan. The chitosan/PLA/chitosan (CPC) scaffolds were fabricated by a sequential electrospinning process and their properties were evaluated for their suitability for urethral tissue engineering. The physical and biological properties of the CPC scaffolds were evaluated in comparison to electrospun PLA scaffolds and acellular dermis (Alloderm) as controls for a synthetic and a natural scaffold, respectively. Compared to the controls, the CPC scaffolds exhibited higher elastic modulus and ultimate tensile strength, while maintaining extensibility and suture retention strength appropriate for clinical use. The CPC scaffolds displayed significant hydrophilicity, which was associated with a higher water absorption capacity of the chitosan nanofibres. The degradation products of the CPC scaffolds did not exhibit cytotoxicity and promoted wound closure by fibroblastsin vitro. In addition, CPC scaffolds showed increased growth of smooth muscle cells, an essential component for functional regeneration of urethral tissue. Furthermore, in a chicken embryo-based assay, CPC scaffolds demonstrated significantly higher angiogenic potential, indicating their ability to promote vascularisation, a crucial aspect for successful urethral reconstruction. Overall, these results suggest that CPC hybrid scaffolds containing both natural and synthetic components offer significant advantages over conventional acellular or synthetic materials alone. CPC scaffolds show promise as potential candidates for further research into the reconstruction of the urethrain vivo.

Exploring the in vivo anti-cancer potential of Neosetophomone B in leukemic cells using a zebrafish xenograft model.

Neosetophomone B (NSP-B) is a unique meroterpenoid fungal secondary metabolite that has previously demonstrated promising anti-cancer properties against various cancer cell lines in vitro. However, its in vivo anti-cancer potential remaines unexplored. To fill this gap in our knowledge, we tested NSP-B's in vivo anti-cancer activity using a zebrafish model, an organism that has gained significant traction in biomedical research due to its genetic similarities with humans and its transparent nature, allowing real-time tumor growth observation. For our experiments, we employed the K562-injected zebrafish xenograft model. Upon treating these zebrafish with NSP-B, we observed a marked reduction in the size and number of tumor xenografts. Delving deeper, our analyses indicated that NSP-B curtailed tumor growth and proliferation of leukemic grafted xenograft within the zebrafish. These results show that NSP-B possesses potent in vivo anti-cancer properties, making it a potential novel therapeutic agent for addressing hematological malignancies.

The Role of p90 Ribosomal S6 Kinase (RSK) in Tyrosine Kinase Inhibitor (TKI)-Induced Cardiotoxicity.

Targeted therapy, such as tyrosine kinase inhibitors (TKIs), has been approved to manage various cancer types. However, TKI-induced cardiotoxicity is a limiting factor for their use. This issue has raised the need for investigating potential cardioprotective techniques to be combined with TKIs. Ribosomal S6-kinases (RSKs) are a downstream effector of the mitogen-activated-protein-kinase (MAPK) pathway; specific RSK isoforms, such as RSK1 and RSK2, have been expressed in cancer cells, in which they increase tumour proliferation. Selective targeting of those isoforms would result in tumour suppression. Moreover, activation of RSKs expressed in the heart has resulted in cardiac hypertrophy and arrhythmia; thus, inhibiting RSKs would result in cardio-protection. This review article presents an overview of the usefulness of RSK inhibitors that can be novel agents to be assessed in future research for their effect in reducing cancer proliferation, as well as protecting the heart from cardiotoxicity induced by TKIs.

Disturbed hemodynamics and oxidative stress interaction in endothelial dysfunction and AAA progression: Focus on Nrf2 pathway.

Hemodynamic shear stress is one of the major factors that are involved in the pathogenesis of many cardiovascular diseases including atherosclerosis and abdominal aortic aneurysm (AAA), through its modulatory effect on the endothelial cell's redox homeostasis and mechanosensitive gene expression. Among important mechanisms, oxidative stress, endoplasmic reticulum stress activation, and the subsequent endothelial dysfunction are attributed to disturbed blood flow and low shear stress in the vascular curvature and bifurcations which are considered atheroprone regions and aneurysm occurrence spots. Many pathways were shown to be involved in AAA progression. Of particular interest from recent findings is, the (Nrf2)/Keap-1 pathway, where Nrf2 is a transcription factor that has antioxidant properties and is strongly associated with several CVDs, yet, the exact mechanism by which Nrf2 alleviates CVDs still to be elucidated. Nrf2 expression is closely affected by shear stress and was shown to participate in AAA. In the current review paper, we discussed the link between disturbed hemodynamics and its effect on Nrf2 as a mechanosensitive gene and its role in the development of endothelial dysfunction which is linked to the progression of AAA.

Latest Developments in Adapting Deep Learning for Assessing TAVR Procedures and Outcomes.

Aortic valve defects are among the most prevalent clinical conditions. A severely damaged or non-functioning aortic valve is commonly replaced with a bioprosthetic heart valve (BHV) via the transcatheter aortic valve replacement (TAVR) procedure. Accurate pre-operative planning is crucial for a successful TAVR outcome. Assessment of computational fluid dynamics (CFD), finite element analysis (FEA), and fluid-solid interaction (FSI) analysis offer a solution that has been increasingly utilized to evaluate BHV mechanics and dynamics. However, the high computational costs and the complex operation of computational modeling hinder its application. Recent advancements in the deep learning (DL) domain can offer a real-time surrogate that can render hemodynamic parameters in a few seconds, thus guiding clinicians to select the optimal treatment option. Herein, we provide a comprehensive review of classical computational modeling approaches, medical imaging, and DL approaches for planning and outcome assessment of TAVR. Particularly, we focus on DL approaches in previous studies, highlighting the utilized datasets, deployed DL models, and achieved results. We emphasize the critical challenges and recommend several future directions for innovative researchers to tackle. Finally, an end-to-end smart DL framework is outlined for real-time assessment and recommendation of the best BHV design for TAVR. Ultimately, deploying such a framework in future studies will support clinicians in minimizing risks during TAVR therapy planning and will help in improving patient care.

Evaluation of cadmium and mercury on cardiovascular and neurological systems: Effects on humans and fish.

Chemicals are at the top of public health concerns and metals have received much attention in terms of toxicological studies. Cadmium (Cd) and mercury (Hg) are among the most toxic heavy metals and are widely distributed in the environment. They are considered important factors involved in several organ disturbances. Heart and brain tissues are not among the first exposure sites to Cd and Hg but they are directly affected and may manifest intoxication reactions leading to death. Many cases of human intoxication with Cd and Hg showed that these metals have potential cardiotoxic and neurotoxic effects. Human exposure to heavy metals is through fish consumption which is considered as an excellent source of human nutrients. In the current review, we will summarize the most known cases of human intoxication with Cd and Hg, highlight their toxic effects on fish, and investigate the common signal pathways of both Cd and Hg to affect heart and brain tissues. Also, we will present the most common biomarkers used in the assessment of cardiotoxicity and neurotoxicity using Zebrafish model.

Use of Animal Models for Investigating Cardioprotective Roles of SGLT2 Inhibitors.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors represent one type of new-generation type 2 diabetes (T2DM) drug treatment. The mechanism of action of an SGLT2 inhibitor (SGLT2i) in treating T2DM depends on lowering blood glucose levels effectively via increasing the glomerular excretion of glucose. A good number of randomized clinical trials revealed that SGLT2is significantly prevented heart failure (HF) and cardiovascular death in T2DM patients. Despite ongoing clinical trials in HF patients without T2DM, there have been a limited number of translational studies on the cardioprotective properties of SGLT2is. As the cellular mechanism behind the cardiac benefits of SGLT2is is still to be elucidated, animal models are used to better understand the pathways behind the cardioprotective mechanism of SGLT2i. In this review, we summarize the animal models constructed to study the cardioprotective mechanisms of SGLT2is to help deliver a more comprehensive understanding of the in vivo work that has been done in this field and to help select the most optimal animal model to use when studying the different cardioprotective effects of SGLT2is.

Experimental assessment of cardiovascular physiology in the chick embryo.

High resolution assessment of cardiac functional parameters is crucial in translational animal research. The chick embryo is a historically well-used in vivo model for cardiovascular research due to its many practical advantages, and the conserved form and function of the chick and human cardiogenesis programs. This review aims to provide an overview of several different technical approaches for chick embryo cardiac assessment. Doppler echocardiography, optical coherence tomography, micromagnetic resonance imaging, microparticle image velocimetry, real-time pressure monitoring, and associated issues with the techniques will be discussed. Alongside this discussion, we also highlight recent advances in cardiac function measurements in chick embryos.

How does hemodynamics affect rupture tissue mechanics in abdominal aortic aneurysm: Focus on wall shear stress derived parameters, time-averaged wall shear stress, oscillatory shear index, endothelial cell activation potential, and relative residence time.

An abdominal aortic aneurysm (AAA) is a critical health condition with a risk of rupture, where the diameter of the aorta enlarges more than 50% of its normal diameter. The incidence rate of AAA has increased worldwide. Currently, about three out of every 100,000 people have aortic diseases. The diameter and geometry of AAAs influence the hemodynamic forces exerted on the arterial wall. Therefore, a reliable assessment of hemodynamics is crucial for predicting the rupture risk. Wall shear stress (WSS) is an important metric to define the level of the frictional force on the AAA wall. Excessive levels of WSS deteriorate the remodeling mechanism of the arteries and lead to abnormal conditions. At this point, WSS-related hemodynamic parameters, such as time-averaged WSS (TAWSS), oscillatory shear index (OSI), endothelial cell activation potential (ECAP), and relative residence time (RRT) provide important information to evaluate the shear environment on the AAA wall in detail. Calculation of these parameters is not straightforward and requires a physical understanding of what they represent. In addition, computational fluid dynamics (CFD) solvers do not readily calculate these parameters when hemodynamics is simulated. This review aims to explain the WSS-derived parameters focusing on how these represent different characteristics of disturbed hemodynamics. A representative case is presented for spatial and temporal formulation that would be useful for interested researchers for practical calculations. Finally, recent hemodynamics investigations relating WSS-related parameters with AAA rupture risk assessment are presented. This review will be useful to understand the physical representation of WSS-related parameters in cardiovascular flows and how they can be calculated practically for AAA investigations.

Developmental Toxicity of Surface-Modified Gold Nanorods in the Zebrafish Model.

BACKGROUND: nanotechnology is one of the fastest-growing areas, and it is expected to have a substantial economic and social impact in the upcoming years. Gold particles (AuNPs) offer an opportunity for wide-ranging applications in diverse fields such as biomedicine, catalysis, and electronics, making them the focus of great attention and in parallel necessitating a thorough evaluation of their risk for humans and ecosystems. Accordingly, this study aims to evaluate the acute and developmental toxicity of surface-modified gold nanorods (AuNRs), on zebrafish (Danio rerio) early life stages. METHODS: in this study, zebrafish embryos were exposed to surface-modified AuNRs at concentrations ranging from 1 to 20 µg/mL. Lethality and developmental endpoints such as hatching, tail flicking, and developmental delays were assessed until 96 h post-fertilization (hpf). RESULTS: we found that AuNR treatment decreases the survival rate in embryos in a dose-dependent manner. Our data showed that AuNRs caused mortality with a calculated LC50 of EC50,24hpf of AuNRs being 9.1 µg/mL, while a higher concentration of AuNRs was revealed to elicit developmental abnormalities. Moreover, exposure to high concentrations of the nanorods significantly decreased locomotion compared to untreated embryos and caused a decrease in all tested parameters for cardiac output and blood flow analyses, leading to significantly elevated expression levels of cardiac failure markers ANP/NPPA and BNP/NPPB. CONCLUSIONS: our results revealed that AuNR treatment at the EC50 induces apoptosis significantly through the P53, BAX/BCL-2, and CASPASE pathways as a suggested mechanism of action and toxicity modality.

Computational Modeling of Motile Cilia-Driven Cerebrospinal Flow in the Brain Ventricles of Zebrafish Embryo.

Motile cilia are hair-like microscopic structures which generate directional flow to provide fluid transport in various biological processes. Ciliary beating is one of the sources of cerebrospinal flow (CSF) in brain ventricles. In this study, we investigated how the tilt angle, quantity, and phase relationship of cilia affect CSF flow patterns in the brain ventricles of zebrafish embryos. For this purpose, two-dimensional computational fluid dynamics (CFD) simulations are performed to determine the flow fields generated by the motile cilia. The cilia are modeled as thin membranes with prescribed motions. The cilia motions were obtained from a two-day post-fertilization zebrafish embryo previously imaged via light sheet fluorescence microscopy. We observed that the cilium angle significantly alters the generated flow velocity and mass flow rates. As the cilium angle gets closer to the wall, higher flow velocities are observed. Phase difference between two adjacent beating cilia also affects the flow field as the cilia with no phase difference produce significantly lower mass flow rates. In conclusion, our simulations revealed that the most efficient method for cilia-driven fluid transport relies on the alignment of multiple cilia beating with a phase difference, which is also observed in vivo in the developing zebrafish brain.

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.