Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper β-cell function.β-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support β-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate β-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in β-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse β-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of β-cell genes, including  In addition to providing biomechanical support to the islets, ECM components are required for β-cell development, proliferation, survival, and proper insulin secretion. The ECM regulates β-cell gene expression directly through binding specific receptors on these cells, and indirectly, through the storage of signaling molecules, such as growth factors5. Loss of the ECM during the islet isolation process is considered to be a significant factor in the poor engraftment rate of transplanted β-cells, thus limiting cell replacement therapy to diabetes6. Hence, recapitulating the islet ECM in both isolated islets and stem-cell-derived β-cells is of high therapeutic value. However, while many studies demonstrated the importance of the ECM, its interactions with β-cells are still not fully understood.β-Cells depend on the islet niche for their mass and functionality. A central constituent of the islet niche is the extracellular matrix (ECM)7. IMs are found in the space between the tissue stroma cells and contain, among other components, fibrillar collagens and fibronectin. BMs are specialized structures that underlie epithelia and surround organs and mainly comprise collagen IV, laminins, and heparan sulfate proteoglycans (HSPG). Islets have two distinct BMs: the peri-islet membrane and the vascular BM, which differ in both their anatomic location and molecular composition4. The peri-islet membrane, which encapsulates the islets, separates the endocrine cells from the exocrine pancreas and serves as a barrier from immune cell infiltration to the islets. The vascular BM surrounds the islet capillary network and directly contacts mouse endocrine cells. Human islets are characterized by a double-layered BM, consists of a vascular BM layer covered by an invagination of the peri-islet membrane9. Thus, human endocrine cells are not in direct contact with the vascular BM components but with the invaginated peri-islet membrane.Two major ECM classes exist, the interstitial matrix (IM) and the basement membrane (BM)5. For example, β1 integrin, a significant component of the integrin dimer expressed by β-cells, is required for these cells' development and expansion, as well as for insulin production and secretion13. While integrins bind multiple ECM components, including collagens and fibronectin, their interactions with laminins are potentially more significant4. The interactions with the various BM components differentially affect β-cells, although the underlying mechanism is largely unknown.β-Cells directly respond to BM components through both integrin and non-integrin receptors -mediated signaling2. The islet BM composition displays temporal and spatial patterns. For example, HSPGs can be found in the adult islets, but not the embryonic or newborn pancreata14. On the other hand, Laminin (LM)-111 is the primary isoform expressed in the embryonic pancreas but is absent from the adult tissue15. Laminins are heterotrimers composed of α, β, and γ chains, when the α chain largely dictates receptor specificity16. Three different types of α , two of β (β1 and β2), and one of γ (γ1) laminin chains were detected in the adult islets, potentially forming six different isoforms8. The various laminin isoforms in the islets are found at different locations. In humans, the islet vascular BM comprises α4 and α5 laminin chains, while the peri-islet membrane contains α2 and α5 laminin chains8. In the mouse, α2 laminins can also be found in the vascular BM18. Similarly, the β1 and γ1 laminin chains comprise both the peri-islet membrane and vascular BM, whereas the β2 chain is specific to the latter18. The additional key component of the islet ECM, collagen IV, constitutes both the peri-islet and vascular BM5. Which cells produce the various islet BM components is still unclear.The specific expression and distribution of the different BM components in the islets were yet to be fully characterized. It has been shown, however, that β-cells do not produce BM components2. However, an additional source of the islet BM has been suggested9. Pericytes are found within and around islets, where they are associated with ECs. Pancreatic pericytes were shown by others and us to control insulin secretion, both indirectly through the modulation of the islet blood flow and directly, through the production of signaling molecules that regulate β-cell gene expression and function22. The identity of the pericyte-produced β-cell regulatory factors is only now begun to unveil. While pericytes are considered to contribute to the vascular BM throughout the body, their level of contribution is unclear16. Furthermore, whether pancreatic pericytes are capable of producing components of the vascular and peri-islet BM is unknown.Endothelial cells (ECs) were long known to contribute to the islet BM. Pancreatic ECs produce some components of the islet BM, including collagen IV and the α4 and α5 laminin chains, to regulate insulin productionHere, we set to determine the contribution of pericytes to the islet ECM. First, we utilized RNAseq analysis to profile their expression of ECM components. Our analysis pointed to the production of multiple constituents of both pancreatic IMs and BMs by pericytes. We then focused on the main components of the islet BM, collagen IV and laminins. While both ECs and pericytes produce collagen IV, we show that pericytes are the primary source of two of the three pancreatic laminin α chains: α2 and α4. To determine the effect of these laminins, we cultured mouse islet cells in their presence. Pericytic laminins, in particular α2 laminins, promoted islet cell clustering. The α4 laminin LM-421, but not other tested laminin isoforms, induced the expression of β-cell genes and stimulated glucose-stimulated insulin secretion. Thus, our analysis indicated a significant contribution of pericytes to the pancreatic and islet ECM and pointed to the differential role of pericytic laminins in β-cell organization and function.7. To assess pericytes' contribution to the pancreatic IM, we profiled their gene expression employing an RNAseq analysis (previously described in Ref.21). In this array, we analyzed pancreatic mural cells, labeled by Nkx3.2/YFP, which primarily constitute pericytes (~ 85%) in addition to the closely related vascular Smooth Muscle cells (vSMCs)23. Islets, which contain primarily pancreatic endocrine cells, served as controls. Indeed, pericytic genes, including Cd248, Acta2, and Eng 24, were enriched in the pericyte population  was low in the analyzed pericytic population  and did so in significantly higher levels than endocrine cells do  at higher levels than endocrine cells do  were also expressed by pancreatic pericytes  and collagen XV (encoded by Col15a1) by pancreatic pericytes at significantly higher levels than in islets . Pericytes further express the two islet β chains, β1 and β2 , and the single detected γ chain, γ1 (encoded by Lamc1) of Col4a2 than pericytes did. Pancreatic pericytes expressed Lama4 at significantly higher levels than pancreatic ECs did , α4 laminins (a mixture of recombinant LM-411 and LM-421), or control PDL. To determine potential changes in β-cell proliferation, cells were stained for insulin and the proliferating cell marker Ki67, and the percentage of proliferating β-cells was determined by flow cytometry. As shown in Fig. Whereas islet cells cultured on PDL remained dispersed, these cultured on laminins clustered Fig. D. CulturTo conclude, laminins naturally produced by pancreatic pericytes do not support β-cell expansion and survival but promote the clustering of islet cells, thus potentially fostering cell–cell interactions.33. RNA was extracted from dispersed islet cells cultured on PDL, α2 (LM-211 and LM-221), and α4 Laminins (LM-411 and LM-421) for 72 h, and Ins1 and MafA transcript levels were analyzed by qPCR  and high (16.7 mM) glucose concentrations, and the level of secreted insulin was measured and compared. As shown in Fig. To conclude, our analysis indicates that β-cells respond differently to the various laminin isoforms naturally produced by pancreatic pericytes, and each of these BM components likely plays a different role in supporting islet structure and function. We further showed that LM-421, predominantly produced by pancreatic pericytes, induces β-cell gene expression and function in cultured islet cells.ECM components, and more specifically BM components, are long known for their ability to support β-cell physiology. However, the source of these components was yet to be fully characterized. Here, we show that alongside ECs, pericytes are a significant source of the pancreatic IM and BM, including major constituents of the peri-islet membrane and the islet vascular BM. Specifically, we show that pericytes are the primary source of two of the three pancreatic laminin α chains, α2 and α4, and express these chains at significantly higher levels than pancreatic endothelial, endocrine, and exocrine cells do. Laminins produced by pericytes differentially stimulated the clustering of dispersed mouse islet cells. We found that the pericytic-produced laminin LM-421 induces the ex vivo expression of β-cell genes, including insulin, and improved the function of dispersed, cultured β-cells. Thus, our study points to the production of BM components as a potential mechanism through which pericytes regulate β-cells.24. Our results point to a similar interaction between these two cell populations in the pancreas. Interestingly, while some BM components, as collagen IV and the α4 laminin chain, are produced by both cell types, albeit at different levels, other components are produced by a single cell population. ECs, but not pericytes, produce the laminin α5 chain2 35, here we did not observe this effect when culturing on recombinant human α2 laminins Wez) mice were a generous gift from Warren Zimmer (Texas A&M). R26-YFP (Gt(ROSA)26Sortm1 (EYFP)Cos) mice were obtained from Jackson Laboratories. Wild-type mice were purchased from Envigo Ltd. .All experimental protocols were approved by the Tel Aviv University Institutional Animal Care and Use Committee (IACUC). For islet isolation, Collagenase P  dissolved in RPMI (Gibco) was injected through the common bile duct into the pancreas of a euthanized mouse. Dissected pancreatic tissue was incubated for 11–15 min at 37 °C, followed by a gradient separation with Histopaque 1119 (Sigma) for 20 min at 1300 g. Islets were collected from the gradient interface, followed by their manual collection.26. Briefly, dissected pancreatic tissues were digested with 0.4 mg/ml collagenase P (Roche) and 0.1 ng/ml DNase (Sigma) diluted in HBSS for 30 min at 37ºC, followed by cells filtration. For cell sorting, pericytes were collected using FACS Aria (BD) based on their YFP expression in Nkx3.2-Cre;YFP mice. After immunostaining with PE-conjugated anti-PECAM1 antibody, ECs were similarly collected based on their fluorescent labeling. For cell counting and proliferation measurement, dispersed islet cells were fixed in 4% PFA for 15 min, immunostained with AlexaFlour 647-conjugated anti-insulin and FITC- conjugated anti-Ki67 antibodies, and analyzed using the volumetric counting feature of CytoFLEX flow cytometer (Beckman Coulter). For quantification of dead cells, unfixed cells were incubated with DAPI (Sigma) and analyzed using the volumetric counting feature of CytoFLEX flow cytometer (Beckman Coulter), when cells that uptake this dye were considered dead. Antibodies are listed in Supplementary Table Pancreatic cell isolation was performed as described21 that was deposited in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5325/). To characterize gene expression of pancreatic pericytes, cells were purified by flow cytometry from 10-week-old mice, as described above, and islets were isolated from age-matched non-transgenic mice. RNA was extracted using the PureLink RNA micro kit (Invitrogen) and subjected to RNA sequencing to obtain 1.5–2 × 107 reads from each sample. RNA amplification, cDNA library preparation, deep RNA sequencing, and bioinformatics analysis were performed using commercial services . Raw reads (Fastq files) for each sample were aligned to the Bacteroides Fragilis using STAR v2.4.0 with default parameters, using the DNAnexus platform. After alignment, estimation of transcript abundance measures as FPKM values was performed using Cufflinks in the Tuxedo protocol. Gene expression FPKM values were then used for differential expression analysis using Cuffdif, a part of the Cufflinks package, to compare the different groups.We utilized a previously described RNA sequencingDissected pancreatic tissues were fixed in 4% paraformaldehyde for 4 h. Tissue was transferred to 30% sucrose solution overnight at 4ºC, followed by embedding in Optimal Cutting Temperature compound  and storage at -80ºC. Tissues were sectioned by a cryostat (Leica) into 11 μm thick section and stained with primary antibodies (Supplementary Table RNA was extracted from isolated tissue and cells using PureLink RNA micro kit (Invitrogen) according to the manufacturer's protocol, followed by cDNA generation using a high capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression levels were determined with Taqman and SYBR green assays (Applied Biosystems), using specific assays as listed in Supplementary Table Islets were isolated as described above. 50–70 Islets were dispersed to single-cell by incubation with 0.05% Trypsin and 0.02% EDTA solution  at 37 °C for 5 min with agitation. Cells were culture for either 48 or 72 h in CMRL medium (Gibco) containing 10% FCS (Hyclone) and 1% Penicillin–Streptomycin solution  on human recombinant laminins (BioLamina) or Poly-D-Lysine  -coated 96-well plates . Clusters size was measured by ImageJ software (NIH). Clusters smaller than a cell size were excluded from the analysis.Cells were collected, rinsed twice with RPMI, and pre-incubated for 30 min in RPMI 1640 (Gibco) supplemented with 0.1% BSA and 25 mM HEPES containing 1.67 mM glucose. Islets were then incubated in 1.67 mM glucose for 1 h, followed by additional incubation with 16.7 mM glucose for 1 h. After each incubation period, supernatant samples were collected, and secreted insulin levels were determined using mouse Ultrasensitive Insulin ELISA . The ratio between insulin secreted in response to 1.67 and 16.7 mM glucose was determined per each well.t-test.Paired data were evaluated using a two-tailed Student Supplementary Information.


Segmental fusion is not necessarily needed in treatment of thoracolumbar unstable burst fracture requiring surgery. Our objective was to report the results of follow-up for at least 10 years in patients with thoracolumbar unstable burst fracture requiring surgery in which fractured segment was healed following temporary posterior instrumentation without fusion, and in whom implants were subsequently removed.Retrospective Cohort Study.Nineteen patients in whom union of fractured vertebra was observed following surgery and in whom implants were removed within an average 12.2 months, and who could be followed up for at least 10 years, were enrolled.At the last follow-up, we evaluated the segmental motions, anterior body height ratio, progress of further kyphotic deformity, Oswestry Disability Index, Rolland Morris Disability Questionnaire and Short Form 36.Results: The follow-up period after implant removal surgery was 151 months on average. The local kyphotic angle was 26.89 ± 6.08 degrees at the time of injury and 10.11 ± 2.22 degrees at the last follow-up. The anterior body height ratio was 0.54 ± 0.16 at the time of injury and 0.89 ± 0.05 at the last follow-up. Thus, the fractured vertebra was significantly reduced after surgery and maintained till last follow-up. The segmental motion was 9.84 ± 3.03, Oswestry Disability Index was 7.95 ± 7.38, Rolland Morris Disability Questionnaire was 2.17 ± 2.67, short form 36 Physical Component Score was 77.50 ± 16.61, and short form 36 Mental Component Score was 79.21 ± 13.32 at last follow-up.We conducted at least 10-year follow-up and found that temporary posterior instrumentation without fusion should be considered one of the useful alternative treatments for thoracolumbar unstable burst fracture in place of the traditional posterior instrumentation and fusion. There may be various surgical options on the proper treatment modality, from simple non-surgical treatment to complicated posterior-anterior-posterior approach considering the postoperative stability of the fracture fragment. However, there is consensus that acute, unstable, severe kyphotic deformity or those with damage to the middle osteoligamentous complex and posterior ligament complex require surgical treatment in most cases. However, it has also weak points, including early fixation failure by insufficient anterior support with only posterior fixation.–20 McCormack et al have tried to address this weakness and contended that anterior support is required for patients with a load-sharing score of seven points or more, since posterior fixation alone was not enough to ensure stability. Besides early fixation failure, reduction loss in the intervertebral disc following implant removal has also been reported recently. Although there are several strong and weak points, some spine surgeons have suggested that temporary posterior instrumentation without segmental fusion may replace conventional posterior instrumentation with segmental fusion.–25 However, they evaluated only the stability of the fracture site by short segment fixation and the functional outcome for not-long period. In addition, there were little researches on whether segmental motion is lost in long-term follow-up after removal of implant, and whether additional kyphotic deformity does not progress over time.In the case of thoracolumbar unstable burst fracture requiring surgery, temporary posterior instrumentation without fusion has a number of strong points. That is, it can align fractured spines and retain vertebral height and canal dimension by repositioning posteriorly displaced bone fragments through indirect reduction by ligamentotaxis, as well as is less invasive. And, this technique prevent many problems caused by fusion of mobile segment by preserving motion segments even after union of fractured vertebra and subsequent implant removal.The purpose of this study was to evaluate the outcome of at least 10 years follow-up after union of the fractured spine by posterior instrumentation without fusion in thoracolumbar unstable burst fractures requiring surgery, to confirm whether mobile segment was preserved, and whether functional outcomes or quality of life deteriorated. This is the first report of a long-term follow up of more than 10 years using this non-fusion technique.22.1Patients with thoracolumbar unstable burst fracture requiring surgery owing to definite posterior ligament injuries detected with magnetic resonance imaging (MRI) or computed tomography (CT) before surgery, and who could be followed up for at least 10 years after healing of fractured segments and removal of implants were enrolled. This study excluded patients who had severe neurological deficit requiring anterior decompression, who were child or aged 50 years or older at surgery, who were unable to tolerate surgical treatment because of other severe medical illness, who had burst fracture of two segments or more, who could not receive primary instrumentation within 1 week after injury, and those who had secondary gains such as worker's compensation. We also excluded patients who had a McCormack load-sharing score of seven points or more requiring additional anterior support. McCormack load sharing classification score is composed of comminution = 1, More(30%–60%) = 2, Gross(>60%) = 3), apposition of fragments  = 2, Wide(>50%) = 3) and reducibility of sagittal deformation = 1, More (4°∼9°) = 2, Most (≥10°) = 3). Severely comminuted fractures scoring 7 points or more supposed to be repaired by an anterior approach with partial corpectomy and strut grafting and excluded in this study.From March 1, 2004 to January 31, 2007, total 27 patients who met the inclusion and exclusion criteria were eligible. In one case, at the 2-year follow-up, nonunion of the fractured vertebral body was found by an intervertebral disc inserted in the fracture vertebral body, which additional posterior segmental fusion surgery was required. In one case, implant removal surgery was rejected due to spontaneously anterior segmental fusion. Four cases had follow-up loss (2 cases moved to another city and 2 cases were telephone and address unknown). One patient died of another problem such as pancreatic cancer. One patient refused to complete the questionnaire and check the X-ray due to death of spouse. Thus, 19 patients out of a total 27 patients were enrolled.2.2Posterior instrumentation without fusion was performed within an average of 2.8 days (range: 0–6 days) after injury. The midline skin is incised and bilateral fascia is incised separately. We used a paramedian approach accessing the posterolateral aspect of the spine through a passage between the multifidus and longissimus muscles using finger dissection for minimum invasion to nearby soft tissues. We basically instrumented the upper 2 levels and lower one level in the fracture site using pedicle screws Fig. A. If the2.3 The facet joints were decided to become worse if four joints adjacent of fractured vertebra progressed more than twice as compared preoperative CT finding. After removal surgery, plain radiography consisting of standing antero-posterior images, standing lateral image, and standing flexion-extension dynamic image was annually carried out in order to measure anterior body height ratio (ABHR)  Fig. A and locHR) Fig. B. At leaHR) Fig. C.In order to evaluate functional outcomes related to the spine in all patients, the Oswestry Disability Index (ODI) and Rolland Morris Disability Questionnaire were used. To evaluate quality of life in all patients, short form 36 (SF-36) Physical Component Score (PCS) and SF-36 Mental Component Score (MCS) were measured. The functional outcomes and quality of life were assessed 3 times before removal, 1 year, and at least 10 years after removal.2.4P value of less than .05 was considered statistically significant.All statistical analyses were performed using a paired t-test in SPSS 18.0 . The result of functional outcome and quality of life were analyzed using a repeat measure one factor analysis. Post-hoc test was analyzed by multiple comparison result by contrast. A 2.5This study was approved by Daegu Catholic Medical Center IRB. Approval number is CR-14–055. Waiver of Documentation of Consent was confirmed by IRB, because, this study is retrospective study based on medical record and data obtained was protected in secured storage. This study also has no possibilities to benefit or harm patients who involved in.33.1Patients enrolled for this study consisted of 8 males and 11 females. The mean age was 34.8 years (range: 18–49 years). The mean duration to implant removal surgery following primary surgery was 12.2 months (range: 8–15 months). The mean follow-up duration in implant removal surgery was 151 months (range: 120–168). Fracture sites included T11 in 1 case (5.26%), T12 in 4 cases (21.05%), L1 in 8 cases (42.11%), L2 in 5 cases (26.32%), and L3 in 1 case (5.26%). Causes of injury were traffic accident in 4 cases (21.05%), fall from height in 12 cases (63.16%), and direct injury in 3 cases (15.79%). AO classification by preoperative imaging study was 6 cases in B2, 8 cases in A3, and 5 cases in A4. According McCormack load sharing classification score system, score 3 was 2 case, score 4 was 3 cases, score 5 were 6 cases, and score 6 was 8 cases. Demographic characteristics of all patients were listed in Table 3.2P < .05). However, there was no significant different in LKA value between 1 year after the removal surgery and the last follow-up (P = .71). Average ABHR was 0.54 ± 0.16 at the time of injury, 0.89 ± 0.04 at 1 year after the removal surgery, and 0.89 ± 0.05 at the last follow-up. These results confirmed that fracture vertebra was significantly repositioned compared to that at the time of injury (P < .05). However, there was no significant difference in ABHR value between 1 year after the removal surgery and the last follow-up (P = .87). The segmental motion was measured to be 10.43 ± 3.32 (range: 1–16) degrees at the 1 year after removal surgery, 9.27 ± 3.34 (range: 3–16) degrees at the last follow up after removal surgery, on average, respectively, showing that it was statistically significantly decreased with the passage of time (P = .028)  degrees at the time of injury, 10.37 ± 1.98 (range: 7–14) degrees at one year after implant removal surgery, and 10.11 ± 2.22 (range: 6–14) degrees at the last follow-up, showing significantly reduction state compared to that at the time of injury (3.3P < .001). Rolland Morris Disability Questionnaire was 7.23 ± 4.47 at the time of removal surgery, 4.83 ± 4.18 one year after implant removal surgery, 2.17 ± 2.67 at the last follow up, respectively, showing statistically significant improvement over time (P < .001). In addition, for the evaluation of quality of life the SF-36 PCS was 40.71 ± 18.67 at the time of removal surgery, 56.58 ± 21.56 one year after implant removal surgery, 76.73 ± 17.24 at the last follow up, respectively, showing statistically significant improvement over time (P < .001). SF-36 MCS was 50.24 ± 21.32 at the time of removal surgery, 62.08 ± 20.31 one year after implant removal surgery, 78.58 ± 13.27 at the last follow up, respectively, showing that SF-36 MCS was statistically significantly improved over time (P < .001) . It occurs more frequently in some conditions, including old age, high body mass index, degenerative alteration in the facet joint before surgery, and a longer period from fixation to implant removal surgery, with an overall frequency of 3.6%. Similarly, in the present study, spontaneous fusion occurred in one case of B type fractures (according to AO classification). Pedicle screws were inserted via a convergent path from the lateral to the medial aspects of facet joints to minimize the risk of facet joint violation through the paramedian approach. These joints were immobilized in just 12 months after fixation. For flexion-extension movement, there were no problems caused by the level of preservation for degenerative osteoarthritis of facet joints, since subjects were relatively young (mean age of 35 years) at the time of fixation surgery. At the last follow-up, there were pain-free joint motions with an angle of approximately 9.84 ± 3.03 degrees. Facet joint deterioration was observed by Tromme et al during average follow-up period of 12.3 months. In our study, although the exact degeneration may be not easy to assess due to lack of further CT scans, there were no decrease in joint motion range and patients did not complain of pain during motion. On this basis, degenerative changes are thought to progress no further. However, additional studies are thought to be needed about this subject.There may be a problem with the integrity of facet joints owing to long-term immobilization. Tromme et al In addition, for the evaluation of quality of life in general patients, SF-36 PCS and MCS were 77.50 ± 16.61 and 79.21 ± 13.32, respectively. Both indices confirmed that they had excellent quality of life corresponding to nearly the top 80% on a normal distribution. However, SF-36 is not enough to evaluate quality of life because it represents relative values, not absolute values. Notably, we had trouble making a comparative study with other studies due to the absence reporting functional outcomes measured at least 10 years after implant removal or functional outcomes of the fusion group due to other factors that should be considered such as surgical method, long-term follow-up results, instability of adjacent segments related to fusion, and disc degeneration of adjacent segments.Of spine-related functional outcomes measured at least 10 years after the removal surgery, ODI was 7.95 ± 7.38. This represents minimal disability (scores from 0% to 20%) based on a classification proposed by Fairbank et al.This study has some limitations. First, results of temporary posterior instrumentation without fusion should be compared with those of posterior instrumentation with fusion in studies for patients with thoracolumbar unstable burst fracture. However, there was no control group for this comparison. In the future, more specific results should be collected in a randomized comparison study for the 2 surgeries. Second, at the last follow-up, we measured spinal alignment based on plain radiographs while, joint motion and progress of additional kyphotic deformity were based on flexion-extension images. Additional MRI or CT scans were not taken to examine more clear results for facet joint integrity or disc degeneration. Third, more patients are required for more accurate analysis. In fact, based on inclusion/exclusion criteria, patients with the following conditions were considered ineligible for this study. Fourth, in nonfusion surgery, screw loosening may be frequent. In this study evaluation of screw loosening was not performed but definite screw loosening or rod breakage was not noticed during removal surgeries. Fifth, work ability or occupation change due to disability was not checked during follow-up.5In patients with thoracolumbar unstable burst fracture who had a McCormack load-sharing score of less than 7 points, injured posterior ligaments, and operation indication, we found that the fracture site was healed after temporary posterior instrumentation without fusion and implants were subsequently removed. As a result of at least 10 years of follow-up, there were no additional crucial local kyphotic alterations or reduction loss of the fracture site. In addition, the joint motion range was maintained at an angle of nearly 10 degrees even after implant removal without adjacent segment complications. Furthermore, patients’ functional outcome and quality of life improved over time and showed minimal disability at last follow-up, with all patients being in the 80th percentile. Therefore, among the alternative treatments of posterior instrumentation with segmental fusion, temporary posterior instrumentation without segmental fusion should be considered as an excellent treatment option for patients at least 10 years after removal surgery.Conceptualization: Sangbong Ko.Data curation: Sangbong Ko, Sukhan Jung, Jun-Young Kim.Formal analysis: Sukkyoon Song.Investigation: Sangbong Ko, Sukhan Jung.Methodology: Sangbong Ko, Sukhan Jung, Sukkyoon Song.Supervision: Sangbong Ko, Sukhan Jung, Jaibum Kwon.Validation: Sangbong Ko, Sukhan Jung, Sukkyoon Song, Jaibum Kwon, Jun-Young Kim.Writing – original draft: Sangbong Ko.Writing – review & editing: Sukhan Jung, Sukkyoon Song, Jaibum Kwon, Jun-Young Kim.Sukhan Jung orcid: 0000-0001-8966-1486.


This suggested CACNA1D variants as a potential cause for this condition.Congenital hyperinsulinemic hypoglycemia is the most frequent cause of persistent and recurrent hypoglycemia in the first years of life and in many patients rare genetic variants can be identified. Recently a case of congenital hyperinsulinemic hypoglycemia and a severe neurodevelopmental syndrome due to a mutation in the voltage-gated Cav1.3 Cade novo CACNA1D missense mutation p.L271 H, replacing a highly conserved residue in a functionally relevant region of the voltage-gated Cav1.3 Ca2+ channel. The patient was treated with diazoxide and nifedipine with adequate control of glucose metabolism and blood pressure, and with improvement in muscle tone.Here we support this observation by presenting the case of a female child with congential hyperinsulinemic hypoglycemia and primary hyperaldosteronism, aortic insufficiency, pronounced developmental delay, muscle hypotonia, and facial dysmorphias but without seizures. Sequencing of the exome of the child and its parents identified a novel CACNA1D for congentital hyperinsulinemic hypoglycemia and primary aldosteronism. Moreover, we provide evidence that the dihydropyridine Ca2+ channel blocker nifedipine, although not considered a first-line treatment for congenital hyperinsulinism, may be beneficial to control blood pressure and neurological symptoms in patients with CACNA1D mutations.Our findings further confirm the pathogenic role of  CACNA1D gene has been identified in a subject with diazoxide-dependent congential HH. CACNA1D encodes the pore-forming α1 subunit of the voltage-dependent L-type Cav1.3 Ca2+ channel [Congenital hyperinsulinemic hypoglycemia (HH) is the most frequent cause of persistent and recurrent hypoglycemia in the first years of life. It represents a heterogeneous group of diseases in which there is a dysregulation between plasma glucose concentration and insulin secretion –4. Recen channel .2+ channels [2+ channel blockers, including dihydropyridines (DHPs) such as nifedipine and isradipine. Cav1.3 channels are widely expressed in the mammalian organism, including neurons, endocrine cells , sinoatrial node and cochlear inner hair cells [de novo variants in humans causing abnormal channel gating permitting enhanced channel activity have been identified as high risk pathogenic mutations leading to autism spectrum disorder with and without intellectual disability. In many patients these variants cause an even more severe neurodevelopmental syndrome mainly reflecting abnormal functioning in neurons and endocrine cells [de novo G403D variant was found in a female with normal aldosterone levels but with congential HH requiring continuous diazoxide treatment for the first five years of life [CACNA1D to the list of potential congential HH genes but reports of additional cases is needed to substantiate this finding.Cav1.3 is one of four members of the so-called L-type Cachannels ,6 which ir cells ,6. Homozir cells ,7,8. In ne cells –11. Sympne cells –13. Some of life . Other c of life . These oCACNA1D variant in a second case of a patient with congential HH requiring continuous treatment with diazoxide. In addition this subject exhibited clinical overt primary aldosteronism and therefore represents the first case with both endocrine disorders. Moreover, we also found that the L-type Ca2+ channel blocker nifedipine not only controlled high blood pressure but also improved muscle hypotonia. This suggests that nifedipine may be a valuable tool to improve also neurological symptoms in selected patients affected by CACNA1D gain-of-function mutations.Here we report a novel de novo To identify a genetic cause for the patient’s condition we performed sequencing using a Custom Focused Exome gene panel (Agilent Technologies) and Illumina technology (NextSeq 500). The coding region and 10 base pairs of flanking intronic sequences of the following gene panel related to hyperinsulinism and syndromic forms of hyperinsulinism was sequenced: ABCC8, GCK, GLUD1, HADH, HK1, HNF1A, HNF4A, INSR, KCNJ11, PGM1, SLC16A1, UCP2, APC2, APPL1, BLK, CDKN1 C, CEL, DIS3L2, H19, HNF1B, HYMAI, IGF1 R, INS, KCNQ1OT1, KDM6A, KLF11, KMT2D, LMNA, MAFA, MPI, NEUROD1, NSD1, PAX4, PDX1, PLAGL1, PMM2, RAP1A, RAP1B and SETD2 genes. In addition we analyzed nuclear genes with mitochondrial involvement, syndromic and non-syndromic neurodevelopmental disorders, that could explain the pathology of the patient  as well as genes causing Nemaline Myopathy .Heterozygous variants were filtered by removing non-coding and synonymous variants, and variants in control populations, including the IIS-La Fe Genomic Unit’s own database general population (<0.01) and other databases including the Human Gene Mutation Database, Clin Var Database and the Leiden Open Variation Database.Copy Number Variation Analysis was performed using the Affymetrix Array-CGH (Array-based Comparative Genomic Hybridization). The results were analyzed using Affymetrix’s Chromosome Analysis Suite software, reviewed by at least two specialists.The genetic study was conducted in accordance with the Declaration of Helsinki with informed parental consent given on behalf of the child.2 = 0.21, which was maintained for 2 minutes until spontaneous crying started. She was maintained with PEEP ventilation and transferred to the neonatal ward. Artificial ventilation  was continued until spontaneous breathing 6 h after birth. She had an edematous appearance, short neck, low-set ears, a wide nasal bridge and left parietal subgaleal hematoma , intravenous glucose perfusion was increased up to 12 mg/kg/min. Ketonemia was absent and a glucagon test was compatible with hyperinsulinism: at a blood glucose of 27 mg/dl , plasma insulin was 10 microU/ml (3–25), peptide C 2.71 ng/ml (1.10–4.40), cortisol 9.39 µg/dl (6.20–19.40), and growth hormone 13 ng/ml (0.06–5). At 20 hours of life treatment with diazoxide was started . Upon diazoxide therapy blood glucose normalized but she developed intestinal symptoms including vomiting, abdominal distension and absence of stool evacuation without the help of physiological serum enemas. This clinical picture of an intestinal pseudo-obstruction was likely a consequence of a diazoxide-mediated decrease in bowel motility because spontaneous fecal deposition only happened when the dose of diazoxide was decreased. Symptoms persisted until 4 months of life. Abdominal ultrasound, opaque enema and rectal biopsy were normal.From birth she also exhibited very pronounced axial hypotonia without improvement, trembling and a tendency to lingual protrusion and to maintain closed fists. Therefore an array of tests was performed all with negative results including analysis of blood amino acids, very long chain fatty acids, acylcarnitines, lactic acid, alanine and organic acids in urine). Cerebrospinal fluid study showed a normal profile of neurotransmitters, pterins and folate. In brain MRI punctate hemorrhagic foci were seen in the cerebellum, periatrial areas, hippocampus and semioval centers. At 6 months of age spontaneous stool deposition started without the help of enemas and blood glucose was normal with diazoxide at 5 mg/kg/day. The patient smiled, laughed and babbled, maintained good eye contact with her parents, but still showed pronounced axial hypotonia.Genetic analysis: The genetic analysis of genes involved in hyperinsulinism, nuclear genes with mitochondrial involvement, syndromic and non-syndromic neurodevelopmental genes, and genes causing Nemaline Myopathy  revealed normal results. No genetic variants were observed that could account for the patient’s pathology.de novo CACNA1D missense mutation [CACNA1D gene, which replaces the amino acid leucine by a histidine in position 271 , is absent in the gnomAD database and not present in the parents strongly reinforces the pathological character of this variant.Since severe hypotonia combined with congenital hyperinsulinism had been described in a single subject with a mutation , we perfCACNA1D de novo variants [+ was 142 mEq/l , K+ 4.7 mEq/l (3.5–5.5),), aldosterone 88.5 ng/dl , renin 16.3 mU/l (sitting 4.2/standing 45.6 mU/l), aldosterone/renin ratio = 5.4 .Since primary aldosteronism was also described in three previous patients carrying pathogenic variants ,14 we alAt 6 months of life treatment with nifedipine 1 mg/kg/day orally at three divided doses every 8 h  was added to diazoxide with adequate response on blood pressure. Blood pressure decreased from 100/65 mmHg at 1–3 months to 69-85/42-57 mmHg after the start of nifedipine treatment (months 6–18). Interestingly, this treatment was also associated with a clinically relevant improvement of muscle tone.At 18 months of life she follow therapy with nifedipine and diazoxide, she is able to bring her hands to the middle line and support her head. She is still unable to sit or crawl. She has oral dyskinesias and continuous movements of the mouth and lingual protrusion. She keeps closed fists and has dystonic hand movements.CACNA1D variant who required long-term treatment with diazoxide to control the condition, with adequate clinical response during the first 18 months of life.This case report adds important clinical information for the diagnosis of individuals affected by congenital HH. First, we report the second patient affected by congenital persistent HH with a pathogenic CACNA1D as the causative gene was important because it guided the further clinical diagnosis of primary aldosteronism and hypertension. Primary aldosteronism was not reported in the first case of congential HH affected by a CACNA1D variant G403D [CACNA1D variants G403D, I750 M and V259Ahad primary aldosteronism but, despite transient short episodes of postnatal hypoglycemia, did not require long-term diazoxide treatment [CACNA1D variants. Like most previously reported CANCA1D variants [Second, identification of nt G403D . Three ireatment ,14. Thervariants –13, L271CACNA1D variant as the most likely cause for the patient’s conditions also had important therapeutic consequences suggesting a “personalized” approach by treatment with the L-type Ca2+ channel blocker nifedipine. There is strong evidence from previous functional studies [de novo CACNA1D variants inducing gating changes that permit a gain of Cav1.3 channel function are pathogenic. Therefore therapeutic intervention with a nonselective L-type channel blocker appeared reasonable. Since nifedipine is licensed since decades to treat hypertension, we used it to successfully treat the patient’s hypertension (by blocking Cav1.2 L-type channels in arterial smooth muscle [Third, diagnosing a  studies –11 that h muscle . In addih muscle ,17. IndeCACNA1D as a risk gene for congential HH as well as for a severe neurodevelopmental syndrome. Further therapeutic trials are required to confirm our observation that some CNS symptoms, such as muscle hypotonia in our patient, are responsive to treatment with DHP Ca2+ channel blockers.Taken together our data strongly support


GFAT and PFK were highly expressed in the wing bud of Nilaparvata lugens and were increased significantly during molting. RNAi of GFAT and PFK both caused severe malformation rates and mortality rates in N. lugens. GFAT inhibition also downregulated GFAT, GNPNA, PGM1, PGM2, UAP, CHS1, CHS1a, CHS1b, Cht1-10, and ENGase. PFK inhibition significantly downregulated GFAT; upregulated GNPNA, PGM2, UAP, Cht2-4, Cht6-7 at 48 h and then downregulated them at 72 h; upregulated Cht5, Cht8, Cht10, and ENGase; downregulated Cht9 at 48 h and then upregulated it at 72 h; and upregulated CHS1, CHS1a, and CHS1b. In conclusion, GFAT and PFK regulated chitin degradation and remodeling by regulating the expression of genes related to the chitin metabolism and exert opposite effects on these genes. These results may be beneficial to develop new chitin synthesis inhibitors for pest control.Glutamine:fructose-6-phosphate aminotransferase (GFAT) and phosphofructokinase (PFK) are enzymes related to chitin metabolism. RNA interference (RNAi) technology was used to explore the role of these two enzyme genes in chitin metabolism. In this study, we found that  In insects, chitin is a major component of the exoskeleton, trachea, and the peritrophic matrix that lines the midgut epithelium4. Chitin enhances the mechanical strength of the insect exoskeleton and protects intestinal epithelial cells from abrasion by rough foods, penetration by toxins, and infection by microorganisms3. However, as the size of insects increases, their growth and development depend on the periodic synthesis and degradation of chitin5.Chitin is a linear polymer composed of N-acetylglucosamine units connected by β-1, 4-glycoside bonds and is the second most abundant biopolymer in nature. It is widely distributed in fungi, nematodes, and arthropods3. In insects, chitin synthesis can be carried out in the microvilli of epidermal cells or intestinal epithelial cells, beginning with trehalose followed by a series of enzymatic reactions to eventually form chitin6. Previous studies have shown that the chitin synthetic pathway consists of at least eight enzymes2. The first enzyme involved in chitin synthesis is trehalase (TRE), which hydrolyzes trehalose to β-D-glucose7. When TRE gene expression was silenced in Leptinotarsa decemlineata, the chitin content decreased accordingly8. Chitin biosynthesis occurs via the hexosamine pathway (HP)9. β-D-glucose can be phosphorylated by hexokinase (HK); it is then converted to fructose-6-phosphate (F-6-P) through isomerization by glucose-6-phosphate isomerase (GPI) and amino and acetyl groups are added via glutamine:fructose-6-phosphate aminotransferase (GFAT) and glucosamine-6-phosphate N-acetyltransferase (GNPNA) to form N-acetylglucosamine-6-phosphate. Phosphoacetylglucosamine mutase (PGM) transforms its phosphate group to form N-acetylglucosamine-1-phosphate. Subsequently, N-acetylglucosamine-1-phosphate is combined with uridine nucleoside triphosphate (UTP) to form UDP-N-acetylglucosamine (UDP-GlcNAc), the final product of HP as well as the precursor substance for chitin, under the action of UDP-N-acetylglucosamine pyrophosphorylase (UAP). Finally, N-acetylglucosamine (GlcNAc) polymers, also known as chitin, is formed by the action of chitin synthase (CHS)11. Two kinds of CHS, CHS1 and CHS2, were identified in insects12. Insect CHS1 and CHS2 are differentially expressed and regulated during growth and development. CHS1 is mainly responsible for chitin synthesis in cuticle and trachea cells11, while CHS2 is only responsible for chitin synthesis on midgut peritrophic membrane11. However, hemipteran insects lack peritrophic membrane, and CHS2 has not been found in hemipteran insects at present.In nature, chitin exists in three crystal structures and is classified as α-, β-, and γ-chitin according to the arrangement of chitin chains13. Chitin polymers can be enzymatically digested by chitinases, which are divided into endochitinases, exochitinases, and β-1, 4-N-acetyl-glucosaminidases14. Endochitinases and exochitinases are responsible for degrading long-chain chitin into short-chain chitooligosaccharides; β-1, 4-N-acetyl-glucosaminidases then hydrolyze short-chain chitooligosaccharides into GlcNAcs15.The physical properties of chitin itself are soft and form the hard exoskeleton of insects by combining with other proteins, but it limits the growth of insects. Therefore, along with chitin synthesis, its timely degradation is also necessary16. In human keratinocytes, GFAT1 silencing reduced the content of UDP-GlcNAc and hyaluronan17. The protein sequence of the GFAT gene is very similar in different types of organisms such as bacteria, insects, yeast, and mammals. GFAT is expressed in different mammalian tissues and may have different functions18. Phosphorylation plays an important role in regulating enzyme activity19; Li et al. have shown that the increase in GFAT activity can be achieved by phosphorylation of AMP-activated protein kinase at Ser243, a new regulation in addition to phosphorylation of protein kinase A20. In addition, a lot of experimental data demonstrate that GFAT activity can be inhibited by UDP-GlcNAc, the last product of HP21. GFAT plays an important regulatory role in shrimp's resistance to environmental stress, proliferation of mammalian cardiomyocytes and cancer cells through catalyzing hexosamine synthesis24. However, little is known about GFAT in insects, especially the relationship between GFAT and insect chitin metabolism.GFAT is the first and rate-limiting enzyme of HP, and converts fructose 6-phosphate and glutamine into glucosamine 6-phosphate and glutamate25. Both PFK in prokaryotic and eukaryotic cells catalyze phosphorylation of F-6-P to fructose-1,6-bisphosphate, but eukaryotic PFK is more than twice the size of bacterial PFK26. In vertebrates, PFK is activated by ADP, AMP, and fructose-2,6-diphosphate, whereas it is inhibited by physiological concentrations of ATP and citrate27. However, citrate does not inhibit PFK in insects28. PFK catalyzes the irreversible reaction in the glycolytic pathway and plays an essential role in glycometabolism, then is involved in the regulation of locomotion of different animals and other physiological activities29. However, there are few studies on the relationship between PFK and chitin metabolism in insects.Unlike GFAT, which is directly involved in chitin synthesis, phosphofructokinase (PFK) is a highly conserved and the main rate-limiting enzyme in the glycolytic pathway. Its activity significantly affects glucose consumption and energy production30. However, its production and storage has long been threatened by approximately 800 species of insect pests31. Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a hemimetabolous brown planthopper (BPH), is one of the most destructive and notorious insect pests32. It possesses high fecundity, sucks rice sap, oviposits in rice tissues, and transmits viruses such as grassy stunt virus and rugged stunt virus33. Use of chemical pesticides such as etofenprox has led to resistance in N. lugens; thus, integrated pest management is required to develop selective and environmentally safe biopesticides34. There is no chitin production in plants and vertebrates including humans and thus, the chitin metabolism pathway can be used as a safe target for pest control. During the past few years, some third-generation insecticides such as benzoylphenylurea related to the inhibition of arthropod pest chitin synthesis have been developed and commercialized, which can result in abortive molting and egg hatching as a consequence of chitin synthesis inhibition in the course of cuticle formation, thereby achieving the purpose of controlling pests37. A series of enzymes are involved directly or indirectly in the synthesis and hydrolysis of chitin. Therefore, we have selected the most critical enzymes in the HP and glycolytic pathway, GFAT and PFK, respectively, and have explored their exact effects on chitin metabolism in N. lugens using RNA interference (RNAi) technology in this study to evaluate their potential as targets for novel pesticide.Rice serves as a staple food for more than half the world’s population, and is a source of calories for billions of people worldwideGFAT and PFK expression levels in different developmental stages of N. lugens were determined by qRT-PCR. These results showed that the expression trends of NlGFAT and NlPFK were relatively similar. Expression of GFAT increased on the 3rd day of the fourth instar nymph but decreased on the 2nd day of fifth instar nymph, subsequently remained at relatively low levels before molting, and significantly increased (p < 0.05) during the adult stage  increased, whereas the expression of PFK in female adults showed no changes   Fig. A,B.FigurGFAT and PFK were both the highest in the wing bud and were significantly (p < 0.05) higher than their expression levels in the head, leg, ovary, cuticle, midgut, and fat body  after dsGFAT or dsPFK injection alone; both GFAT and PFK thus have a great influence on the growth and development of N. lugens individuals.As shown in the figures, after 48 h or 72 h of GFAT, GNPNA, UAP and two PGM genes were identified in N. lugens6. When the PFK of N. lugens was inhibited, the relative expression levels of GFAT were decreased extremely significantly (p < 0.01) at 48 h and 72 h  at 48 h and an extremely significant decrease (p < 0.01) at 72 h  at 72 h  at 48 h and 72 h after dsGFAT injection  at 48 h but showed little change at 72 h , one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase)1. At 48 h and 72 h after inhibiting the expression of NlGFAT using RNAi, the relative expression levels of all chitin degradation-related genes showed extremely significant declines (p < 0.01)  at 48 h. But their subsequent trends were not the same. The expression levels of Cht2, Cht3, Cht4, Cht6, and Cht7 decreased significantly (p < 0.05)    at 48 h and showed an extremely significant increase (p < 0.01) at 72 h  in N. lugens38. At both 48 h and 72 h after GFAT gene silencing by RNAi, the relative expression of CHS1, CHS1a, and CHS1b showed an extremely significant (p < 0.01) decline  increase at 48 h and 72 h , but N. lugens possesses only CHS1 with two transcript variants (CHS1a and CHS1b)38. RNAi against NlCHS1 and NlCHS1a causes high mortality rates and severe morphological malformations51. Knockdown of NlTRE1 could downregulate CHS1 and CHS1a and cause abnormal phenotypes44, and knockdown of TPS1 could downregulate the expression of CHS1, CHS1a, and CHS1b, resulting in extremely high malformation and mortality rates in N. lugens52, as well as HK-knockdown also could result in the downregulation of CHS1, CHS1a, CHS1b53. In our study, the mRNA levels of CHS1, CHS1a, and CHS1b were acutely decreased at 48 h and 72 h after dsGFAT injection , one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase)1. Among these 12 genes, RNAi targeting Cht1, Cht 5, Cht 7, Cht 9, and Cht 10 caused a lethal phenotype in N. lugens, whereas RNAi against Cht2, Cht3, Cht4, Cht6, Cht8, IDGF, ENGase had little effect on the morphology and survival of N. lugens1. Our results show that the relative expression levels of Cht1, Cht5, Cht7, Cht9, and Cht10 were decreased extremely significantly at 48 h and 72 h after dsGFAT injection , and the variety of all rice (Oryza sativa L.) cultivars was Taichung Native 1 (TN1) planted in cement tanks from April to October and in a greenhouse or growth chamber during winter. Insects were reared on fresh TN1 rice seedlings in an artificial climate chamber at 26 ± 1 ºC, 70% relative humidity, and 16 L:8 D (light:dark) photoperiod55. All experiments were performed under the same conditions. Developmental stages were synchronized by collecting new eggs laid by N. lugens, and the instar was judged based on the hind foot and antennae of the nymph.The N. lugens individuals used in gene expression stage analyses were obtained from the 4th instar nymphs on their first day, and after every 24 h until they reached the adult stage; 10 individuals were taken from each stage. Besides, female adults and male adults were also collected separately. The N. lugens used to detect the tissue expression level of genes were collected from 50 individuals of adults, and with a 1:1 ratio of male to female. The head, leg, ovary, cuticle, fat body, midgut, and wing bud of N. lugens were dissected in a saline solution (0.75% NaCl) under an EZ4 microscope . Three biological replicates were used for each developmental stage and tissue sample. All samples were kept at − 80 °C until RNA extraction.N. lugens were used to extract the total RNA with TRIzol reagent , following the manufacturer’s instructions. Total RNA integrity was determined by 1% agarose gel electrophoresis, and the RNA concentration and purity were determined by measuring the sample absorbance at 260 nm on a Nanodrop 2000 spectrophotometer 44; the purified RNA was stored at -80℃ for future experiments. First-strand complementary DNA (cDNA) synthesis was performed using the PrimeScript RT reagent kit with gDNA Eraser  following the manufacturer’s instructions and was stored at − 20 ℃.Tissues and whole bodies of NlGFAT and NlPFK using gene-specific primers  in a Bio-Rad CFX96 Real-Time PCR Detection System . Each PCR was performed in a 20 µL volume, containing 1 µL cDNA, 1 µL (10 µM) of each primer, 7 µL ultrapure water, and 10 µL SYBR buffer55. The reactions were performed under the following conditions: preincubation at 95 ºC for 2 min; 39 cycles of 95 ºC for 5 s and annealing at 59 °C for 30 s; and a melting curve at 65–95 °C. Amplification of 18S RNA was used as an internal control55.The expression of N. lugens cDNA template and specific primers (Table NlGFAT and NlPFK genes with reverse transcription polymerase chain reaction (RT-PCR). The reaction procedure is set as follows: preincubation at 95 ºC for 3 min, 35 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. Purified GFAT and PFK amplicons were transcribed to synthesize dsRNA using the T7 RiboMax Express RNAi System 44. A green fluorescence protein (GFP) gene amplicon was also used to synthesize dsRNA for being control group. Sense and anti-sense strands were separately produced using PCR and were then mixed for annealing. Reactions were incubated for 10 min at 70 °C and then placed on ice for 20 min. Finally, dsRNAs were purified with 95% ethanol and 4.4 M sodium acetate (pH 5.2), then washed with 70% ethanol, air dried, and redissolved with DEPC. The integrity and quantity of dsRNAs were determined by spectrophotometer with Nanodrop 2000 (Thermo Fisher Scientific) and agarose gel electrophoresis44.The rs Table  were useN. lugens on the 1st day of the 5th instar nymphs. Control groups were injected with dsGFP.Using an IM-31 microinjector , dsGFAT and dsPFK (3000 ng of each) were injected into the abdomen of N. lugens, the malformation rates and mortality rates of N. lugens were counted at 48 h and 72 h, respectively. In addition, insects were randomly collected  at 48 h and 72 h after injection to detect the relative expression of chitin metabolism-related genes. Collected samples were stored at -80℃. Photographs of abnormal insects were taken in different dsRNA injection treatments.After dsRNA was injected into fifth-instar larvae of N. lugens treated with dsRNA were used to extract the total RNA using the TRIzol reagent , then first-strand cDNA synthesis was performed using the PrimeScript RT reagent kit with gDNA Eraser . Relative expression levels of chitin metabolism-related genes were estimated by qRT-PCR using gene-specific primers . Other data was analyzed using the Tukey’s test. In Tukey’s test, a double asterisk indicates an extremely significant difference in mRNA levels (p < 0.01), and an asterisk indicates a significant difference (p < 0.05).In this study, all data were analyzed using one-way analysis of variance (ANOVA) and are shown as the mean ± standard error (SE) of three biological replicates. Data on developmental and tissues expression patterns were analyzed using Duncan’s test. In Duncan’s test, different letter indicates a significant difference (


Crassostrea virginica. Sperm motility was activated in ambient seawater with salinity 4–32 PSU with highest motility and velocity observed at 12–24 PSU. In artificial seawater (ASW) with salinity of 20 PSU, sperm motility was activated at pH 6.5–10.5 with the highest motility and velocity recorded at pH 7.5–10.0. Sperm motility was inhibited or totally suppressed in Na+, K+, Ca2+, and Mg2+-free ASW at 20 PSU. Applications of K+ (500 μM glybenclamide and 10–50 mM 4-aminopyridine), Ca2+ (1–50 μM mibefradil and 10–200 μM verapamil), or Na+ (0.2–2.0 mM amiloride) channel blockers into ASW at 20 PSU inhibited or suppressed sperm motility and velocity. Chelating extracellular Ca2+ ions by 3.0 and 3.5 mM EGTA resulted in a significant reduction and full suppression of sperm motility by 4 to 6 min post-activation. These results suggest that extracellular K+, Ca2+, and Na+ ions are involved in regulation of ionic-dependent sperm motility in Eastern oyster. A comparison with other bivalve species typically spawning at higher salinities or in full-strength seawater shows that ionic regulation of sperm motility is physiologically conserved in bivalves. Elucidating sperm regulation in C. virginica has implications to develop artificial reproduction, sperm short-term storage, or cryopreservation protocols, and to better predict how changes in the ocean will impact oyster spawning dynamics.Oyster aquaculture is expanding worldwide, where many farms rely on seed produced by artificial spawning. As sperm motility and velocity are key determinants for fertilization success, understanding the regulation of sperm motility and identifying optimal environmental conditions can increase fertility and seed production. In the present study, we investigated the physiological mechanisms regulating sperm motility in Eastern oyster,  Similar to vertebrates –4, spermOnce sperm motility in aquatic animals is activated by environmental osmolality or ions –13, sper+), calcium (Ca2+), potassium (K+), magnesium (Mg2+), and sodium (Na+) play critical roles in regulation of sperm motility e) ions or application of Ca2+ channel blockers resulted in suppression or inhibition of sperm motility i) and intracellular Ca2+ ([Ca2+]i) i) ,40,55,56seawater . Taken tThe Eastern oyster is native to benthic habitats along the Atlantic coast of America from Canada to the Gulf of Mexico, the Caribbean, and the coast of South America . It is a+, Ca2+, Na+, and Mg2+-free ASW or in ASW with various ion channel blockers. Thereafter, sperm kinematics (motility and velocity) were quantified using a CASA system. Our results provide valuable information to develop methods for artificial reproduction, short- or long-term preservation of sperm, and to understand species specificity of sperm motility regulation in bivalves that might be related to environmental changes.In the present study, our goal was to investigate regulation of sperm motility in the Eastern oyster, where specific objectives were to study the contributions of salinity, pH, and ions. In this context, we evaluated sperm motility and velocity after activating sperm in ambient seawater and artificial seawater (ASW) with different salinity and pH, respectively. To understand the contribution of ions in regulation of sperm motility, sperm were activated in KAll investigations using male Eastern oyster were conducted in accordance with the Auburn University Animal Care and Use Program (IACUC).2 × 2H2O, MgCl2 × 6H2O, and MgSO4 × 7H2O were purchased from VWR Analytical, Ward’s Science, EMD Millipore Corp, and Ward’s Science, respectively.Buffers including MES, HEPES, and Tris were obtained from VWR Life Science as well as ethylenediaminetetraacetic acid (EDTA). Nifedipine and choline chloride were from Beantown Chemical. Pluronic F-127 and mibefradil dihydrochloride hydrate were from Sigma Aldrich. Verapamil hydrochloride, glybenclamide, 4-Aminopyridine (4-AP), amiloride hydrochloride dehydrate, NaOH, HCl, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and DMSO were purchased from AdipoGen, InvivoGen, Acros Organics, ENZO, Fisher Scientific, EMD, Alfa Aesar, and Macron Fine Chemicals, respectively. KCl, CaClOysters were collected in batches from floating cages and longline systems at the Auburn University Shellfish Laboratory (AUSL) research oyster farm site in Grand Bay, AL (30°22’34.8” N 88°18’52.8” W), and transported to the AUSL in Dauphin Island, AL  or the Aquatic Reproductive Physiology Laboratory (ARPL) at Auburn University, Auburn, AL. At AUSL, the oysters were held in a flow-through system at 19°C and 10 PSU. The incoming seawater flowed through a 50-micron bag filter and was chilled with a 1/3 HP Trimline Delta Star Water Chiller. At the ARPL, oysters were acclimated and held in a recirculation aquaculture system, equipped with two 794 L polyethylene round open top tanks (122 cm diameter × 81 cm height) . Each day, the oysters were fed ~40 mL of Shellfish Diet 1800 . Seawater was made by mixing Crystal Sea Marinemix  into tap water  filtered through a reverse osmosis system  to reach a salinity of 10 PSU that was identical to environmental salinity at the collection site. Water temperature was also maintained at 19°C.Oysters were opened with a shucking knife, cup-side down, at the hinge, and sex was determined by observation of the gonad samples under a microscope. Each male was then thoroughly rinsed with distilled water to flush away any excess seawater. The mantle tissue around the gonad was dried with paper towels before semen collection by pipette. Collected semen was stored in a 1 mL Eppendorf tube and placed into a chilling block  set to 19°C. An aliquot of each sample was examined to ensure the sperm was not moving prior to the start of each experiment. If the sperm was motile (an indication that semen was contaminated with seawater) the sample was discarded. Morphometrics of oysters including length, width, and height (± 0.01 mm) were recorded as well as sperm density using a Neubauer hemocytometer .Osmolality of testicular fluid  was determined in triplicate for each sample using a Vapor Pressure Osmometer . The pH of testicular fluid  was measured using a benchtop pH meter  and an electrode . Osmolality and pH were also measured for all activation media.In all experiments, sperm activating solutions (500–1000 μL) were pipetted into 1.5 mL Eppendorf tubes placed into a chilling block set to 19°C for the duration of the experiment. To activate sperm motility, 0.2–1.0 μL of semen was then diluted with sperm activating solutions in the Eppendorf tube and inverted several times to mix thoroughly. At different times post-activation, 5 μL of suspension was pipetted into a 20 μm deep 2X-CEL chamber , placed under a light microscope , and sperm motility was recorded at 20× magnification. Percentage of motile sperm and curvilinear velocity (VCL) were determined. All solutions contained 0.4% Pluronic F-127 to prevent sperm from sticking to the slides .Semen of 5 males were used to study the effect of salinity on sperm motility. Ambient seawater from the AUSL was boiled to make a high salinity stock solution (40 PSU). This stock solution was then cooled and diluted with ultrapure water to make salinities ranging from 4 to 32 PSU. A refractometer and a Vapor Pressure Osmometer were used to determine salinity and osmolality, respectively . Seawate2, 34 mM MgCl2, and 22 mM MgSO4 e) exchanger. Under this condition, [pH]i is equivalent to [pH]e if [K+]e is equivalent to [K+]i [+ efflux occurs in sperm following activation in a solution with [pH]e higher than [pH]i [+-free ASW or ASW containing 4-AP suggests that K+ ions might be involved in regulation of [pH]i.We also observed that sperm velocity was decreased in King 4-AP , which wto [K+]i , and H+ an [pH]i . Resultsan [pH]i . Therefo2+-free ASW or in ASW containing EGTA, however they were rapidly decreased compared to ASW  and pH (6.5–10.5). Our results show that when sperm spawned into seawater, Kectively ,36. TakeUltimately, this study can be used to improve bivalve aquaculture by optimizing activation or immobilizing medium. For instance, to preserve sperm for short or long-term storage (cryopreservation), it can be maintained in the quiescent state in ASW close to that of testicular fluid. This study also has implications that surpass the scope of assisted reproduction into a more comprehensive understanding of the natural ecology of the Eastern oyster. For instance, our results provide ecologists with valuable information to better predict how changes in the ocean will impact oyster spawning dynamics.S1 FigEffect of potassium (K+) ions on sperm velocity (μm/s) in Eastern oyster, Crassostrea virginica at 30 (A), 45 (B) and 60 (C) min post-activation. Sperm was activated in K+-free artificial seawater (ASW) and ASW containing a voltage-gated  or an ATP-sensitive  K+ channel blocker. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 5). Treatments with different superscripts significantly differ (P < 0.05).(TIF)Click here for additional data file.S2 FigEffect of calcium (Ca2+) ions on sperm motility (%) in Eastern oyster, Crassostrea virginica at 6 (A), 10 (B), 12 (C), 14 (D), 16 (E), 18 (F), and 20 (G) min post-activation. Sperm was activated in artificial seawater (ASW), Ca2+-free ASW and ASW containing Ca2+ channel blockers: mibefradil (M), nifedipine (N), or verapamil (V). Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (P < 0.05). A motility of 0% was indicated by asterisk.(TIF)Click here for additional data file.S3 FigEffect of calcium (Ca2+) ions on sperm velocity (μm/s) in Eastern oyster, Crassostrea virginica at 6 (A), 10 (B), 12 (C), 14 (D), 16 (E), 18 (F), and 20 (G) min post-activation. Sperm was activated in artificial seawater (ASW), Ca2+-free ASW, and ASW containing Ca2+ channel blockers: mibefradil (M), nifedipine (N) or verapamil (V). Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (P < 0.05). A velocity of 0 μm/s was indicated by asterisk.(TIF)Click here for additional data file.S4 FigSperm motility  in Eastern oyster, Crassostrea virginica after activation in artificial seawater containing EGTA. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (P < 0.05). Motility of 0% was indicated by asterisk.(TIF)Click here for additional data file.S5 FigSperm velocity  in Eastern oyster, Crassostrea virginica after activation in artificial seawater containing EGTA. Average velocity at each EGTA concentration (B) and time post-activation (C) is displayed. Data were analyzed using a repeated measures ANOVA and shown as mean ± SE (n = 4). Treatments with different superscripts significantly differ (P < 0.05). Velocity of 0 μm/s was indicated by asterisk.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table2 value, and absolute maximum or peak (vertex) are shown in columns for both motility and velocity. Significant values are denoted by ***P < 0.0001 and **P < 0.01.For each time, the equation of the line, R(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


However, patient management with central venous oxygen saturation  and central venous pressure (CVP) was monitored, and the change of VO2 was evaluated. After delivery, her APCO was almost unchanged. However, her ScvO2 increased dramatically from 42.1 to 67.3% and her CVP increased from 9 to 11 mm Hg. The calculated mean maternal VO2 changed from 443 to 295 mL/min.A 30-year-old woman, who had received a total cavopulmonary connection for tricuspid atresia, was planned to undergo cesarean section at 38 weeks’ gestation. During combined spinal-epidural anesthesia, ScvO2 dramatically increased and maternal VO2 decreased by more than 25% after delivery.In a cesarean section for a Fontan-palliated parturient, ScvO The long-term survival rate of patients with congenital heart disease (CHD) has been improved through progress in surgical techniques and multidisciplinary perioperative management methods , 2. Acco2) and oxygen consumption (VO2) during this procedure has never been reported. Here, we present the first case of a cesarean section during which ScvO2, arterial pressure-based cardiac output (APCO), and CVP were monitored in a patient with Fontan circulation. Furthermore, we examined perioperative changes in maternal VO2. Our report will contribute to the understanding of the hemodynamic and VO2 changes that occur during cesarean delivery in patients with Fontan circulation.During pregnancy, dynamic physiological changes occur, including an increase in cardiac output, oxygen consumption, and intravascular volume, making both pregnancy and delivery challenging for these patients . Previou2; 90%) and presented an exacerbation of symptoms, including cyanosis, clubbed fingers, and hepatomegaly. She underwent an extracardiac total cavopulmonary connection. Her postoperative CVP was 15 mm Hg. This procedure improved her New York Heart Association (NYHA) functional classification to class I. At 28 years old, her peak VO2, assessed by a cardiopulmonary exercise test, was 62.2% of that of healthy age-mates. Echocardiography indicated that her left ventricular ejection fraction (LVEF) was 64% without atrioventricular valvular regurgitation.A female infant, diagnosed with tricuspid atresia (TA) type 1B , underwent a balloon atrioseptostomy at 17 days of age and a Fontan operation at the age of 4 years. At age 12, she developed low percutaneous oxygen saturation , although she had an LVEF of 61% without left ventricular dilation. Her NYHA classification changed to class II. She was admitted to our hospital at 36 weeks’ gestation. Her blood hemoglobin decreased from 13.9 to 10.8 g/dL antenatally. Because her exercise capacity was 60% of that of healthy age-mates, a cesarean section was performed at 38 weeks’ gestation. Her anticoagulation therapy  was discontinued 6 h before surgery.At age 30, she became pregnant and was referred to our hospital at 15 weeks’ gestation. Her usual anticoagulation therapy was changed from warfarin to aspirin because of the former’s teratogenic potential. A Holter electrocardiogram revealed a sinus rhythm of 91 beats/min without any episodes of arrhythmia. At 35 weeks’ gestation, she developed exertional dyspnea, followed by cyanosis , and APCO were monitored via a left radial artery catheter . Additionally, a central venous catheter  was inserted via the right internal jugular vein to monitor CVP and ScvO2 continuously. The tip of the central venous catheter was located just cephalad to the connection between the superior vena cava and the right pulmonary artery by chest X-ray examination. On room air, the patient’s SpO2, CVP, and ScvO2 levels were 92% , 10 mm Hg, and 52.2%, respectively  = 1.34 × Hb (g/dL) × (SaO2 − ScvO2) × CO (L/min) × 10.Ten minutes after the induction of anesthesia, she developed hypotension and her ScvOSurgery was completed without complications. For postoperative analgesia, 0.25% levobupivacaine was administered via the epidural catheter at 4 mL/h. Anesthesia and surgery lasted 69 and 50 min, respectively. During anesthesia, the estimated blood loss was 895 mL including amniotic fluid, and urine output was 220 mL. These were replaced with 750 mL of crystalloids.The patient was transferred to the intensive care unit, where her CVP ranged from 11 to 15 mm Hg. Her brain natriuretic peptide levels increased by 14.2 to 26.7 pg/mL, although the cardiac silhouette on her chest X-ray did not show any dilation. For postoperative anticoagulation therapy, heparin was administered intravenously by 10,000 U/day. Her wound pain was well managed by epidural anesthesia. Heparin was stopped 6 h before the removal of the epidural catheter to prevent a hemorrhagic complication. She returned to the general ward on her first postoperative day and was discharged on the 9th postoperative day on 100 mg aspirin and 2.5 mg enalapril, daily.2 , APCO, and CVP.Here, we reported a case describing the use of combined spinal-epidural anesthesia for the management of cesarean section in a Fontan-palliated parturient. Because some adult CHD patients are at high risk for developing heart failure during pregnancy and postpartum , we focuDuring the perinatal period, intravascular volume increases and systemic vascular resistance substantially decreases, increasing cardiac output up to 7.0 L/min 12]. For a Fontan-palliated parturient, it is difficult for cardiac output (CO) to be adequately increased, making this type of aggressive volume challenge potentially dangerous for this population . To trea2 decreased to 52.2% as a result of a substantial VO2 increase. At the time, the VO2 level (393 mL/min; Table 2 may have been caused by maternal stress. Furthermore, the critically low ScvO2 levels observed indicate that she was at the limit of her cardiac capacity. If her systemic ventricle had been the morphologic right ventricle, she would probably have had significant systemic heart failure during the perinatal period.Our patient was born with TA, and her systemic ventricle was the morphologic left ventricle after Fontan procedure. Before pregnancy, her heart function and exercise capacity were preserved. Before anesthesia induction, her APCO was 7.02 L/min, which is equivalent to 140% of the CO of non-pregnant women and the CO of normal parturients , our pat2 levels further decreased to 38.6%, while VO2 levels further increased to 543 mL/min (Table 2 and VO2 levels may have resulted from further maternal psychological stress. After the delivery, her CO increased by 10% and CVP increased from 10 to 14 mm Hg. Additionally, her ScvO2 increased from 42.1 to 67.3% and the calculated VO2 decreased to 256 mL/min. The latter is equivalent to the VO2 of a non-pregnant woman at rest. This change in VO2 suggests that fetus delivery and subsequent stress relief may substantially decrease maternal VO2 and restore it to normal levels.After anesthesia induction, the patient’s arterial blood pressure slightly decreased to 109/53 mm Hg and CO was 6.73 L/min. ScvO2, APCO, and CVP was presented. Our report revealed maternal circulatory dynamics and VO2 changes during a cesarean section in a Fontan-palliated patient. Maternal VO2 was found to decrease substantially after the delivery of the fetus.The combined spinal-epidural anesthesia management in a cesarean section for a Fontan-palliated parturient with monitoring of ScvO


O-methylated anthocyanidin, has not been well documented. In this study, we investigated metal chelation of petunidin derivatives in a wide pH range and its effects on color and stability. Purple potato and black goji extracts containing >80% acylated petunidin derivatives (25 μM) were combined with Al3+ or Fe3+ at 0 μM to 1500 μM in buffers of pH 3–10. Small metal ion concentrations triggered bathochromic shifts (up to ~80nm) at an alkaline pH, resulting in vivid blue hues (hab 200°–310°). Fe3+ caused a larger bathochromic shift than Al3+, producing green colors at pH 8-9. Generally, metal ions increased the color stability and half-life of petunidin derivatives in a dose-dependent manner, particularly at pH 8. Petunidin derivative metal chelates produced a wide range of colors with enhanced stability.Anthocyanins with catechol (cyanidin) or pyrogallol (delphinidin) moieties on the B-ring are known to chelate metals, resulting in bluing effects, mainly at pH ≤ 6. Metal interaction with petunidin, an  Anthocyanins are water-soluble pigments that provide brilliant red, purple and blue colors to vegetables and fruits . They un2+, Fe3+, Ga3+, Al3+, Mg2+, Sn2+, Ti2+, and Cu2+ , purple potato extracts developed a variety of blue colors. At pH 7, brilliant blue with a hue angle of ~261° appeared, starting from [Al3+] of 750 μM. Darker blue (~240°) and greenish blue (~200°) were obtained when [Al3+] was 50 μM at pH 8 and pH 9, respectively. Fe3+ also triggered bluing shifts, as the increasing [Fe3+] led to the transition of Pt-Fe3+ from quadrant IV (purple to blue region) to quadrant III (green to blue region) in the CIELAB color space , while more [Fe3+] was needed. In purple potato extracts, Al3+ in small concentrations ([Al3+] = 12.5 μM) could produce blue colors at pH 8 and 9, and it was the same for Fe3+ at pH 7 and 8  seemed to have limited impact on Pt colorimetric properties, as the ΔE*ab were all smaller than 5 (data not shown). In addition, the color of petunidin derivatives faded quickly at pH 10. Therefore, our following analysis focused on the Pt-M3+ at pH 7–9.At pH 3–6, however, Pt-Almax, and absorbance at the λmax of Pt-M3+, are shown in 3+ at pH 7–9, similar to the findings in previous studies on cyanidin or delphinidin–M3+ chelates at acidic pHs  that resulted in maximum λmax and absorbance at each pH level was closely related to the M3+ source. In purple potato extracts, the largest λmax of Pt-Al3+ was accomplished when [Al3+] equaled 1500 μM, 250 μM, and 125 μM at pH 7, 8, and 9, respectively; while it was 25 μM, 50 μM, and 750 μM in Pt-Fe3+ at these same pH levels  are shown in 3+ generally exhibited enhanced color stability in a [M3+] dose-dependent manner. Higher [Al3+] produced bluer hues that were significantly better retained over time for ~7 days at all pH values tested (pH 7–9), while petunidin derivatives without metal chelation started losing color rapidly, with the blue color only formed at pH 9 and gone in less than 1 day. Interestingly, brown hues steadily formed in higher [Fe3+] chelates after mixing with petunidin derivatives and were more severe at pH 9. Fe3+ is known for its reductive–oxidative capabilities, and has been reported to catalyze the oxidative degradation of flavonoids  = 1500 μM and 98.5 h in [Fe3+] = 50 μM). Because of its reductive–oxidative capabilities, excessive Fe3+ started to reduce the half-life of Pt-Fe3+ chelates at pH 7–9.The half-lives (h) of the pigments and their metal chelates at pH 7–9 were calculated and are presented in 3+ still showed a promising stabilization capability. At neutral and alkaline pH, anthocyanin is supposed to exist predominantly in quinoidal base forms, and would take on increasingly negative charges with further increases in pH [a2 and pKa3, the disassociation constant for the transition from the quinoidal base configuration to that with one negative charge, and the transformation from one negative charge to two negative charges. A previous study reported that the pKa2 and pKa3 of petunidin aglycone were 6.99 and 8.27, respectively [a for petunidin-3-rutinoside-(p-coumaroyl)-5-glucose would be different, it is reasonable to postulate that the purple potato anthocyanin carries negative charges at pH 8 and thus is more stable, compared to other close neutral and alkaline pHs.Among the pH values (pH 7–9) in the stability study, petunidin derivatives and their metal chelates displayed the highest color stability at pH 8. At this pH, a lower concentration of Mes in pH . This prectively . Althougab and increased the pigments half-life, though this stabilization effect is dependent on the metal source and pH. Fe3+ in high concentrations, because of its oxidative capability, accelerated the color degradation and promoted the browning effect.Overall, metal ions helped to stabilize petunidin derivatives in a dose-dependent manner, and significantly slowed down the escalation of ΔE*trans-p-coumaroyl)-rutinoside-5-glucoside as the predominant pigment. The extracts displayed vivid red, purple, violet-blue colors from acidic, neutral, to alkaline pH, respectively. In the presence of metal ions, Al3+ and Fe3+, the petunidin derivatives experienced bathochromic (up to 79 nm) and hyperchromic shifts (increased up to 0.399 in absorbance at λmax), resulting in various blue to green hues and intensified colors in neutral and alkaline conditions. The magnitudes of the metal chelation effect were dependent on metal ions, pH, and M3+ concentration. Fe3+, with a higher spin configuration in the outer electrons, triggered further bathochromic shifts than Al3+, and its unique yellow color in the aqueous system led to green hues when combined with petunidin derivatives’ blue colors. The oxidative capability of Fe3+ towards flavonoids limited its application in larger concentrations (>125 μM). Al3+, in low concentrations (~12.5 μM), was able to chelate Pt and produce blue colors at pH 8 and 9. The concentration of Al3+ needed for blue color production decreased upon the increase in pH. The chelation of metal ions significantly enhanced the color stability of petunidin derivatives in neutral and alkaline conditions.Purple potato and black goji extracts contained abundant petunidin derivatives, with petunidin-3-(3+ and Fe3+, in neutral and alkaline pH, resulting in enhanced color and stabilities. Purple potato and black goji, containing abundant petunidin derivatives, are promising sources for natural colorants over a wide range of pH. Chelated with metals, these pigments could provide the food industry with various vivid violet, blue, and green colors with enhanced stability. They could be potentially applied in food products with neutral or slightly higher pH, such as milk, milkshakes, ice cream, and creamers. This is important to the food industry, as the elucidation of the anthocyanin–metal interaction can bring innovative technologies to obtain a wide range of vivid colors from natural sources, fulfilling the current clean label trend.This study demonstrated that petunidin derivatives could chelate with metal ions, Al


Patch clamp recording was conducted to assess L-type Ca2+ current (ICa-L), and the expression of proteins involved in signaling pathways was measured by western blotting. Myocardial cytoskeletal protein and nuclear translocation of target proteins were assessed by immunofluorescence. The results indicated that siCaMKII suppressed Ang II-induced CH, as evidenced by reduced cell surface area and ICa-L. Notably, siCaMKII inhibited Ang II-induced activation of CnA and NFATc4 nuclear transfer. Inflammatory signaling was inhibited by siCaMKII and WXKL. Interestingly, CsA inhibited CnA-NFAT pathway expression but activated CaMKII signaling. In conclusion, siCaMKII may improve CH, possibly by blocking CnA-NFAT and MyD88 signaling, and WXKL has a similar effect. These data suggest that inhibiting CaMKII, but not CnA, may be a promising approach to attenuate CH and arrhythmia progression.Previous studies have demonstrated that calcium-/calmodulin-dependent protein kinase II (CaMKII) and calcineurin A-nuclear factor of activated T-cell (CnA-NFAT) signaling pathways play key roles in cardiac hypertrophy (CH). However, the interaction between CaMKII and CnA-NFAT signaling remains unclear. H9c2 cells were cultured and treated with angiotensin II (Ang II) with or without silenced CaMKII CH is a prerequisite marker of heart failure (HF) and usually occurs after myocardial infarction and stress overload. Although the initially adaptive response can maintain cardiac output, sustained hypertrophic growth can lead to a pathological state that leads to decreased compliance, HF, and sudden death –5. Thereeceptors .β-adrenergic-induced CH [2+-binding subunit (CnB) [δb (located in the nucleus) and CaMKIIδc (located in the cytosol) promotes CH and dilated cardiomyopathy, respectively. However, the complete pathological molecular mechanism is not fully understood, which hinders the development of improved treatments for CH [2+-dependent signaling through CaMKII and CaN has been suggested to contribute to adverse CH [Calcineurin (CaN), a calcium- and CaM-dependent serine/threonine phosphatase, is a well-established mediator of duced CH , 8. CaN it (CnB) , 10. It it (CnB) , 12. Morit (CnB) . Transges for CH , 15. Ca2verse CH . Howeverqi, supply yin, promote blood circulation, and remove blood stasis according to the theory of traditional Chinese medicine. The main components of WXKL consist of Nardostachys jatamansi (D.Don) DC (Gansong), Codonopsis pilosula (Franch.) Nannf (Dangshen), Panax notoginseng (Burkill) F.H.Chen (Sanqi), Succinum (Hupo), and Polygonatum cyrtonema Hua (Huangjing). According to the national pharmacopoeia [47H80O18), ginseng saponin Rg1 (C42H72O14), and ginseng saponin Rb1 (C54H92O23). Panax notoginseng and Codonopsis pilosula. Previous studies have shown that WXKL may inhibit HF and arrhythmia by regulating the CaMKII signaling pathway [Wenxin Keli (WXKL) is the first Chinese medicine approved by the Chinese state for its antiarrhythmic effects; the medicine can tonify acopoeia , WXKL ma pathway –23. WXKL pathway –26. HoweGiven the pivotal roles of the CaMKII and CaN signaling pathways in the regulation of abnormal hypertrophy, we selected H9c2 cells induced by angiotensin II (Ang II) and used CaMKII silencing and treatment with cyclosporine A  to explore the correlation between the CaMKII and CnA-NFAT signaling pathways. Additionally, we sought to determine whether WXKL could improve HF by regulating CaMKII and CnA-NFAT.δ (Rat), was performed. According to the target gene sequence site, four sequences with the target gene were inserted into the vector pCDNA6.2 emGFP, and miRNA reverse primer was used as the sequencing primer  RNA interference cell line. The H9c2-1632 cells were cultured in an incubator containing 5% CO2 in high-sugar Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C.H9c2 rat embryonic cardiomyocyte cells were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences . First, RNA interference at the site of the target gene, CaMKIIg primer . Sequencg primer , and theg primer . CaMKIIδ632 site . Combine−5 mol/L; 20 μL of this solution was added to the culture medium (2 mL), reaching a final concentration of 10−7 mol/L. CsA (Sigma Co.) was dissolved in ethyl alcohol at a concentration of 10−4 mol/L; 20 μL of this solution was added to the culture medium (2 mL) at a final concentration of 10−6 mol/L. WXKL  was dissolved in saline at a concentration of 5 g/L.Ang II  was dissolved in deionized water at a concentration of 10n-butanol was added, and the flask was tightly stoppered and weighed. After soaking for 12 hours and ultrasonic treatment  for 1 h, the flask was weighed again after cooling, water-saturated n-butanol was added to make up for the lost weight, and the flask was shaken well. After filtration, 25 ml of the filtrate was collected. The n-butanol solution was evaporated to dryness, and methanol solution was added to the residue to 10 ml and shaken. According to the proportion of prescription Chinese medicine, notoginseng and Codonopsis were removed separately to make negative samples of Panax notoginseng and Codouopsis pilosula, and the negative control solution was prepared according to the previously mentioned methods. Methanol was added to prepare reference substances containing notoginsenoside R1 0.247 mg, ginsenoside Rg1 0.422 mg, ginsenoside 0.042 mg, ginsenoside Rb1 0.847 mg, and ginsenoside Rd 0.255 mg per 1 mL.In HPLC, 1 g of finely ground WXKL was added to a conical flask with a stopper, 50 ml of water-saturated 18 column . The gradient elution used acetonitrile (A) and water (B) as a mobile phase at a flow rate of 1 mL/min. The gradient program was as follows: 0–14 min, 22%–30%A; 14–35 min, 30%–38%A; 35–45 min, 38%–38%A; 45–47 min, 38%–95%A; 47–62 min, 95%–95%A; 62–65 min, 95%–22%A. The column temperature was maintained at 27°C, and the chromatogram was monitored at a wavelength of 210 nm.Analyses were performed on an Agilent 1260 HPLC system consisting of a quaternary delivery system, an autosampler, and a DAD detector. All the separations were carried out on Amethyst CH9c2 rat embryonic cardiomyocytes were split into 13 different treatment groups: Proteins were extracted from H9c2 cells, subsequently lysed with lysis buffer containing phenylmethylsulfonyl fluoride in ice for 40 min, and shaken once every 8 min during this period. The lysates were centrifuged at 1200 rpm for 20 min at 4°C, and the supernatants were collected. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, blocked in 5% bovine serum albumin (BSA) or milk, and then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were washed three times with tris-buffered saline/Tween 20 at the specified time intervals and finally incubated with secondary antibody at room temperature. ECL visualization was performed, and the resulting images were captured using the GeneGnome Gel Imaging System . The gel images were analyzed using ImageJ software . The antibodies used in the present study are listed in μL) and added to 200 μL of the working fluid, fixed for 20 min, and washed with PBS three times for 5 min each. Cells were subjected to confocal laser microscopy. The cells of each group were removed and washed with cold PBS (precooled at 4°C) twice, and the residual PBS was aspirated. Each group was fixed with 4% paraformaldehyde for 10 min at −20°C and washed twice with PBS. Cells were permeabilized with 0.25% Triton X-100 for 10 min and washed three times with cold PBS for 5 min each wash and then treated with 1% BSA for 30 min to block nonspecific antigen recognition. Primary antibodies were diluted with 1% BSA in PBS and added at 100 μL to each group. Cells were plated at 4°C for 12 h and then washed thrice with PBS for 5 min each. The secondary antibody was diluted with 1% BSA in PBS with 100 μL per sample at room temperature for 60 min away from light. The secondary antibody was removed and cells were washed thrice with PBS for 5 min each. Cells were incubated with DAPI for nuclear staining; finally, an antifluorescence quencher was added. Later, a transparent nail polish seal was used, and cells were observed using a confocal microscope.For the cytoskeletal assay, H9c2 cells were cultured in a confocal laser culture dish until reaching a moderate density. Upon reaching 70–80% confluence, the cells were washed with phosphate-buffered saline (PBS) twice, fixed with 4% paraformaldehyde for 15 min, and washed with PBS three times for 5 min each. Cells were incubated with 0.1% Triton X-100 penetrating fluid at room temperature for 5 min and washed thrice with PBS for 5 min each. Rhodamine-phalloidin was diluted with PBS , and the total number of cells was counted.2+ current was recorded by an Axon-700B amplifier  with the pCLAMP 9.2 software (Axon Instruments). Borosilicate glass microelectrodes had tip resistances of 3.0–5.0 MΩ, which adjusts the three-dimensional manipulator for GΩ sealing and breaks the membrane absorption in the whole-cell recording mode. The membrane capacitance and ICa-L current were recorded after stabilization. To eliminate the errors resulting from cell size, the I value was expressed as the current density (pA/pF). Cells were superfused with extracellular fluid containing : 125 NaCl, 10.8 BaCl2, 1 MgCl2, 5.4 CsCl, 10 HEPES, and 10 glucose . A pipette solution was used containing (mmol/L): 120 CsCl, 3 MgCl2, 5 Na2ATP, 10 EGTA, and 5 HEPES .Using the whole-cell patch clamp technique, the whole-cell CaP < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. Data acquisition and analysis were performed using pCLAMP 9.2 software (Axon Instruments), Origin 6.1 software , and GraphPad Prism 5 .Data were expressed as mean ± SD. One-way ANOVA was used to compare multiple groups with a normal distribution. Statistical analysis was performed using the SPSS program (version 20.0). 2+ current (ICa-L); expression of CaMKII, CnA-NFAT, and inflammatory signaling pathways in cardiomyocytes induced by Ang II; and the protein expression and nuclear transfer of NFATc4.We explored the interaction between CaMKII and CnA-NFAT signaling in CH induced by Ang II and the effect of WXKL. Therefore, we sought to characterize the role of CaMKII and CnA-NFAT signaling in cardiomyocytes using siRNA-mediated silencing (siCaMKII) and CsA. We analyzed the results of cytoskeletal enlargement; L-type CaWe used fluorescence confocal microscopy to observe cytoskeletal enlargement  to assess whether the CH model had been successfully established and ensure the feasibility of the experiment.Immunofluorescence staining showed that Ang II effectively induced CH, as evidenced by an increase in cell width and length . CsA andCa-L is present in many cardiomyocytes, and it plays a key role in the formation of an action potential plateau, intracellular Ca2+ elevation, and muscle contraction. CaM, as a Ca2+ receptor, plays a major role in Ca2+-dependent inactivation and facilitation of ICa-L.ICa-L within the range of −20 mV to +10 mV. Pretreatment with siCaMKII reduced the elevated ICa-L amplitude. Similarly, WXKL decreased the Ang II-induced elevated amplitude of ICa-L , sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, ryanodine receptor 2 (RyR2), and PLB proteins. CaMKII phosphorylates the LTCC that leads to increased ICa-L and forms a Ca2+ overload in the cell, which causes early afterdepolarizations and triggers arrhythmia. Hyperphosphorylation of the sarcoplasmic reticulum (SR) results in the consumption of SR Ca2+ stores, leading to damaged cytosolic Ca2+ transients, which in turn induces systolic and diastolic dysfunction. Moreover, hyperphosphorylation events at RyR2 cause abnormal release of SR Ca2+ that activates the electrogenic Na+/Ca2+-exchanger (NCX), which can cause delayed afterdepolarizations [Excess CaMKII activity leads to phosphorylation of the L-type Caizations . Interesizations , 36. Conizations . These dizations , 39. Acuizations . The curizations .α and CnAβ, which are universally expressed, and CnAγ [β1 may have a cardioprotective action that decreases inflammation and scar formation [β leads to increased cell death and reduced cardiac function [α lacking the autoinhibitory domain show strong CH and develop HF within the first few weeks of life, which is a response phenocopied by overexpression of a constitutively active form of NFAT [β show smaller hearts at baseline and exhibit reduced hypertrophy in response to pressure overload, Ang II, or isoproterenol [β.Three different CnA subtypes are found in mammals: CnAand CnAγ . Furtherormation . Moreovefunction , 43. In  of NFAT . Other soterenol . Notablyoterenol , CsA augoterenol , 45–47. κB, TLR2, and TLR4 inflammatory response pathways in pathological states induces an increase in CaMKII protein expression, causes calcium homeostasis, and promotes the occurrence of potentially malignant arrhythmias [There have been some studies on the mechanism of CaMKII and inflammatory pathway regulation –53. Incrhythmias , 57.WXKL is the first antiarrhythmic Chinese medicine to be approved by the state. Our previous studies indicated that WXKL treatment considerably preserves cardiac function and inhibits arrhythmia by modulating the CaMKII signaling pathway , 23. MorIn conclusion, the present study demonstrated that siCaMKII attenuates Ang II-induced CH, which may be partially associated with the downregulation of the CnA-NFAT and MyD88 signaling pathways, and WXKL had a similar effect. Furthermore, siCaMKII may be a promising approach to attenuate the progression of CH and arrhythmia. Our studies define the crosstalk between the CaMKII and CnA-NFAT signaling pathways in vitro, so numerous biological studies are needed for further research in vivo. This will improve the understanding of the mechanisms underlying hypertrophy and may provide evidence for drug application in the treatment of CH.