Endothelial dysfunction (ED) is the initiation of atherosclerosis (AS). Our previous studies have found that cholesterol metabolism and the Wnt/β-catenin pathway can affect endoplasmic reticulum stress (ER stress), which ultimately leads to ED. However, the effects of cholesterol efflux on ED, which are caused by oxidative stress and the correlation among ER stress, Wnt/β-catenin pathway, and cholesterol efflux, are not clear during ED. To uncover them, the expressions of liver X receptors (LXRα and LXRβ) and ATP-binding cassette protein A1 (ABCA1) and G1 (ABCG1) in HUVECs  were measured under oxidative stress. Moreover, HUVECs were treated with LXR-623 (LXR agonist), cholesterol, tunicamycin, and salinomycin alone or together. The results indicated that oxidative stress-induced ED could deregulate the expressions of LXRα and LXRβ and trigger the ER stress and Wnt/β-catenin pathway, resulting thereafter in the accumulation of cholesterol. Furthermore, similar results were shown after treatment with cholesterol; however, the activation of liver X receptor (LXR) could reverse these changes. Furthermore, other results demonstrated that tunicamycin-induced ER stress could stimulate the accumulation of cholesterol and the Wnt/β-catenin pathway, further leading to ED. Inversely, salinomycin could reverse the above effects by deregulating the Wnt/β-catenin pathway. Collectively, our results showed that cholesterol efflux is partly responsible for the oxidative stress-induced ED; in addition, ER stress, the Wnt/β-catenin pathway, and cholesterol metabolism can interact with each other to promote ED. N2O2 .The endoplasmic reticulum (ER) is an organelle with functions such as protein folding, calcium ion storage, and the synthesis of lipids and carbohydrates , which aThe Wnt/β-catenin pathway is another inducement to ED. For instance, increased serum leptin levels in chronic kidney patients can lead to ED via the MTA1-WNT/β-catenin pathway . Under o2O2 to explore whether the oxidative stress could affect the cholesterol metabolism of HUVECs. The results . The results indicated that LXR was activated successfully, and the impacts of Hnt group A. Additi LXR-623 B. These  LXR-623 C found t LXR-623 C,D. Howe or H2O2 C,D. The 2O2, and the extracellular LDH and intracellular ROS were decreased in the LXR-623-treated group . Nevertheless, there were opposite results in the LXR-623 treated group , VCAM-1(Vascular cell adhesion molecular-1), and MCP-1(Monocyte chemoattractant protein-1) were upregulated under Hed group A,B. Therto THP-1 C,D. In g2O2, whereas LXR-623 reversed the above trends with the decreased expressions of GRP78, CHOP, and β-catenin and the increased level of p-β-catenin protein . Next, LRP6 was exclusively knocked down using shRNA interference to further explore the effects of Wnt/β-catenin signaling on the ER stress and cholesterol metabolism A. Intere2O2 was 500 μmol/L. However, Ransy et al. believed that this dose could release O2 from the catalase reaction, which was disproportionally high with regard to physiological oxygen concentration [2O2 concentration (from 100 to 700μmol/L) for the secretion of LDH. Therefore, the mechanisms of HUVEC injury are very complicated, which may be related to the Fenton’s reaction, a large excess in antioxidant defense, etc. [2O2. Among them, the levels of cholesterol efflux-related proteins (ABCA1 and ABCG1) were significantly downregulated, and cholesterol synthesis-related protein HMGCR was upregulated  and the expressions of ICAM-1, VCAM-1, and MCP-1 had no significant difference and showed slight downregulation. The reasons may be related to the feedback regulation, because cholesterol was treated earlier than H2O2 by about 2 h , LXR-623 , cholesterol , tunicamycin , and salinomycin .HUVECs , within 30 generations, were cultured with DMEM medium , and the THP-1 cells  were incubated with RPMI-1640 medium . All media were filled with 10% FBS, and all cell cultures were carried out in an environment at 37 °C containing 5% COThe LRP6 gene was cloned into the pLKO.1 puro vector, and further the resulting vector was transferred into TransStbl3 Chemically Competent Cells . The plasmid was extracted with an EasyPure HiPure Plasmid MiniPrep Kit  according to the instructions. We cotransfected psPAX.2, pMD2.G and the target plasmid into 293 T cells. TurboFect  was used to transfect cells according to the protocol. The lentivirus in the supernatant of the medium was concentrated after culturing for 48 h using Lentivirus Concentration Kit . Finally, the concentrated lentivirus was used to infect HUVECs at the confluency of 50% for 48 h. Two pairs of shRNA were created. One of the LRP6 shRNA sequences was 5′-CCGATGCAATGGAGATGCAAA-3′, and the other was 5′-GATAGCCTTCAGTTAACTAAC-3′.3 per well. With cell confluency reaching about 60%, HUVECs were treated with drugs and cultured for 24 h. Afterwards, we discarded the medium and then washed the cells with PBS buffer. Then, after being fixed with 4% paraformaldehyde at 37 °C for 35 min, HUVECs were rinsed three times with PBS buffer and further immersed in glycine (1.5 mg/mL) for 10 min to neutralize the remaining formaldehyde. Finally, after rinsing off the excess dye with PBS after dyeing with filipin solution  for 2 h, we observed the fluorescence intensity using a LeicaDM1000  and an Olympus FV3000  and then calculated the average fluorescence intensity of all the signal regions of the entire image by Image J 1.53e software, excluding the black background area.HUVECs were seeded on 24-well plates and cultured into an incubator at a density of 2 × 10HUVECs were planted in 6-well plates and then cultured with drugs together for 24 h with the cell density attaining about 60%. The supernatant was taken for the determination of LDH activity by a lactate dehydrogenase (LDH) assay kit . Furthermore, HUVECs homogenated with PBS buffer were taken for the determination of SOD and NOS activity. The superoxide dismutase (SOD) assay kit  was used to test SOD content, and the nitric oxide synthase (NOS) assay kit  was used to detect NOS content. All of the above experiments were measured strictly according to the corresponding instructions.3 per well. A DCFH-DA working solution (15 μmol/L) was prepared with DCFH-DA stock solution  and buffer. With the medium aspirated carefully, the cells were rinsed once with buffer solution. Then, at about 70% growth, the HUVECs and the DCFH-DA working solution were cultured together for 50 min. Finally, when HUVECs were rinsed three times with buffer solution, the fluorescence degree was observed using an Olympus IX71 fluorescence microscope , and then the mean fluorescence intensities of all the signal regions of the entire picture were analyzed with Image J 1.53e software.The level of intracellular ROS was determined by the fluorescence probe 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA). HUVECs were tiled in a 24-well plate with a density of 2 × 104 THP-1 cells, labeled with BCECF/AM  for 1 h in the dark cell incubator, were then transferred into drug-treated HUVECs in 24-well plates when the HUVECs had grown to about 70% and cultured for 1 h in the dark. Afterwards, the THP-1 cells and HUVECs were dyed with Hoechst33342 for 40 min after being removed from the previous medium. Finally, PBS buffer was added to rinsed cells three times to gently wash away the THP-1 cells that had not adhered. Images were taken with an Olympus inverted fluorescence microscope . The average number of THP-1s of three independent tests was calculated.The HUVECs were incubated in 24-well plates. Furthermore, 5 × 10The HUVECs were harvested with trypsin  after being treated with drugs for 24 h. RIPA lysate  was used to lyse cells, and then the total protein of supernatant was collected through centrifugation. The collected supernatant was boiled for 5 min after adding 1 × loading buffer  and then electrophoretically isolated on 8–12%SDS–PAGE gel. Then we used 5% skimmed milk to block the PVDF membrane for 1 h. Firstly, the PVDF membrane was incubated with the primary antibodies at 4 °C for 12 h and washed with 1 × TBST for 30 min. Next, the PVDF membrane was incubated with the secondary antibodies at 37 °C for 1 h, further washed with 1 × TBST for 30 min, with 10 min each time at 100 rpm/min. At last, ECL ultra-sensitive luminescent liquid was added on the PVDF film equably, which was imaged on the BIORAD gel imager. The antibodies are as follows: MCP-1 , 1:500; CD106/VCAM-1 , 1:500; GRP78 , 1:10000; GAPDH , 1:40000; β-catenin , 1:4000; ICAM-1 , 1:1000; ABCA1 , 1:500; ABCG1 , 1:500; LXRα , 1:2000; LXRβ , 1:1000; LRP6 , 1:1000; HMGCR , 1:1000 and p-β-catenin , 1:2000. In these experiments, the protein bands normalized to GAPDH. p > 0.1, suggesting that all date conformed to a normal distribution. Then, a Student’s t-test was performed to compare between two groups, and one-way analysis of variance (ANOVA) was used to analyze the differences between more than two groups. The fluorescence intensity analysis was processed by Image J 1.53e. All experiments were performed at least three times, with three biological replicates per experiment, and all data were expressed as mean ± standard deviation. p < 0.05 indicates there was a statistical difference.All data were first tested by the Shapiro–Wilk normality test using GraphPad Prism 6.0 software, and we found Our studies suggested that inhibiting the cholesterol efflux of HUVECs could partly induce the cholesterol accumulation by decreasing the activity of LXRs, which led to the oxidative stress-induced ED. Mechanically, both cholesterol accumulation and Wnt/β-catenin pathway in HUVECs can trigger the ER stress; conversely, ER stress can regulate cholesterol metabolism and the Wnt/β-catenin pathway as well. Moreover, cholesterol metabolism and Wnt/β-catenin pathway also can affect each other. In conclusion, ER stress, cholesterol metabolism, and the Wnt/β-catenin pathway can interact with each other to promote ED in HUVECs .


Sweet potato is a widely cultivated crop with storage roots. Although many studies have been conducted on the mechanism of its storage root formation, the details have not been fully elucidated. We screened mutant lines with inhibition of storage root formation to clarify parts of the mechanism. In this study, the process of storage root formation in one of the mutant lines, C20-8-1, was investigated. The inhibition of storage root formation was observed during the early stages of growth. The roots in C20-8-1 did not show histological differences compared to those in wild type. The transition from fibrous roots to pencil roots, which are the developmental stages prior to mature storage root formation, was delayed or inhibited in C20-8-1. The upregulation of starch biosynthesis-related genes and downregulation of lignin biosynthesis genes with storage root swelling were not confirmed in the root of C20-8-1 during the developmental transition stage, suggesting that most of the roots in C20-8-1 are in the pre-transition state toward the storage root swelling. C20-8-1 showed a mutant phenotype during the critical period of storage root swelling initiation, and further clarification of this mutation is expected to provide new insights into storage root formation. Ipomoea batatas [L.] Lam.) is one of the most important food crops and is cultivated worldwide. Storage root (SR) is an economically important component of sweet potato. SR has been used for table, food processing, alcohol, and industrial purposes because it is rich in carbohydrates, vitamins, and micronutrients . After harvesting, the roots of each plant were cleaned with water. Total weight, total shoot weight, total root weight, and leaf area were measured. The leaf area was measured using ImageJ for the three largest leaves of each plant. The roots were immediately frozen in liquid nitrogen and stored at –80°C or fixed in FAA fixative.As the yield test for WT and C20-8-1, the acclimatized plants were cultivated in the field at Kushima, Miyazaki, Japan, according to Paraffin sectioning was performed on fibrous root samples. The fixed roots were immersed in turns for more than 3 hours; distilled water:ethanol:n-butanol = 50:40:10, 30:50:20, 15:50:35, 0:50:50, 0:30:70, 0:0:100, and 0:0:100. Briefly, the samples were stained with safranin and embedded in paraffin. Paraffin blocks containing the samples were sectioned to a thickness of 15 μm using a microtome. Paraffin sections were extended on glass slides and deparaffinized under the following conditions. The slide glasses were immersed for more than 30 min twice in 100% xylene; xylene:ethanol = 1:1; 100%, 90%, 70%, 50% ethanol; and distilled water. After staining with hematoxylin for 1 min, dehydration was performed in the reverse order of deparaffinization. Thereafter, they were sealed with Entellan new  sealant and covered with a micro cover glass. Specimens were observed and photographed using an optical microscope  and AxioCam208color OSD (ZEISS).The samples at 45 and 90 DAT were stained with safranin diluent (50% ethanol 5 mL mixed with Safranin solution 10 μL) for 1 min and washed twice with distilled water. Afterward, the sample was stained with fast green diluent (95% ethanol 5 mL mixed with Fast Green FCF (FUJIFILM) 0.01 g) for 10 s and washed twice with distilled water. In the last step, the sample was re-stained with a safranin diluent for 30 s and washed twice with distilled water. Specimens were observed and photographed using a stereomicroscope  and a MicroStudio system (Wraymer).Total RNA was isolated from all root samples using the RNeasy Plant Mini Kit (Qiagen) and then treated with RNase-free DNase I (Invitrogen). cDNA was reverse-transcribed from 1.5 μg of total RNA in each sample using the SuperScript IV First-Strand Synthesis Kit (Invitrogen) with random hexamers (Invitrogen). The procedures were performed according to the manufacturer’s instructions.–ΔΔCT method  Kit (KAPA Biosystems)), 0.4 μL 10 mM of each primer, and 1 μL of cDNA (20 ng). The PCR procedure was as follows: CFX Connect Real-Time PCR Detection System (Bio-Rad); 95°C for 3 min, 40 cycles of 95°C for 3 s, 60°C for 20 s, and a melting curve was generated ranging from 60°C to 95°C. Three biological replicates and three technical replicates per biological replicate were performed for each experiment. Relative gene expressions were calculated using the 2T method . The prip < 0.05 by Student’s t-test).At first, we confirmed the yield of the mutant line of C20-8-1 cultivated in the field. The average yield (g) and number of the SRs in WT were 960.0 ± 92.5 S.E. and 4.3 ± 0.5 S.E., respectively, and those in C20-8-1 were 430.0 ± 54.5 S.E. and 2.5 ± 0.3 S.E., respectively. The yield and SR number in C20-8-1 were significantly decreased compared to those in WT .At 15 DAT, all the roots were categorized as early fibrous roots in WT and C20-8-1 plant . At 45 DThe inner structure of the roots was observed histologically . We compAPRT gene, which is related to cytokinin activation, in C20-8-1 was 2.4-fold higher than that in the WT, with a significant difference at 15 DAT (IbNAC083, which is a key regulator of SR swelling, increased from 15 DAT to 45 DAT in WT and C20-8-1, and there was no significant difference in the expression levels at each stage (Ib4CL, IbCAD, and IbCOMT genes at 15 DAT and IbPAL at 45 DAT (IbSBEII and Ibα-AMYL showed a higher expression in C20-8-1 than in WT (We focused on the early development of SR in C20-8-1, and qRT-PCR was used to analyze the SR development-related gene expression levels at 15 and 45 DAT. The root samples for RNA extraction at 15 DAT consisted of early fibrous roots from WT and C20-8-1 plants. Those at 45 DAT consisted of late fibrous to late-developing pencil roots in WT and late fibrous to early-developing pencil roots in C20-8-1. We selected the genes based on previous reports and a primer test in WT of ‘Beniharuka’ . In the t 15 DAT . The expch stage . Most ofch stage . Among tt 45 DAT . In the an in WT . Althougan in WT .We applied heavy-ion mutagenesis to ‘Beniharuka’ and obtained mutant candidates with phenotypes of high yield and inhibition of SR formation , 3. The APRT, one of the differentially expressed genes, showed a significantly high expression level at 15 DAT (APRT is involved in cytokinin conversion from active free bases to inactive nucleotides (IbNAC083 in C20-8-1 showed a decreasing trend compared to WT, but not significant. IbNAC083 has been identified as a key regulator of bulking in sweet potatoes, and downregulation of IbNAC083 by RNAi inhibits SR formation (IbNAC083. Ib4CL, IbCAD, and IbCOMT genes at 15 DAT and IbPAL at 45 DAT, showed the higher expression in C20-8-1 than in WT (For molecular characterization of the change in the SR formation process, the expression levels of the genes related to SR formation and development were compared between WT and C20-8-1, and variation in expression was observed in several genes . APRT, ot 15 DAT . APRT isan in WT , suggestan in WT . The staIn addition to the SR phenotype, there was a reduction in leaf area in C20-8-1 , Table 1In this study, we characterized a novel mutant with a delay or suppression of SR swelling initiation. Gene deletions associated with the decrease in DNA content in C20-8-1 may be thought to inhibit SR formation. Knockout of the key genes mentioned above was not observed in C20-8-1. Further clarification of the details of the mutation in the genome and identification of the gene responsible for the phenotype is expected to provide new insights on SR formation.Conceptualization, T.H. and H.K.; methodology, T.H., H.P., T.A., and H.K.; investigation, H.P., T.H., H.K., and T.A.; writing-original draft preparation, H.P. and T.H.; writing-review and editing, T.A. and H.K.Supplemental Table


Immune checkpoint inhibitor (ICI) therapy has caused a paradigm shift in cancer therapeutic strategy. However, this therapy only benefits a subset of patients. The difference in responses to ICIs is believed to be dependent on cancer type and its tumor microenvironment (TME). The TME is favorable for cancer progression and metastasis and can also help cancer cells to evade immune attacks. To improve the response to ICIs, it is crucial to understand the mechanism of how the TME is maintained. Protein arginine methyltransferase 5 (PRMT5) di-methylates arginine residues in its substrates and has essential roles in the epigenetic regulation of gene expression, signal transduction, and the fidelity of mRNA splicing. Through these functions, PRMT5 can support cancer cell immune evasion. PRMT5 is necessary for regulatory T cell (Treg) functions and promotes cancer stemness and the epithelial–mesenchymal transition. Specific factors in the TME can help recruit Tregs, tumor-associated macrophages, and myeloid-derived suppressor cells into tumors. In addition, PRMT5 suppresses antigen presentation and the production of interferon and chemokines, which are necessary to recruit T cells into tumors. Overall, PRMT5 supports an immunosuppressive TME. Therefore, PRMT5 inhibition would help recover the immune cycle and enable the immune system-mediated elimination of cancer cells. The accumulation of genetic changes in cells can lead to cancer development. Cancer cells lose their normal regulatory mechanisms, resulting in increased cell proliferation and invasion rates ,2. AlthoProtein arginine methyltransferases (PRMTs) methylate arginine residues in their substrates. PRMTs regulate gene expression, signal transduction, and the fidelity of mRNA splicing . RecentlThe concept of the cancer-immunity cycle has been proposed to represent the complex and dynamic mechanism used by the immune system to kill cancer cells  3]. In . In 3]. Although the human body has excellent immune system mechanisms against cancer cells, the cycle does not work properly in cancer patients. To explain this, the cancer immunoediting concept was proposed  4,5]. W,5. W4,5]There are several factors that can regulate the cancer-immunity cycle through either promoting or repressing it. Cancer cells and the cancer-associated microenvironment can inhibit this cycle. In step 3 of the cancer-immunity cycle, antigen recognition through T cell receptors and the interaction of CD28 on T cells with CD80/CD86 on DCs are necessary for full T cell activation . CTLA-4 Several factors that contribute to the TME, such as CSCs, are also involved in evading the cancer-immunity cycle. These factors can support the recruitment of immunosuppressive cells into the TME  to the guanidino nitrogen atoms of arginine, resulting in the formation of methylarginine  14]. Th. Th14]. PRMT5 was first identified as a binding protein of Janus kinase 2 (JAK2) . PRMT5 ePRMT5-mediated epigenetic regulation of gene expression is regulated by the arginine methylation of histones H2, H3, and H4. PRMT5 mediates the dual symmetrical di-methylation of H2AR3 (H2AR3me2s) and H4R3 (H4R3me2s). This modification contributes to transcriptional repression by reducing the levels of H4K5ac . In addiThe regulation of mRNA splicing is a crucial role of cytoplasmic PRMT5. The mRNA splicing process is performed by the ribonuclear protein (RNP) complex , which contains small nuclear RNPs (snRNPs) and seven Sm proteins  . PRMT5 mArginine methylation is also part of the regulation of signaling pathways. Examples include PRMT5-mediated arginine methylation of components of the epidermal growth factor receptor (EGFR), AKT, and hedgehog (HH) signaling pathways e.PRMT5 di-methylates the intracellular domain of EGFR (Arg 1175). This modification induces the autophosphorylation of a tyrosine residue at residue 1173 and the binding of protein tyrosine phosphatase, Src homology region two domain-containing phosphatase 1 (SHP1). Then, the EGFR-SHP1 interaction attenuates the extracellular signal-regulated kinases (ERK) . PRMT5-mPRMT5-mediated AKT methylation regulates its activity and cellular localization. PRMT5 methylates AKT at arginine residues 15 and 391. These modifications promote AKT activation, leading to AKT membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the target of rapamycin complex 2 (mTORC2) .PRMT5 also regulates the activity of transcriptional regulators. PRMT5 methylates arginine residues in the oligomerization domain of p53 . PRMT5-mThe glioma-associated oncogene (GLI) family transcriptional regulator is activated downstream of the HH signaling pathway. PRMT5 methylates cytoplasmic GLI1 arginine residues 990 and 1018. These modifications induce GLI1 protein stabilization by preventing the interaction of GLI1 with the itchy E3 ubiquitin protein ligase (ITCH)-NUMB E3 ligase complex . In addiAs described in the previous section, PRMT5 has essential roles in gene expression and mRNA splicing. It has been reported that these functions of PRMT5 have important roles in T cell maintenance and B cell development. Although PRMT5 inhibitors may attenuate T cell or B cell functions, side effects have not yet been reported. In addition, a previous report  did not 2 gene expression after T cell signaling activation [IL-2 is produced during the antigen-specific clonal growth of T cells and is essential for the proliferation and survival of CD4+ T cells and CD8+ T cells . PRMT5 ptivation . IL-2 antivation . These ctivation . PRMT5 etivation . B cells are part of the adaptive immune system and produce high-affinity antibodies that mediate protection from pathogens, but remain tolerant of self-tissues. At first, B cell progenitor cells are generated in the bone marrow. Then, progenitor cells transform into mature B cells. In this stage, progenitor cells repeatedly proliferate and differentiate to undergo successful rearrangement of the immunoglobulin genes. Activated mature B cells enter the germinal center (GC). Then, they undergo expansion, immunoglobulin class switching, and programmed Ig mutation coupled to antibody affinity-based selection. Finally, mature B cells differentiate into memory B cells or plasma cells .Litzler et al. found that PRMT5 is necessary for B cell development . PRMT5 mPRMT5 overexpression has been observed in several cancers see . FurtherThe overexpression of PRMT5 is observed in various cancers and is associated with worse survival rates ,63,64,65PRMT5-mediated histone modification is involved in CSC survival, and deregulated PRMT5 mRNA splicing machinery can induce strange insertions of introns and exon skipping that result in gene mutations. Furthermore, PRMT5 upregulates the expression of various genes that are associated with oncogenic signaling, CSC renewal, metastasis, and metabolic reprogramming . RecentlCDKN2A gene, which encodes p16/INK4A and p19/ARF [Homozygous deletion of methylthioadenosine phosphorylase (MTAP) frequently occurs in cancer because of its proximity to the  p19/ARF . In addi p19/ARF ,69.The stimulator of the interferon gene (STING) is a transmembrane protein in the endoplasmic reticulum that is activated by cytosolic double-stranded DNA (dsDNA). Cyclic GMP-AMP synthase (cGAS) acts as a sensor for dsDNA and is necessary for STING activation. Activated STING upregulates the interferon regulating factor 3 (IRF3)-TANK binding kinase 1 (TBK1) cascade, resulting in the production of cytokines and type I interferon .Kim et al. reported that PRMT5 can attenuate the cGAS-STING pathway, which supports immune evasion in melanoma . PRMT5 sThe authors also demonstrated the correlation between PRMT5 and the anti-tumor immune response by in vivo experiments. PRMT5 knockdown melanoma cell xenograft tumor growth was inhibited in immunocompetent C57BL/6 mice. However, PRMT5 knockdown did not affect tumor shrinkage in immunocompromised NOD-SCID gamma (NSG) mice. These results suggest that PRMT5 inhibition upregulates the anti-tumor immune response. Furthermore, the melanoma cell xenografted tumor was shrunk following combination treatment with a PRMT5 inhibitor (GSK3326595)  and an aTreg cells are essential for maintaining self-tolerance and immune homeostasis under normal conditions. They can also infiltrate into tumor tissues and hamper anti-tumor immune responses . A membeNagai et al. found that PRMT5 can regulate Treg function through FOXP3 methylation . PRMT5 wCancer cell xenograft tumor analysis revealed that PRMT5 inhibitor (DS-437)  treatmenCSCs are a subpopulation of cancer cells that exhibit the characteristics of both stem cells and tumor cells. CSCs drive tumor initiation and self-renewal. CSCs have enhanced drug-efflux pumps, a high DNA repair capacity, and upregulated protection against reactive oxygen species (ROS). Therefore, chemotherapy and radiation therapy have minimal effects on these cells . CSCs caAccumulating evidence shows that suppressing PRMT5 can attenuate the CSC population in tumors ③. PRMT5 Cancer cells can spread from the primary tumor site to nearby tissues, and in some cases to distant tissues, forming a new tumor. This process is called metastasis. EMT is essential for tumor metastasis . EMT is AKT acts downstream of TNF-α and TGF-β to upregulate EMT-associated transcription factors, such as SNAIL and TWIST1 ,93,94. IXenografted tumor analysis revealed that PRMT5 inhibitor treatment can attenuate tumor growth and metastasis. The PRMT5–AKT1 axis is also associated with EMT in lung and colorectal cancer .cadherin 1  and ten–eleven translocation methylcytosine dioxygenase 1 (TET1) gene expression by binding to their promoter regions [CDH1 promoter and suppresses its gene expression in colon cancer [Other previous reports have shown that PRMT5 forms a complex with SNAIL and nucleosome remodeling and deacetylase (NuRD)  or with  regions . The PRMn cancer .cadherin 2  and SNAIL expression, which is involved in EMT and promotes cell migration and lymph-node metastasis [In laryngeal carcinoma, PRMT5 regulates WNT4 expression, and PRMT5–WNT4 activates β-catenin to induce tastasis   would be essential factors for this approach.


The hybrid nanofillers are observed to have synergistic properties as epoxy/hybrid mixtures showed better processability than epoxy/SWCNT, while maintaining high values of electrical conductivity. On the other hand, epoxy/SWCNT nanocomposites present the highest electrical conductivities with the formation of a percolating conductive network at lower filler content, but very large viscosity values and filler dispersion issues, which significantly affect the final quality of the samples. Hybrid nanofiller allows us to overcome the manufacturing issues typically associated with the use of SWCNTs. The combination of low viscosity and high electrical conductivity makes the hybrid nanofiller a good candidate for the fabrication of aerospace-grade nanocomposites with multifunctional properties.In this work, we investigate the processability and the volumetric electrical properties of nanocomposites made of aerospace-grade RTM6, loaded with different carbon nanoparticles. Nanocomposites with graphene nanoplatelets (GNP), single-walled carbon nanotubes (SWCNT) and hybrid GNP/SWCNT in the ratio 2:8 (GNP In recent years, carbon nanoparticles have attracted considerable interest in several industrial fields for their nature of possessing simultaneously superior physical properties with respect the traditional materials. Indeed, carbon nanotubes may have very high electrical and thermal conductivities, of several orders of magnitude greater than copper ,2,3, andThe capability of nanocomposites to simultaneously enhance different properties is particularly relevant in the aerospace field, where multifunctional polymer-based composites can be advantageous to satisfy the lightweight requirements of aircrafts and spacecrafts by replacing the heavy and complex subsystems ,13,14,15From a rheological point of view, nano-reinforced polymer blends may show different rheological behaviors depending on the type of nanofillers used ,32. This2SWCNT8, GNP5SWCNT5 and GNP8SWCNT2) were investigated in terms of electrical and rheological properties and related to material processability. This resin was selected because it is already qualified for aerospace applications. In addition, it has a low viscosity (0.05 Pa·s) at the injection temperature (about 80 °C) so that it can be used in liquid composite molding to manufacture components with complex geometries and large dimensions [The aim of this work is to investigate the potentiality of hybrid nanocarbon systems to overcome the problems of nanocomposite processing, such as poor filler dispersion and increase in viscosity, which typically makes the use of SWCNTs inapplicable in industrial processes despite their exceptional properties. The point of view of processing is generally neglected in the literature and the present work aims to fill this gap. An extensive study of the rheological properties of hybrid CNT/GNP fillers dispersed in a typical aerospace resin before curing is reported. This work aims to determine the optimal ratios between graphene and nanotubes to obtain suitable processability characteristics useful for liquid composite molding while enhancing the electrical properties of the nanocomposites. In particular, the properties of aerospace-grade RTM6, a mono-component epoxy resin, loaded with GNP/SWCNT hybrid nanofiller in the ratio of 2:8, 5:5 and 8:2  and exfoliated graphene nanoplatelets (xGNP) with different specific surface area and aspect ratio were purchased from XG Sciences . In particular, nanoplatelets of grade M5 , grade H5 , and grade C750  were used. The selected resin was the aerospace-grade mono-component epoxy RTM6  formulated for resin-transfer molding processes. All materials were used as received.Single-walled carbon nanotubes with a purity higher than 85% were purchased from OCSiAl Europe . According to the producer datasheet, the average diameter is ~1.8 nm and the average length is >5 µm. Graphene nanopowder of grade AO-4 was from Graphene Supermarket  ≤40 m2SWCNT8, epoxy/GNP5SWCNT5 and epoxy/GNP8SWCNT2 nanocomposites with filler concentrations in the range of 1–5 wt% were prepared. For the hybrid nanofillers, ratios of GNP to SWCNT of 2:8, 5:5 and 8:2 (by weight) were used. Nanocomposite samples with GNP AO-4, xGNP-C750, xGNP-H5 and xGNP-M5, each dispersed in RTM6 epoxy resin at 1 wt%, were also fabricated. The dispersion procedure was tuned considering the dependence of the viscosity from the temperature in order to allow the nanofillers mobility in the resin without incurring premature gelling. In this procedure, the resin was initially pre-heated to 80 °C with a constant rate of 2 °C/min. When the resin was homogenously heated, the desired amount of nanofillers was blended for about 90 min in an ultrasonic bath at 80 °C. According to the RTM6 datasheet, the process window is sufficiently long lasting at 80 °C and the viscosity remains constant during the nanofillers dispersion procedure. During this last step, the mixture was also degassed in order to eliminate any entrapped air bubbles. The same nanoparticle dispersion protocol was used to prepare both nanocomposite mixtures and cured samples for the rheological and electrical testing. The specimens for electrical characterization were realized by pouring the mixture into a silicon mold and curing in an oven at 180 °C for 2 h. The curing parameters were the same for all types of hybrid nanocomposites since the inclusion of 1D fillers does not lead to significant differences in the curing kinetics with respect to the unfilled epoxy mixture [−2 [Epoxy/SWCNT, epoxy/GNPture [−2  whereas d) was averaged over 10 different images across each sample. SEM investigations were carried out using a VEGA II LSH instrument  at an accelerating voltage of 5 kV and 40× magnification. SEM images were processed using the MountainsMap 7 software , which allows a three-dimensional (3-D) reconstruction of the specimen surface and the evaluation of surface roughness from images acquired at different tilt angles (0° and 5°). The roughness was averaged over values measured on profiles extracted every 0.25 mm across the reconstructed 3-D surface. The analysis was performed on the top surface of the specimen, which was exposed to air during the curing process. The electrical conductivity of the cured nanocomposite samples was determined by electrical impedance spectroscopy (EIS) over the frequency range 20 Hz–1 MHz using an Agilent E4980A Precision LCR Meter. The samples (10 mm × 10 mm) were placed in a custom-made Teflon cell with two square copper electrodes measuring 10 mm × 10 mm at the top and bottom surfaces in a two-point configuration. Impedance measurements were performed with the parallel circuit model, providing the equivalent parallel resistance (pR). The electrical resistance data were averaged over 20 measurements. The volumetric conductivity (vσ) of the nanocomposites was determined as vσ = 1/vρ, where vρ is the volumetric resistivity evaluated according to the ASTM D257-07. The rheological properties of the nanocomposite mixtures were measured using a rotational stress-controlled MCR 302 rheometer  equipped with a Peltier heating system. Experiments were conducted in steady shear mode using the 25 mm plate–plate configuration, with gap size in the range of 1.1–1.3 mm depending on the filler concentration. Steady shear measurements were performed at shear rates in the range of 0.1–100 s−1 and at 80 °C, the recommended injection temperature for neat RTM6 epoxy in resin transfer molding processes, with a constant heating rate of 2 °C/min starting from room temperature. The dispersion state of the nanofillers was investigated using a quantitative method based on the analysis of grayscale optical images . Dropletd) with values of 0.532 ± 0.008 for the epoxy/GNP AO-4, 0.509 ± 0.015 for the epoxy/xGNP-C750, 0.478 ± 0.018 for the epoxy/xGNP-H5, and 0.437 ± 0.013 for the epoxy/xGNP-M5 nanocomposites.Optical images showing the dispersion state of the different nanofillers blended into RTM6 resin are in d is 1 in the ideal case of the uniform dispersion state and decreases as the dispersion state deteriorates [The dispersion index was evaluated by comparing the grayscale optical image to the corresponding image with uniformly dispersed pixels. The value of Iriorates . If we lriorates .The volumetric electrical conductivity of the epoxy/graphene samples at 1 wt% of nanofiller over the frequency range of 1.5 kHz–1 MHz is presented in 10 Ω·cm, the electrical conductivity of the neat RTM6 epoxy resin is not reported. As shown in All epoxy/graphene samples exhibit the typical behavior of insulating materials, with electrical conductivity that increases with frequency. It is noted that for these nanocomposites with large resistance values at low frequencies, the EIS data were acquired only in the frequency range above 1.5 kHz. This was due to the lower accuracy of the measured resistance values near the instrument measurement limit. For the same reason, having resistivity values higher than 10From the analysis of the electrical and dispersion properties, it emerges that the GNP AO-4 has the best dispersion degree among the nanofillers considered in this study, as well as high electrical conductivity. 2SWCNT8, GNP5SWCNT5 and GNP8SWCNT2, in order to simultaneously take advantage of the good dispersion state and processability of the GNP AO-4 nanofillers and of the high aspect ratio of SWCNTs. These factors play a crucial role in the development of the 3D conductive network, whilst avoiding the formation of SWCNT entanglements occurring at high loadings of SWCNTs. For these reasons, the GNP-AO4 type was selected for the graphene/SWCNT hybrid nanofiller. In particular, the epoxy/hybrid carbon mixtures were prepared with GNP AO-4 and SWCNT in the ratios of 2:8, 5:5 and 8:2 (by weight) (by weight), respectively, referred to as GNP2SWCNT8, epoxy/GNP5SWCNT5 and epoxy/GNP8SWCNT2 nanocomposite samples, as determined by electrical impedance spectroscopy over the frequency range 20 Hz–1 MHz. All types of nanocomposites exhibit typical resistive behavior with frequency-independent electrical conductivity up to values of the order of 105 Hz. Overall, the epoxy/SWCNT system has higher electrical conductivities than the epoxy/GNP8SWCNT2 and epoxy/GNP5SWCNT5 systems at all concentrations. As regards the epoxy/GNP2SWCNT8 nanocomposites, they show electrical conductivities close to the values exhibited by the epoxy/SWCNT nanocomposites. In general, the conductivity of nanocomposite systems at high filler concentrations is known to follow the percolation law when a certain volume fraction (percolation threshold) is reached.p is the filler volume fraction, cp is the percolation threshold and t is the critical exponent, related to the dimensionality of the system. In theory, the exponent assumes the value of t = 1.3 and t = 2 for two- and three-dimensional percolating networks, respectively [5SWCNT5 and epoxy/GNP2SWCNT8 systems also exhibit such behavior, showing an electrical conductivity at 1 wt% almost identical to the epoxy/SWCNT 1wt% system. For the nanocomposites filled with the GNP8SWCNT2 hybrid, we note the insulating behavior of the 1 wt% mixture at high frequency as well as the remarkable enhancement of electrical properties when increasing the loading fraction up to 2 wt%, meaning that the percolation threshold can be found between these two filler concentrations. In addition, the difference between the electrical conductivity of the epoxy/GNP8SWCNT2 at 4 wt% and 5 wt% is minimal, meaning that further increasing the nanofiller concentration would be ineffective. By analyzing the results obtained for the epoxy/SWCNT samples at different loadings of filler, we observe a saturation of the electrical conductivity at 3.5 wt% of SWCNTs. The increase in nanotube concentration is not followed by the improvement of the electrical properties, contrary to what would be expected. This behavior can be explained by the increase in entanglements at high filler loadings, which do not participate in the improvement of the network structure and inhibit further improvement of the electrical properties. In addition, the agglomeration phenomenon is responsible for the deterioration of the mechanical properties and for limiting the processability of such nanocomposites. This result suggests that the further increase in the SWCNTs loading fraction over 3.5 wt% is not recommended, and this can be considered an upper limit as concerns the electrical properties of the epoxy/SWCNT nanocomposites. When looking at the volumetric electrical conductivity of the epoxy/GNP8SWCNT2, epoxy/GNP5SWCNT5 and epoxy/GNP2SWCNT8 hybrid samples σ=∝ were selected as nanofillers, which readily increase the viscosity of the composite fluid by interacting with each other. However, as the shear rate increases, the network is de-bundled and the nanotubes align in the shear direction, with the consequence of reducing significantly the viscosity of the system. Whereas the viscosity of the SWCNT-loaded epoxy decreases steeply with increasing shear rates, the shear thinning behavior of the hybrid mixtures becomes less pronounced as the GNP:CNT ratio increases. Indeed, the epoxy/GNPK is the consistency index and n is the flow behavior index (dimensionless). The fitted parameters for the RTM6 resin loaded with SWCNTs and with GNP2SWCNT8, GNP5SWCNT5 and GNP8SWCNT2 hybrids are summarized in Different models have been proposed to characterize the rheological behavior of polymer melts. In our case, all the samples analyzed exhibit a shear thinning behavior that can be characterized using the power law model, with the viscosity pression :(2)η γ˙=K is a proportionality constant and can be considered as the value that the viscosity assumes at a shear rate 1 s−1. Depending on the value of the power law index, the fluid exhibits a shear-thickening (n > 1), shear-thinning (n < 1) or Newtonian behavior (n = 1). In addition, the smaller is the value of n, the more shear thinning is the fluid. The rheological behaviors of the epoxy/SWCNT and epoxy/GNP8SWCNT2 mixtures are clearly different, with the second one approaching the Newtonian response more than the first system. More important, the viscosity values of the two nanocomposite mixtures are markedly different: the hybrid system has lower viscosity values than the epoxy/SWCNT system at all concentrations, with variations of one order of magnitude at nanofiller loading of 1 wt% and two orders of magnitude at the higher loadings 4–5 wt% (2SWCNT8 hybrids are similar, showing viscosity values of the same order of magnitude at all nanofiller loadings (5SWCNT5 system, it simultaneously shows high viscosity values at the high loadings of 3 wt% and 4 wt%, and a rheological behavior that approaches that of the epoxy/GNP8SWCNT2 system at the nanofiller loadings of 1 wt% and 2 wt% (8SWCNT2 hybrid system with 3 wt%, 4 wt% and 5 wt% of nanofiller exhibits better electrical properties than the epoxy/SWCNT system at 1 wt% and 2 wt% of SWCNTs, while maintaining lower viscosity values, which translates in better processability for composite manufacturing. Similarly, the epoxy/GNP5SWCNT5 hybrid system with 1 wt% and 2 wt% of nanofiller exhibits electrical conductivity values of the same order of magnitude as the epoxy/SWCNT system at 1 wt% and 2 wt% of SWCNTs, while maintaining viscosity values one order of magnitude lower. Regarding the epoxy/GNP2SWCNT8 hybrid system, it shows electrical and rheological behaviors similar to those of the epoxy/SWCNT system, which translates in similar processability for composite manufacturing. The consistency index  4–5 wt% a,d. On tloadings a,b. As rnd 2 wt% a,c. OverThe rheological behavior of the nanocomposite blends confirms the formation of the 3D filler network in the hybrid systems, where graphene planes are intercalated between 1D single-wall carbon nanotubes. GO platelets can separate the walls of SWCNTs, avoiding the formation of agglomerations among the CNTs. In addition, the graphene layers act as a lubricant reducing the friction between the polymer chains during their relative motion resulting in a decreased polymer viscosity.8SWCNT2 samples with different nanofiller loadings.The morphology of the nanocomposites after a cure was investigated by SEM and the effect of the viscosity on the final quality of the surfaces was studied. 8SWCNT2 and the epoxy/SWCNT sample, which exhibits the roughest surface. For the epoxy/SWCNT system, a large number of asperities are present on the specimen surface due to the formation of entanglements, which is more evident at high concentrations of nanofiller (8SWCNT2 hybrid system, we observe that the epoxy/GNP8SWCNT2 hybrid at 5 wt% (8SWCNT2 hybrid system, which leads to better processability and more homogenous samples. In addition, the epoxy/GNP8SWCNT2 hybrid at 5 wt% has higher electrical conductivity with respect to the epoxy/SWCNT 1 wt% (By comparing the 1 wt% systems, we note that the epoxy/GNP AO-4 sample has the smoothest surface among the analyzed specimens, followed by the epoxy/GNPnofiller c. When cat 5 wt% f shows aat 5 wt% b. This bNT 1 wt% , despite8SWCNT2 at 4 wt% (8SWCNT2 at 4 wt% showing a better surface finish than the epoxy/SWCNT at 1 wt%, and with the epoxy/SWCNT at 4 wt% showing the roughest surface. The calculated mean values of surface roughness were 109.8 ± 17.5 µm and 252.1 ± 35.6 µm for the epoxy/SWCNT at 1 wt% and 4 wt%, respectively, and 97.9 ± 16.8 µm for the epoxy/GNP8SWCNT2 at 4 wt%.In order to obtain a quantitative assessment of the nanocomposite surface morphology, a 3D reconstruction with the evaluation of the surface roughness was performed using the MountainsMap software for the epoxy/SWCNT at 1 wt% and 4 wt% and for the epoxy/GNPat 4 wt% . The rou2SWCNT8, epoxy/GNP5SWCNT5 and epoxy/GNP8SWCNT2 at different filler loadings was analyzed. Results showed that the epoxy/SWCNT system has higher electrical conductivities at all nanotube concentrations with respect to the hybrid systems, except for the epoxy/GNP2SWCNT8 at 2 wt%. However, rheological tests highlighted the large difference in viscosity for the systems analyzed, with the epoxy/hybrid mixtures having lower viscosity values at all loading fractions than the epoxy/SWCNT. In particular, the epoxy/GNP8SWCNT2 5 wt% showed lower viscosity than the epoxy/SWCNT 1 wt%, despite having the same amount of carbon nanotubes in addition to 4 wt% of graphene nanoplatelets. This results in a net difference in the final surface quality of the samples, as revealed by SEM investigations with 3D surface reconstruction. Likewise, the epoxy/GNP5SWCNT5 2 wt% showed similar viscosity to the epoxy/SWCNT 1 wt% despite having the same amount of carbon nanotubes in addition to 1 wt% of graphene nanoplatelets.The dispersion state and the electrical properties of RTM6 epoxy resin loaded with different grades of GNPs and with GNP/CNT hybrid nanofiller were investigated. The electrical conductivity of RTM6/GNP 1 wt% nanocomposites were shown to be of the same order of magnitude within experimental error, with the epoxy/xGNP-H5 showing the lowest electrical properties, whereas the best fillers distribution in RTM6 was achieved using the GNP AO-4 type. Next, GNP AO-4 and SWCNTs were combined in the ratio of 2:8, 5:5 and 8:2 to take advantage of the high aspect ratio of the selected carbon nanotubes and of the dispersion capabilities of GNP AO-4, creating an interconnected hybrid architecture. The electrical and rheological behavior of epoxy/SWCNT, epoxy/GNP8SWCNT2 mixture simultaneously ensure high electrical conductivities and low viscosity values. At 1 wt% and 2 wt% of nanofiller, the epoxy/GNP5SWCNT5 and epoxy/SWCNT nanocomposites exhibited similar electrical properties, with the epoxy/GNP5SWCNT5 hybrids maintaining viscosity values one order of magnitude lower than the SWCNT-loaded epoxy nanocomposites. The better processability along with the good electrical properties of the hybrid mixtures is attributed to the synergistic effect among the selected carbon nanoparticles: the high aspect ratio of SWCNTs is responsible for the increased electrical conductivity and for preventing the restacking of the GNPs, while the GNPs inhibit the aggregation of the SWCNTs. Regarding the electrical properties, it was observed that high loadings of the hybrid GNPThese results lead to the rational selection of the GNP/CNT ratio for the optimal processability of high-performance fiber-reinforced composites exploiting the advantages of carbon-modified matrices. In future work, liquid composite molding may be used for the production of laminates in order to analyze the enhancement of the mechanical and electrical properties of the final component. The interactions among nanoparticles and long fibers during the impregnation phase of the preform will be the subject of future investigation.


Microneedles (MNs) have been widely used in biomedical applications for drug delivery and biomarker detection purposes. Furthermore, MNs can also be used as a stand-alone tool to be combined with microfluidic devices. For that purpose, lab- or organ-on-a-chip are being developed. This systematic review aims to summarize the most recent progress in these emerging systems, to identify their advantages and limitations, and discuss promising potential applications of MNs in microfluidics. Therefore, three databases were used to search papers of interest, and their selection was made following the guidelines for systematic reviews proposed by PRISMA. In the selected studies, the MNs type, fabrication strategy, materials, and function/application were evaluated. The literature reviewed showed that although the use of MNs for lab-on-a-chip has been more explored than for organ-on-a-chip, some recent studies have explored this applicability with great potential for the monitoring of organ models. Overall, it is shown that the presence of MNs in advanced microfluidic devices can simplify drug delivery and microinjection, as well as fluid extraction for biomarker detection by using integrated biosensors, which is a promising tool to precisely monitor, in real-time, different kinds of biomarkers in lab- and organ-on-a-chip platforms. Microfluidic technology is present in lab-on-a-chip and organ-on-a-chip platforms. In order to enable high-throughput screening and automation, lab-on-a-chip (LoC) devices—also known as multitasking devices—combine many (bio)chemical laboratory operations in a single integrated chip that ranges in size from a few millimeters to a few square centimeters . The mosOrgan-on-a-chip (OoC) platforms replicate tissue and miniaturized organs, while preserving tissue/organ-level function and homeostasis . They arMicroneedles (MNs), which are based on the concept of miniaturized needles, are increasingly used in biomedical technology. These have the ability to assess biological information with minimal invasion, and are frequently used as a strategy to deliver drugs , biomoleConcerning the fabrication methods, several have been described in the literature, but the most commonly used are micro-molding, microfabrication technologies , additive manufacturing , and layer-by-layer assembly . BrieflyOverall, the microfabrication techniques to produce MNs and microfluidic devices are identical. Hence, it is expected that microfluidic devices and MNs can be easily combined using those fabrication techniques and in this way to create, in a synergetic way, an advanced microfluidic device for drug screening and/or organ models monitoring ,48. BaseThis work was conducted taking into account the research guidelines for systematic reviews proposed by PRISMA ,50.The search was performed using three different databases: ScienceDirect, PubMed and Scopus, until the 1st of December 2022. The search string used was (“organoids” OR “organ-on-a-chip” OR “organ on a chip”) AND (“microneedle (s)”) AND (”lab-on-a-chip” OR “microfluidics” OR lab on a chip”) AND (“microneedle (s)”).Review articles, conference papers, short communications, and non-English written articles were removed from the search results, either manually or using the filters from the database. After the elimination of duplicates, the articles were selected based on the relevance of their title in the context of this review. Further screening was performed to evaluate which paper presented the defined inclusion or exclusion criteria presented below. To avoid biases, the two first authors screened and selected the research papers separately and then compared the classifications. Disagreements or doubts regarding the classification were solved by a third author.Published since 2000;Use of microfluidic platforms or organs-on-a-chip in combination with MNs;Use of microfluidic platforms or lab-on-a-chip in combination with MNs;MNs for media/ISF collection;MNs for cell injection;MNs for biomarkers detection.MNs for biofluid extraction, microneedle sensors, and analyte-capturing MNs, or combinations thereof.The studies included in this review followed the criteria: The study did not present the excluded criteria.As previously mentioned, the authors followed the PRISMA-recommended guidance to conduct systematic reviews. Based on the title, 91 potentially relevant articles were identified from the three databases selected. In total, 80 studies were included after removing duplicates. After the evaluation of abstracts, 24 articles were dismissed due to a lack of data and different study strategies; thus, 56 full papers were analyzed. In the end, a total of 35 full-text articles were selected. Additionally, a metadata analysis was carried out using the Scopus database with the searched keywords “MNs + microfluidic” and “MNs + organ-on-a-chip” and “MNs + lab-on-a-chip” between 2000 (the year of the first work reported in the literature) and 2022, which shows a total sum in this period of 82 papers (67 articles and 15 reviews) .Among the included studies, 30% corresponded to studies that have addressed the integration of MNs into microfluidic devices. The number of publications tended to increase over the past 20 years, where the majority of the publications were original research articles. This reflects researchers’ increased interest in combining MNs with lab/organ-on-a-chip. Among the most published areas were Engineering, Material Sciences, and Physics and Astronomy, representing a total of 63.4%. Based on the selected articles from the defined criteria, 36 works were included in this systematic revision, which had as main topic MNs applied in advanced microfluidic devices , and subdivided into two main applications: (1) devices for extraction and biomarker detection, and (2) devices for drug delivery and microinjection, as follows in the sub-chapter.An increased research effort has been focused on the use of MNs for direct or indirect sensing. This new trend has germinated naturally from former efforts of the use of MNs for OoC and LoC devices. As is possible to observe in Hollow MNs are regarded as the best choice for extract/release systems, since they provide the exact amount of drug needed at the desired location in a faster and controlled way. Therefore, hollow MNs are the most common type of MN employed in microfluidic devices, mostly made of silicon, but metals and glass are also used . Such deTo identify the presence of a particular target analyte, microneedle-based biofluid extraction products are mainly combined with downstream analytical techniques . For exaUltimately, MNs systems can provide results and detect different biomarkers in real-time, which can be used to monitor in vivo tissues, or in vitro organoids and cell cultures. The main advantage of MNs is that they can be repeatedly used to collect cellular contents without causing cell lysis. They may also promote a decrease in lateral diffusion . Overall, studies show that MNs are mainly used in microfluidic applications for biomarker detection ,66,67,68Another interesting aspect in the design and application of MNs in microfluidic devices is the type of hydrodynamic force mechanism employed, which can be passive, such as capillary force, or active, such as by using micropumps. For instance, the detection of analytes in fluids may be facilitated by the use of capillary action in a microneedle-assisted biosensing . In thisRegarding fabrication strategies and materials, typically porous MNs are fabricated using a PDMS mold followed by a leaching method to remove the porogenic casted materials ,62, or bThe use of microfluidic components combined with MNs for glucose measurement has received the greatest attention ,91. As iAs a possible solution, Najmi et al., 2022, developed and simulated a nonenzymatic glucose detection device by integrating a microfluidic system and a semi-permeable membrane located at the MN base to separate the dialysis fluid from the waste fluid. In this case, an amperometric sensor was used . HoweverMansor et al., 2017, created a device for the detection of cells in suspension. The microfluidic chip consists of two MNs integrated at both sides of the channel to detect impedance measurements of passing cells through the applied electric field. The MNs can be reused, but each PDMS microchip can only be used for one cycle. Due to the low fabrication cost and more than one functionality (solution detection and cell concentration detection), this device was described as suitable for various applications, such as cancer cell detection and water contamination  Figure C.Miller et al., 2014, developed an ion-sensitive microfluidic chip with hollow MNs, one approach that provides an attractive platform for an on-body sensing system for monitoring potassium, which can be easily expanded to other relevant physiological markers for the next generation of point-of-care diagnostic devices. This work was the first ion-selective-electrode MN sensor integrated into a microfluidic device  Figure D. A multAlthough hollow MNs are further explored, some devices incorporate other types of MNs, such as porous MNs. The fundamental benefit of porous MNs is that biodegradable polymers can be used, since their porous structure does not require the micromachining procedures used for hollow structures. Takeuchi et al., 2019, F developMicrofluidic devices integrated with MNs can be used for drug delivery due to the precise control of drugs released by means of microfluidic components, such as micropumps. This strategy enhances continuous-on-chip drug delivery. Zhan et al., 2004, describe a delivery system with in-plane MNs with a microchannel and an outlet. This design allows for the decoupling of the mechanical and fluidic performance of the device A 70]. F. F70]. FThe first flexible microneedle patch incorporating microfluidic components for on-chip loading and delivery control was produced by Xiang et al., 2015 . The easIn another study, Kang et al., 2021, fabricated a flexible array base microfluidic neural interface to add a chemical delivery capability to three-dimensional electrode arrays comprising a collection of MNs (a silicon MN coated with Cr/Au) positioned perpendicular to the array base . In thisThe first microfluidics-based microinjection system was suggested by Adamo et al., 2008, in which single cells were propelled by fluid streams and subsequently injected via MNs with the aid of flexible valve actuation C 74]. I. I74]. IAs described above, the incorporation of MNs is more developed for LoC devices than for OoC devices. Among the OoC examples is the work of Hokkanen et al., 2015, which developed an automatic microfluidic sample system for tissue analysis. In this work, a microscope and robot were used for positioning the MNs and samples for real-time biopsies D. The blIn another OoC study, Choi et al., 2007, described a microfluidic perfusion system that integrates microneedle arrays in a packaged system for fluid containment. The hollow MNs were made with a thin body, a sharp tapered tip, and a microfluidic port along the tower’s side to deliver medium to the inside of the target area E 89]. T. T89]. TIn-plane MN with an open capillary was another strategy developed for LoC. Jung et al., 2015, fabricated in-plane MNs with side openings with a microchannel inside the MN that connected to a reservoir F 77]. T. T77]. TOverall, these papers show the vast potential and versatility of applications for MN in microfluidic devices. However, in general, these devices are made using time-consuming fabrication processes that involve numerous steps and additional sealing layers to enhance the total thickness of the structures. The width of microchannels is frequently constrained by small openings created for successful sealing, making it difficult to reliably seal long microchannels. To overcome this fabrication constraint, Trautmann et al., 2019 described a POCT system combining femtosecond laser-generated microfluidic channels and direct laser-written MN arrays that simplify the fabrication process in only a few processing steps. This is an advantage over multiple processing methods . With thMicrofluidic devices with MNs are a relatively novel and appealing method of fluid transport that offers numerous benefits and applications. Because of the multiple possible applications in the field of biomedicine, the attention to these devices has increased significantly in recent years, as shown by the increasing number of published of lab/organ-on-a-chip systems with MNs. These combined structures are compatible with the current biotech requirements for both operation and assessment, including automated liquid management, plate shuttling, biomarker detection and delivery features. Some of the described LoC devices were tested pre-clinically, which shows the end-use applicability of them. However, systems integration and manufacturing MNs present substantial challenges . This inherent complexity can negatively influence the manufacture of such devices’ dependability and repeatability. One of the challenges in the integration of MNs with microfluidic devices is ensuring that the MNs are precisely aligned with the fluidic channels. This is necessary to guarantee that the fluid flows smoothly through the device and the MNs are able to perform their intended function. Another challenge is ensuring that the MNs’ material corresponds to the practical demands of the application that the device is being developed for, as previously outlined in this work.Overall, the integration of MNs with microfluidic devices is a complex process that requires careful consideration of a number of factors, including design of the MNs, manufacturing techniques, possible functionalization of MN surfaces, and combination or integration with (bio)sensing and actuation systems. As researchers continue to develop new techniques to fabricate MNs-integrated microfluidic devices, the potential application for these devices is expected to expand. As a result, simple fabrication methods and new materials are being explored.Among MNs, hollow MNs are the ones most employed in microfluidic devices, due to their capability to collect and release higher amounts of fluid. Regarding the MN material, different types of materials from metallic to non-metallic were described in the literature reviewed, which are highly dependent on their application. For instance, MNs designed to penetrate the skin or tissue must be fabricated with materials that provide enough strength and biocompatibility.Among the microfluidic type of devices, LoC systems with MNs are being explored for extraction, biomarker detection, microinjection, and drug delivery, which can be combined for two or more of these applications in the same device. On the other hand, MN-fluidic systems for OoC/cell-culture monitoring are in the early stages of development. Nevertheless, its potentiality for cell/tissue monitoring is high, as MN structures can measure protein levels directly inside a living cell, without lyse and at real-time. For that, a thin MN can be inserted into a living cell, or in a cluster of cells (tissue), to assess its microenvironment and homeostasis. This can be used for a variety of purposes, including protein expression monitoring, homeostasis assessment, precise drug delivery, and DNA or RNA therapy. Similarly with LoC, one of the most significant advantages of OoC is the versatility of these devices for multiple applications, which has a wide scope for further development. However, the intricate structure of microfluidic devices, as well as challenges in integrating with other devices and fabrication requirements, all contribute to an untapped potential for MNs in the biomedical field. Nevertheless, work is being conducted to overcome this challenge, as shown by a recent published Patent WO/2022/180595, which presents a multiorgan-on-a-chip containing an MN-(bio)sensing platform for the validation and study of nanomaterials, drugs, or mixtures thereof, intended to be used in biomedical and/or pharmaceutic applications [In addition, several researchers are focusing their efforts on developing simpler and low-cost fabrication methodologies to create powerful MN-microfluidic devices, where the integration of the different components, such as biosensing modules, are more easily achieved. One of the newest approaches is the development of microfluidic devices and biosensors using bioprinting technology, which can fabricate in a few steps. This technology, although promising, is also in its first stages. Therefore, there is plenty of room for the MN-microfluidic technology to progress, from material science and microfluidics to tissue engineering. Furthermore, a wide range of applications can be explored, not only biomedical, but also in the sea and in space.


Cardinalis cardinalis), painted buntings (Passerina ciris), and white‐eyed vireos (Vireo griseus), focusing on the extreme precipitation event of Hurricane Harvey, which caused heavy localized flooding. We found that body condition declined with average daily minimum and maximum temperatures, while precipitation had varied, species‐specific effects in the three species analyzed. Our results also suggest that northern cardinals experienced a notable reduction in average body condition in the 2 years following Hurricane Harvey. Taken together, we conclude that short‐term precipitation and temperature drivers can be important correlates of body condition in songbirds and that severe weather events may reduce body condition in some bird species.Body condition is a frequently used physiological indicator of avian health and can be affected by an array of environmental variables. Although a number of studies have investigated the specific effects of individual weather variables on body condition in birds, few have analyzed the effects of both temperature and precipitation within the context of an extreme weather event such as hurricanes. In this study, we examined the relationship between breeding‐season body condition and daily maximum temperature, daily minimum temperature, and monthly total precipitation for three passerine bird species at the Welder Wildlife Refuge near Rockport, Texas. We also evaluated yearly changes in body condition over a 12‐year period for northern cardinals ( This study demonstrates that the body condition of three songbird species, as quantified by scaled mass index, can be affected by short‐term changes in precipitation and temperature. The continued and expected future effects of climate change render these findings especially relevant and highlight the need for further related research. These species maintain at least a partially granivorous diet , which is largely insectivorous  precipitation would have a positive effect on scaled mass index of northern cardinal  banding station at the Welder Wildlife Refuge, using scaled mass index to identify changes in body condition among three avian species in relation to year, precipitation, and temperature. Scaled mass index is a condition index calculated using both body mass and morphometric measurements  to assess overall body condition  regression line across ln‐transformed data of Mi and Li  with the “mgcv” package Wood,  in the R Figures , 3, 4, 5Using Akaike's Information Criterion (AIC) we performed model selection using the “dredge” function of the “MuMIn” Barton,  package 33.1AICc model selection indicated several competing top models with Δ AICc < 2 in each of the three species analyzed: 10 for northern cardinal, 12 for painted bunting, and 26 for white‐eyed vireo. For each species, the models selected for analysis  ranked higher than the null , such that hatch year, or juvenile, individuals had higher scaled mass index scores than adults (Table p < .001). Neither age, year, nor sex were included in the top model for white‐eyed vireo.The effects of age and sex were present in the selected models for northern cardinal and painted bunting. However, we only found age to be an important predictor of scaled mass index for northern cardinal  and the 10‐year average pre‐2018 . Additionally, we found that average scaled mass index for northern cardinal in 2019, in the second breeding season following Hurricane Harvey, was still significantly lower than that of 2017  and of the 11‐year average  . We did not find any significant differences in the scaled mass index of painted buntings in either 2018 or 2019 compared to the year preceding Hurricane Harvey (2017), or the 11‐year average from 2007 to 2017 (p > .05). For white‐eyed vireos, we found that the average scaled mass index in 2019 was notably lower than that in 2017 . However, results from 2018 showed no notable differences (p > .05).The effect of year was only present in the top model predicting scaled mass index for northern cardinal. Despite this, there was no significant effect of year on scaled mass index Table . However4Previous literature suggests a complex and dynamic response of avian body condition to weather‐related variables , and then reversed. This threshold effect may be due to destruction of cover and food sources because of flooding, and the subsequent displacement of birds  were significantly lower than in previous years Figure . While w and 20195Taken together, our results demonstrate that variation in avian body condition is associated with local temperature and precipitation patterns and that severe weather events may have multiyear impacts on body condition for some species. Body condition is associated with individual fitness for many vertebrate taxa, including birds  may provide further insights as to how local and regional bird populations continue to respond in the years following extreme weather events. Finally, localized studies should also evaluate the effects of weather and reduced body condition on survival and reproductive success.Michael W. D. McCloy: Conceptualization ; data curation (lead); formal analysis (lead); funding acquisition ; investigation ; methodology ; project administration (lead); resources ; software ; supervision ; validation ; visualization (lead); writing – original draft (lead); writing – review and editing (lead). Selma Glasscock: Conceptualization ; data curation (supporting); funding acquisition ; investigation (supporting); methodology (supporting); project administration (supporting); resources ; supervision ; validation (supporting); writing – review and editing (supporting). Jacquelyn K. Grace: Conceptualization ; data curation (supporting); formal analysis (supporting); funding acquisition (supporting); investigation (supporting); methodology ; project administration ; resources (supporting); software (supporting); supervision ; validation (supporting); visualization (supporting); writing – original draft (supporting); writing – review and editing .Funding for this study was made possible through MM's graduate research fellowship with the Rob & Bessie Welder Wildlife Foundation. This manuscript is Welder Contribution Number 737.The authors declare no conflict of interest.Table S1Click here for additional data file.