Great interest and promise are held by human pluripotent stem cell (hPSC) technology to generate functional pancreatic beta cells from diabetic patient-derived stem cells to replace the dysfunctional cells, thereby compensating for insulin deficiency and reducing the comorbidities of the disease and its associated financial and social burden on the patient and society. Beta-like cells generated by most current differentiation protocols have blunted functionality compared to their adult human counterparts. The Ca2+ dynamics in stem cell-derived beta-like cells and adult beta cells are summarized in this review, revealing the importance of proper Ca2+ homeostasis in beta-cell function. Consequently, the importance of targeting Ca2+ function in differentiation protocols is suggested to improve current strategies to use hPSCs to generate mature and functional beta-like cells with a comparable glucose-stimulated insulin secretion (GSIS) profile to adult beta cells.Diabetes mellitus is a chronic disease affecting over 500 million adults globally and is mainly categorized as type 1 diabetes mellitus (T1DM), where pancreatic beta cells are destroyed, and type 2 diabetes mellitus (T2DM), characterized by beta cell dysfunction. This review highlights the importance of the divalent cation calcium (Ca The American Diabetes Association (ADA) has classified diabetes into primarily two categories: type 1 diabetes (T1DM) and type 2 diabetes (T2DM) [Diabetes mellitus, commonly known as diabetes, is a collection of chronic and metabolic disorders typically observed with heightened blood glucose levels over an extended period. Insulin deficiency leading to impaired glucose metabolism is widely deemed to be the mechanism for the onset and effect of the disease ,2. The Is (T2DM) . T1DM iss (T2DM) ,5,6. T2Ds (T2DM) . It is ns (T2DM) . Unhealts (T2DM) . Diabetes (T2DM) ,11,12,13T1DM treatment currently involves controlling glycemia through daily insulin supplementation through insulin injections or insulin pumps with integrated glucose monitors ,15. Alth2+)-dependent process. Therefore, defects in calcium signaling could be one of the underlying reasons for the immature and non-functional phenotypes observed in differentiated hPSC-derived pancreatic beta cells in vitro. In this review, we first describe the role of calcium signaling in pancreatic beta cell function and secondly highlight and summarize recent progress on the implications of impaired calcium signaling in defective insulin secretion in hPSC-derived β cells, and consequently, how targeting calcium signaling might be a new and interesting strategy to improve in vitro differentiation into functional hPSC-derived pancreatic beta cells for diabetes therapy. The methods used in this review are as follows: First, we carried out a comprehensive search of peer-reviewed original and review articles using the PubMed database and Google Scholar based on a wide range of key words linking the involvement of calcium signaling in insulin secretion, hPSCs, and diabetes. Secondly, the reference section for each selected article was searched to find additional articles to further develop this review.Recent progress in the field of regenerative medicine has focused on the generation of surrogate and transplantable functional pancreatic beta cells from human pluripotent stem cells (hPSCs) to treat diabetes. Patient-specific pluripotent stem cell (PSC)-derived beta cells, also known as autologous PSC-derived beta cells, would help circumvent the inadequate islet supply and/or allogeneic immune rejection, thereby being an unlimited source of beta cells for diabetes therapy. More importantly, patient-specific stem cell-derived beta cells can also be used in vitro to study diabetes-related mutations such as inherited monogenic diabetes as well as disease progression ,25. To t2+ has a critical function in the controlled release of insulin from beta cells.Systemic glycemia is carefully regulated by the precise production and release of insulin by beta cells. The interplay between blood glucose and numerous other secretagogues such as neurotransmitters, hormones, and other compounds regulates this function ,28. As w2+ into the cell. This wave of Ca2+ triggers insulin exocytosis from secretory granules [In the postprandial state, an elevation of blood glucose is detected by beta cells, which in turn take glucose into the cell through glucose transporters (GLUT1 in humans and GLUT2 in rodents). Glycolysis is triggered as glucose is phosphorylated by glucokinase into pyruvate. Pyruvate thus formed is transported into the mitochondria and enters the tricarboxylic acid (TCA) cycle. A cascade of events in the TCA cycle enhances ATP production. This increase in the ATP/ADP ratio within the cell inhibits ATP-sensitive K+ channels, causing membrane depolarization. L-type calcium channels are opened because of this depolarization, leading to an influx of Cagranules ,30.2+ is due to the influx of extracellular Ca2+, several intracellular organelles, including the endoplasmic reticulum (ER), nucleus, Golgi apparatus, and mitochondria, contribute to the regulation of Ca2+ within beta cells [2+ homeostasis in β-cells is shown in Although a major share of the intracellular Cata cells ,32. A su2+ store, and effects its release [2+ uptake and release are mainly dependent on inositol 1,4,5-triphosphate receptors (IP3R), ryanodine receptors (RyR), and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA).The endoplasmic reticulum performs crucial functions such as protein folding and lipid synthesis, acts as a Ca release . Endopla2+ from the ER through a positive feedback process named “calcium induced calcium release” (CICR). An increase in cytosolic Ca2+ triggers the opening of RyRs, which leads to an efflux of Ca2+ from the ER into the cytoplasm. The Ca2+ thus released by the ER facilitates the opening of more RyRs, hence amplifying the ER-dependent Ca2+ release [2+ in human beta cells without compromising GSIS activity [RyR receptors release Ca release . Three i release ,36. Know release ,38. Alth release ,40. Addiactivity . RyR stuactivity ,43.2+ from Ca2+-stores and plays a significant role in the release of Ca2+ from the ER [2+ release from the ER. Heightened cytoplasmic Ca2+ levels achieved by the inhibition of SERCA have been shown to accelerate CICR due to the activation of IP3Rs [2Y1, which promoted ER Ca2+ release through IP3Rs, thereby increasing cytoplasmic Ca2+ levels [2+ release is through the activation of the G-protein-coupled receptor GPR40 by free fatty acids. Fatty acid binding to GPR40 appears to activate the Gαq/11-protein complex, leading to the production of IP3 and subsequent ER Ca2+ release. However, studies have also attributed the production of diacylglycerol as a determining factor in the second phase of insulin secretion [2+ release from the ER [IP3R are IP3-sensitive receptors found on the ER membrane of many cell types, such as the cerebellum, smooth muscles, cardiac and skeletal muscle, as well as beta cells. IP3 is formed by the activation of phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 and diacylglycerol. IP3 is capable of mobilizing Cam the ER ,45. Glucm the ER . Studiesof IP3Rs . CICR, iof IP3Rs . In rode+ levels . Anotherecretion ,51,52,53m the ER . Geneticm the ER .2+ released from the ER is sequestered back into the ER lumen through the activity of SERCA pumps in an ATP-dependent manner. This helps maintain a resting cytosolic Ca2+ concentration of 25–100 nM and a high ER Ca2+ concentration (200–500 µM). Alternative splicing of the genes encoding SERCA generates fourteen different isoforms of SERCA. In beta cells, the predominant isoforms are SERCA2b and SERCA3 [2+ reuptake [Cad SERCA3 ,57,58. Treuptake . The impreuptake . SERCA dreuptake .2+ mobilizing messenger produced by the ADP-ribosyl cyclase CD38 by a base exchange reaction between nicotinamide adenine dinucleotide phosphate (NADP) and nicotinic acid [2+ spiking, proving NAADP to be an influential player in glucose-mediated insulin release [2+ release from acidic compartments such as lysosomes and insulin granules [2+. Their main function is the degradation of macromolecules, but they also play a role in Ca2+ regulation in cells. The nanomolecular concentration of NAADP triggers lysosomal Ca2+ release, while micromolecular NAADP concentrations inhibit Ca2+ release [2+ from the lysosome through two-pore channels (TPCs), particularly TPC2. However, recent studies have questioned TPC2 as the major NAADP receptor and suggested TPC1 as the main NAADP receptor instead. Although TPC1 and TPC2 are both known to show high sensitivity to NAADP, studies on knockout mouse models have revealed conflicting results, confounding the identity of NAADP’s primary target receptor [Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Canic acid . Elevatenic acid ,64. In pnic acid . Inhibit release ,67. The granules . Lysosom release . NAADP ireceptor ,69,70,71receptor .2+ second messenger, cADPR, by the cyclization of beta-nicotinamide adenine dinucleotide. Early studies have suggested that glucose stimulation generates cADPR in pancreatic islets. Follow-up studies by the same group observed that overexpression of CD38 in transgenic mice increased glucose and ketoisocaproate-mediated insulin release, while this effect was impaired in CD38 knockout mice [2+ release from the ER through RyRs. However, evidence suggests that cADPR does not directly interact with RyR, indicating the presence of accessory proteins that act as links between cADPR and RyR. FK506-binding protein 12.6 (FKBP12.6), calmodulin, and GAPDH have been proposed as potential accessory proteins that evoke Ca2+ release by cADPR [2+ from the ER is currently unknown. cADPR has also been shown to trigger Ca2+ influx and subsequent insulin secretion by activating the plasma membrane channel TRMP2 [2+-releasing messenger, its Ca2+ mobilizing effects are controversial since numerous studies have failed to find an action of cADPR in beta cells [Adenylyl cyclases such as CD38 are also responsible for the production of another Caout mice ,74,75. cby cADPR ,77,78,79el TRMP2 . Althougta cells ,81,82. Ita cells . Further2+ influx and insulin exocytosis. A close association between Ca2+ homeostasis and beta cell function has been connected to mitochondrial function [2+ showed impaired insulin response to high glucose challenges, thereby linking the tight regulation of Ca2+ and mitochondrial energy metabolism [2+, namely, pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase, and serve as crucial factors for insulin release in beta cells. The influx of Ca2+ into the mitochondria is believed to be essential for the stimulation of Ca2+-dependent dehydrogenases of the TCA cycle to control ATP production under increased glucose conditions [Another organelle crucial for effective GSIS is the mitochondria. The major energy-producing mechanism of the mitochondria is the TCA cycle, where the electron transport chain produces ATP. ATP generated by mitochondria promotes plasma membrane depolarization, leading to Cafunction . Studiestabolism ,86. Threnditions ,87,88,892+ entry into the mitochondria occurs in the outer mitochondrial membrane and inner mitochondrial membrane through voltage-dependent anion channels (VDACs) and mitochondrial Ca2+ uniporters (MCU), respectively [2+ uptake, leading to impaired ATP production and insulin secretion. Moreover, the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption were also lowered. These studies underlined the importance of MCU-mediated Ca2+ regulation for sustained ATP production and metabolism-secretion coupling in beta cells [2+ uptake and GSIS were impaired [2+ by beta cell mitochondria have been implicated in the tone and frequency of cytosolic Ca2+ oscillations and might perhaps contribute to insulin release pulsatility [2+ has also been linked to the activation of the inner membrane channel, the mitochondrial permeability transition pore (PTP). PTP is believed to perform two distinct roles in beta cells, depending on its activation or inhibition. The activation of PTP is required to promote glucose-dependent insulin secretion, while its inhibition is necessary to protect against glucotoxicity and hypoxia [2+ extrusion from mitochondria is also key to mitochondrial and cell function. Persistent accumulation of Ca2+ in the matrix space can cause Ca2+ overload in the mitochondria, leading to the opening of PTPs and, thereby, initiating cell death signals [2+ homeostasis, Ca2+ extrusion from the mitochondria occurs via the Na2+/Ca2+ exchanger (NCLX) [2+ responses and the rate of GSIS during the first phase of insulin secretion [Caectively ,91. Expeta cells ,93. Simiimpaired . The uptsatility ,95. Mito hypoxia ,96,97,98 signals . To mainr (NCLX) . Studiesecretion .Insulin-positive beta cells are first seen around embryonic day 13.5 in mice and weeks 8–9 in humans. During the earlier days of fetal growth, endocrine progenitor cells differentiate into beta cells. However, during the late neonatal stages, beta cells are formed by replication ,103,104.ATP channel-opening drug, reduced the replicative capability of beta cells, thereby suggesting that the requirement for the closure of KATP channels and subsequent depolarization is critical for beta cell replication. Reciprocally, when mice were injected with the KATP channel-blocking drug glyburide, beta cell proliferation was rescued. Therefore, Ca2+ influx appears to play an important role in beta cell replication [2+ and calcium channels in beta cell replication.A study revealed that a signaling pathway linking membrane depolarization was a key factor for beta cell replication in mice. Diazoxide, a Klication . Another2+ concentrations switches on calcineurin, which in turn dephosphorylates NFAT. This leads to the translocation of NFAT into the nucleus and the transcription of multiple genes responsible for beta cell function, glucose sensing, and proliferation [NFATC1, NFATC2, NFATC3, and NFATC4 are present, with NFATC2 and NFATC3 being the most abundant [2+/calmodulin-dependent kinase (CaMK). A study observed that CaMK4 inhibition eliminated beta cell proliferation, whereas constitutively active CaMK4 expression increased beta cell proliferation via a cAMP response element binding protein (CREB) and insulin receptor substrate 2 (IRS-2) mechanism [A known mechanism of beta cell replication is via calcineurin/nuclear factor of activated T-cells (NFAT). Calcineurin/NFAT signaling is central to the regulation of neonatal pancreatic development in mice and human islets. NFATs are activated by the serine/threonine phosphatase, calcineurin. Increasing Caferation ,107,108.abundant . In mousabundant . Interesabundant ,112. Anoabundant . The calabundant ,114. Betechanism .2+ handling and beta cell survival in pancreatic beta cells. High glucose enhances beta cell survival in a Ca2+-mediated manner. In cultured mouse islet beta cells and MIN6 insulinoma cells, a three-fold decrease in apoptosis was found in the presence of high glucose concentrations compared to low glucose concentrations. Blocking of the L-type Ca2+ channel with nifidepine reversed the protective effects of high glucose, suggesting the requirement of Ca2+ influx for beta cell survival at high glucose concentrations [2+ in beta cells, along with an insulin dose-dependent decrease in SERCA2 expression. Prolonged high insulin exposure also triggered apoptosis in beta cells through Caspase-12  and a known mediator of ER stress) and correlated with an increase in intracellular Ca2+. This suggests that prolonged insulin exposure can disrupt ER function and induce apoptosis in beta cells. However, further studies need to be undertaken to deduce the mechanisms of these processes and the interplay between the protective and destructive effects of high glucose and insulin, respectively [2+ depletion-mediated ER stress has been associated with beta cell apoptosis in numerous studies. A study reported that Ca2+ depletion from the ER through dysfunctional SERCA2b, IP3Rs, and RyRs led to beta cell death and suggested a calpain-2-mediated pathway as the mechanism. The authors discussed the possibility of pharmacological modulations of the ER channels as a potential for beta cell survival [2+ depletion and increase cytosolic Ca2+ in beta cells. This led to beta cell death, marked by an increase in caspase-3 and caspase-7 activity levels. In INS-1 cells, cytokine-mediated beta cell apoptosis was inhibited by treating the cells with the calcium-modulating compounds dantrolene and sitagliptin, which restored the function of SERCA and the ER Ca2+ pump and decreased the levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP) [2+ homeostasis. Heightened palmitate levels increase oxidative conditions, leading to Ca2+ depletion from the ER, which interferes with chaperones and results in misfolded proteins. This triggers a series of events where cytosolic and mitochondrial Ca2+ homeostasis is disrupted, thereby leading to beta cell apoptosis [A multitude of studies have found a close association between Catrations . A recenectively . Ca2+ desurvival . Inflamm (TXNIP) . Free fapoptosis ,121. 2+ in beta cell survival was seen when CAMK4 was shown to regulate apoptosis. Constitutively active CAMK4 was shown to significantly improve beta cell survival as a reduction in caspase-3 and caspase-7 was observed. Moreover, increased apoptosis was observed in the presence of the dominant negative form of CAMK4. The authors proposed CAMK4 and its association with CREB as the primary mechanisms for improved beta cell survival. The activation of CAMK4 under intracellular Ca2+ elevations led to the upregulation of IRS-2 via CREB, leading to the regulation of beta cell survivability [Calcineurin-dependent mechanisms have also been implicated in beta cell viability. Pharmacological inhibition of calcineurin is necessary to prevent organ transplant rejection and is associated with post-transplant beta cell failure. A study on human islets revealed that beta cell apoptosis was significantly increased in the presence of the calcineurin inhibitor tacrolimus. The mechanism of these effects linked calcineurin to IRS-2 and the phosphoinositide-3-kinase protein kinase B/Akt (PI3K-PKB/Akt) pathway. It was suggested that the effect of calcineurin on IRS-2 was also mediated partly through NFAT . More evvability .2+ and its signaling pathways are critical to the proper function and survivability of pancreatic beta cells. As important as Ca2+ is for beta cell function, survival, and proliferation, it also plays an important role in insulin production and secretion. A plethora of studies have strongly linked Ca2+ dysfunction to diabetes in both human and animal models [As clearly highlighted in the earlier sections of this review, Cal models ,126,127.Abcc8 (−/−Abcc8 mice), a key component of the beta cell KATP channel, showed sustained membrane depolarization, leading to continued elevation of Ca2+ associated with dysfunctional genes involved in Ca2+ transport, signaling, and beta cell maintenance. Additionally, beta cells were observed to dedifferentiate and transdifferentiate into pancreatic polypeptide cells, as seen through the increased expression of the Aldh1a3 gene (a marker for dedifferentiating beta cells). Interestingly, the expression of Aldh1a3 was reduced by treating the cells with Ca2+ channel blockers, solidifying the link between sustained depolarization and beta cell functional and identity loss [2+ influx, and calcineurin signaling that could also be reversed by the normalization of glycemia [HNF1A gene, whose deficiency impaired a network of genes crucial to calcium signaling, GSIS, and beta cell fate. These cells were then target-differentiated into beta cells. Stem cell-derived beta cells from HNF1A knockouts showed a reduction in the SYT13 gene, which is responsible for calcium-regulated granule exocytosis. HNF1A deficiency also showed a developmental bias toward the generation of alpha cells, as an upregulation of glucagon was observed in the knockout cells. The observations from the knockout cells were comparable with those from the induced pluripotent stem cell (iPSC) line with HNF1A-MODY patient-specific mutations (R200Q) [2+ influx. These pools of insulin granules were absent in T2DM donors and INS-1 cells mimicking diabetes, implicating the role of these Ca2+-triggered granules in the loss of rapid first-phase insulin secretion seen in T2DM patients [vβ3 (a voltage-gated Ca2+ channel subunit) expression, coupled with impaired calcium signaling and insulin secretion, and these effects were reduced in high-fat diet-induced diabetes in Cavβ3 deficient mice. Overexpression of Cavβ3 in human islets also showed impaired GSIS [Studies have suggested the deleterious effects of long-term activation of calcium signaling pathways in beta cells. Calcineurin is a vital component of the calcineurin/NFAT pathway, which is critical to beta cell replication. However, chronic depolarization of beta cells increased calcineurin activity, which in turn reduced glucose tolerance, insulin secretion, and beta cell replication in mouse models . Anotherity loss . Anotherglycemia . A more  (R200Q) . High-repatients . Diabetired GSIS ,133.2+ overload was the main mechanism of glucolipotoxicity-mediated apoptosis and dysfunction, which was inhibited by compounds that decreased glucolipotoxicity-mediated Ca2+ influx into the cells [2+ [db/db mice and cultured human islets [2+ stores in INS-1 cells [Glucolipotoxicity is an umbrella term to describe the detrimental effects of elevated glucose and fatty acid levels on beta cell function and survival. Glucolipotoxicity-induced beta cell failure and subsequently T2DM are associated with calcium dysregulation and ER stress, along with other non-calcium-related complications. A phenotypic screening study of compounds showed that glucolipotoxicity stress had a close association with calcium flux modulation and apoptosis. It was suggested that a Cahe cells . The ubihe cells ,137,138.ells [2+ . A phaseells [2+ . Free fan islets ,142. The-1 cells .As discussed in the introduction section above, insulin-secreting beta cells from the pancreatic islet of Langerhans play a crucial role in the body by maintaining glucose homeostasis. It is well established that destruction or loss of beta cell mass leading to a negative impact on effective insulin secretion has emerged as the main pathogenic factor in both T1DM and T2DM . CurrentDirected differentiation protocols are created from an extensive study of the developmental phases of cell types. The conversion of pluripotent stem cells into target cells is achieved by the timely addition of small molecules and growth factors in a stagewise manner that mimics the embryonic development of the desired cell type. The success of the protocol is usually determined by the extent to which these cells mirror their in vivo counterparts with respect to functional maturity and efficiency. Indeed, over the past decade, these strategies have been applied to differentiate human iPSCs and ESCs into pancreatic beta cells and islets. Pioneering work from the Melton laboratory at Harvard University has paved the way for protocols that generate beta cells with moderate efficiency. However, multiple labs have published protocols with more optimized culture conditions for differentiation media and small molecules, with the resulting protocols applicable to multiple iPSC cell lines for improved differentiation efficiency and beta cell functionality ,150,151.2+ concentration, indicating that increased Ca2+ influx is a cellular function required for GSIS. Since evidence has highlighted that Ca2+ dysregulation in beta cells has a key role in diabetes, it is possible that functionally defective Ca2+ dynamics play a key role in the generation of immature and non-functionally differentiated hPSC-derived beta cells in vitro. A study used the human ESC (hESC) cell line H1 to create a seven-stage protocol to generate beta cells. The cells thus obtained expressed key markers of mature beta cells and displayed GSIS capabilities. However, as compared to adult beta cells, the differentiated cells showed reduced amplitudes and a slower time to peak of the Ca2+ signals. Sustained Ca2+ transients were observed even after the glucose stimulation ceased, a marked difference from mature beta cells. Similar effects were seen with respect to insulin release on glucose challenges, in which insulin secretion did not return to pretreatment levels and KCl-mediated insulin release was blunted. Direct depolarization with high KCl concentrations revealed the presence of voltage-gated Ca2+ channels and normal Ca2+ efflux pumps, thereby suggesting that the generated beta-like cells had functional immaturity compared to adult beta cells [2+ into the ER, thereby impairing ER function and thereby Ca2+ homeostasis in beta cells [v subunit gamma 4 (Cavγ4) in a CaMKII-mediated manner in controlling beta cell glucose homeostasis [2+ levels and ATP production, leading to impaired GSIS in islets [2+ handling in the differentiating cells and might explain the difficulty in generating beta cells functionally similar to adult human beta cells. One cannot assume that impaired calcium signaling is the major contributor to the formation of immature insulin-producing cells from iPSCs. Impaired expression of genes responsible for potassium channel regulation and mitochondrial function was also found in immature iPSC-derived beta-like cells [Beta cells facilitate GSIS through increased cytosolic Cata cells . Anotherta cells . The autta cells ,156,157.ta cells . A threeta cells . MafA, ata cells ,160,161.ta cells . A receneostasis . Insulineostasis . Anothereostasis . Studiesn islets . Interesn islets . The altke cells ,26. Perh2+ channels in the eBCs. Transcriptome analysis comparing non-enriched clusters and eBCs showed an upregulation of the SCGN gene, which is responsible for calcium sensing. Moreover, genes upregulated in neonatal mouse and human fetal beta cells were seen to be upregulated in the immature beta cells, providing further evidence that immature iPSC-derived beta cells resemble fetal cells more than adult beta cells [Nevertheless, more recent publications have generated beta cells with comparable functionality to human islets by incorporating enrichment and reaggregation of late-stage differentiated beta-like cells for better GSIS functionality ,166. Cel2+ handling in the maturation of beta cells during their development phase in in vitro conditions, suggesting that the calcium signaling pathway is an important puzzle piece in the generation of iPSC-derived pancreatic beta-like cells. Careful considerations need to be made in the differentiation protocols to include small molecules and growth factors in the differentiation media that target the calcium signaling pathways and their components in a timely manner to generate fully functional beta cells in vitro. Current strategies have been built upon accumulating knowledge over the past decade to produce functional beta cells, and more research needs to be performed to generate high-quality iPSC-derived beta cells that resemble human beta cells.Taken together, these studies that compare the differences between iPSC-derived immature beta-like cells and iPSC-derived beta cells functionally closer to human beta cells show the importance of appropriate CaHNF1A gene on the onset of MODY. CRISPR/Cas9 was used to generate HNF1A mutations in the hESC Mel1, which was subsequently differentiated into beta cells. The resulting beta cells were observed to have impaired GSIS and intracellular calcium levels [ATP channel in congenital hyperinsulinism. These cells were target-differentiated into beta cells and used as controls to study the effect of the mutation on beta cell function [WFS1) in IPSCs generated from a patient with Wolfram syndrome. The IPSCs were differentiated into beta cells, and it was observed that the corrected WFS1 IPSC-derived beta cells showed strong dynamic insulin secretion and reversed streptozocin-induced diabetes following transplantation into mice [An important tool to better understand diabetes and its progression is the integration of gene editing technology (CRISPR/Cas9) with stem cell technology. Several studies have reported the use of CRISPR/Cas9 to create stem cell-based disease models of monogenetic diabetes, MODY, and neonatal diabetes, among others, to better understand the mechanisms of disease progression ,169,170.m levels . Moreovem levels . A studynto mice . Thus, C2+ in the functioning of pancreatic beta cells. The role of several Ca2+-handling organelles have been discussed through recent studies, which highlight that the disruption of key Ca2+ pathways could lead to beta-cell dysfunction and thereby play a role in the onset of diabetes. We examined the key advances made in the field of stem cell technology in the study of diabetes. A comparative study of Ca2+ handling characteristics of stem cell-derived beta-like cells and their adult human counterparts was performed, showcasing the pitfalls of the current differentiation protocols and underlining key research strategies targeting Ca2+ dynamics to improve the identity and functionality of directed stem cell differentiation. Massive strides are being made to achieve the goal of generating functional beta-like cells from stem cells. Advances in the efficiency of iPSC differentiation protocols can close the gaps in the structural and functional differences between stem cell-derived pancreatic and human islets. This opens the door to fully using stem cell-derived beta cells for drug screening due to their enhanced kinetics, and the mechanisms of beta cell failure can also be studied, paving the way to greatly improve our knowledge of the intricate aspects of diabetes and its treatment, including transplantation in patients with diabetes [In this review, we emphasize the role of Cadiabetes ,166,172.


Overweight is a growing problem in dogs worldwide and negative health effects associated with excess body weight are common. The body condition score (BCS) scale is a time- and resource-effective method to assess if a dog is overweight, but its precision among dog owners has been found to vary. The aim of this study was to investigate dog owners’ perceptions of various body compositions in dogs and evaluate if a short education on how to use the 9-point BCS scale might change these perceptions. This study included one survey and one clinical study of Swedish dog owners. In the indirect assessment based on photos, normal-weight dogs were underestimated by three-quarters of dog owners, and about half of the dog owners underestimated overweight dogs. Before receiving the standardized education, one-third of the owners underestimated the body composition of their own dogs, mainly for dogs with excess adiposity. The dog owners responded well to the practical education given and, thereafter, performed assessments comparable to veterinary health care personnel. These results indicate that perception of what an “ideal weight” dog should look like is sliding and that the ability to identify overweight dogs might be limited when owners evaluate body composition without previous education.p < 0.0001) and performed as accurately as the veterinary health care personnel (p = 0.99). The results should be verified in the broader dog owner population based on a randomized selection of participants. “Weight blindness”, defined here as an underassessment of normal-weight dogs and an inability to identify overweight dogs, is likely to have a negative impact on canine overweight prevalence. Deeper knowledge about dog owners’ perceptions can inform the development of new strategies to help prevent and manage canine overweight, whereof standardized practical education on BCS assessment is shown here to be one example.Overweight in dogs is an increasing problem, with a prevalence of about 30% in Sweden. To prevent the negative health effects of overweight, it is important to identify and treat canine overweight. Dog owners are essential for such interventions. The aim of this study was to evaluate dog owners’ perceptions of various canine body compositions via indirect assessment based on photos and direct assessment of their own dogs. A second aim was to evaluate the effect of a standardized practical education for dog owners on body condition score (BCS) assessment of their own dogs. The 9-point BCS scale was used, and two study samples were recruited: one was a survey sample where 564 dog owners assessed the BCS of dogs using photos, and one sample was a separate clinical sample where 82 dogs were assessed by their owners and by veterinary health care personnel. The initial BCS assessment by the dog owners in the clinical sample (mean ± SD) was significantly lower (4.6 ± 1.0) than the BCS assessed by the veterinary health care personnel (5.2 ± 1.1), but the owners improved significantly after receiving the standardized education (5.1 ± 1.0) (both  Canine obesity is highly prevalent and an increasing problem in various countries ,3,4,5,6.Body weight alone is not sufficient to conclude if a dog has an ideal body condition or is under- or overweight as body weight is constituted of both lean and fat mass ,18, and The definition of canine obesity as a disease is a relatively new concept. In 2019, the Global Pet Obesity Statement proposed a uniform classification of canine obesity and emphasized the importance of spreading education on how to use the 9-point BCS scale to accurately assess body condition . It has Dog owners are an important and innate target group for educational efforts not only because they are the everyday caretakers of dogs but also because of the shared lifestyle with their pets ,29,30,31This study included two different study samples of Swedish dog owners: one sample where data were collected through an online survey (“survey population”), and one sample where dog owners and their respective dog/dogs participated in an experimental study . All human participants in the survey, as in the clinical population, had to be 18 years or older. Both study samples were non-probability samples  based on voluntary participation.An online survey was distributed to Swedish dog owners and was available online through social media for seven days. It contained questions on the responders’ knowledge about the BCS system  The dogs had a BCS of 5–8  and were photographed from above in a standardized setting (in the same room and position and by the same photographer).A clinical study that investigated the accuracy of in vivo body condition assessment by dog owners was completed with Swedish dog owners and their dogs, who were recruited through social media from three regions located in southern, middle, and northern Sweden . The participating dog owners participated with one or several dogs. The dogs were allowed to be of any breed, sex, age, neuter status, and health status, but they were obligated to show no signs of aggression whilst being handled by strangers. The clinical study was performed by two veterinary nursing students in their last semester of their Bachelor Veterinary Nursing Program at the Swedish University of Agricultural Sciences, Uppsala. The students had received previous education on BCS assessment and specific BCS calibration with an experienced BCS assessor (J.S.) before data collection. All individual dogs were assessed for BCS three times: two times by their respective owner (before and after the standardized education) and one time by of one of the two students. The students will hereafter be referred to as “veterinary health care personnel” in this publication.The clinical sample’s data collection started with investigating the owners’ perceptions through a direct assessment of their own dogs based on predefined oral descriptions, where the dog owners described the body composition of their dogs as “underweight”, “slightly underweight”, “normal weight”, “slightly overweight”, “overweight”, or “obese”. Thereafter, the dog owners received a standardized BCS education where they were shown the 9-point BCS scale from World Small Animal Veterinary Association  in SwediThe data related to dog owners (survey population) or to dog owners and their dogs  were analyzed descriptively as proportions, percentages, and/or by using statistical models.For the survey sample, the expert who had previously performed the in vivo BCS assessment of the dogs in the photos was considered “the primary investigator”, and the expert’s scores were compared with the indirect assessment based on photos performed by the dog owners (the survey population did not receive any education). Free-text answers in the survey regarding negative consequences for overweight dogs stated by the dog owners were grouped as “the locomotor apparatus”, “heart- and coronary disease”, “insulin resistance/diabetes mellitus”, “organ related disease”, “less physically active”, “increased risk for diseases or injuries”, “shortened lifespan”, “reduced quality of life”, and “other negative effects”.For the clinical sample, the veterinary health care personnel were considered to be the primary investigators, and their scores were compared with the direct assessment performed by the owners on their own dogs based on predefined oral descriptions and the owners’ BCS assessment after the standardized education. The predefined oral descriptions were translated into BCS scores for data processing to enable statistical analyses. The translation was made as follows: “underweight” (BCS = 1.5), “slightly underweight” (BCS = 3), “normal weight” (BCS = 4.5), “slightly overweight” (BCS = 6), “overweight” (BCS = 7), and “obese” (BCS = 8.5) . The datp < 0.05, and the results are presented as mean ± SD or mean ± SEM.For statistical analyses, the software GraphPad Prism  and SAS  were used. All data were normally distributed based on an evaluation of the appearance of residuals in SAS or based on D’Agostino and Pearson omnibus normality test in GraphPad Prism. The level of significance for all statistical analyses was set to For the indirect assessment based on photos, a Chi-square test for trend was used to analyze differences in proportions  among all survey participants and all four dogs shown in the photos . In this analysis, the exact score set by the primary investigator was considered the “equivalent assessment”.A Chi-square test for trend was also used to analyze differences in proportions  between owner age groups  regarding the normal-weight dog (BCS = 5) and the obese dog (BCS = 8). A Chi-square test for trend was also used to analyze differences in proportions  between groups of owners with varying knowledge of the BCS scale (answers were grouped as “Yes” or “No” based on familiarity) regarding the normal-weight dog (BCS = 5) and the obese dog (BCS = 8). In the analyses including the factors “owner age” and “previous knowledge”, BCSs of 4–5 and BCSs of 8–9, defined based on the scoring scale of the BCS for normal-weight and obese dogs , were coThe body condition assessments collected from the clinical sample were evaluated using a mixed model random analysis in SAS , where bA Chi-square test for trend was used to analyze differences between the selected explanatory factors: “number of previously owned dogs”  and “owner age” . Linear regression analysis was used to analyze the correlation between owner age and number of previously owned dogs as continuous variables.t-tests were used to analyze the effects of dog intrinsic factors, such as age, gender, and neuter status, on BCSs. Linear regression analysis was used to analyze the association between dog age and BCS . Student’s t-tests were used to compare the BCSs of male and female dogs as well as the BCSs of intact and neutered dogs.Linear regression analysis and Student’s In total, 952 dog owners started the online survey and 564 dog owners completed the questionnaire. The respondents were predominantly women (96%), and the most represented age group was 41 to 60 years of age (45%). Upper secondary school (50%) was the most common educational level, and the largest professional categories were human health care (21%) and veterinary health care (12%). The respondents had previously owned (mean ± SD) 7.0 ± 6.7 dogs (range 1–30). Forty percent (224/564) answered “yes” to the question if they knew what the BCS is and what it is used for. Of those respondents with self-perceived knowledge of the BCS, 84% correctly described the term related to body fat content in an open-ended follow-up question. Nine percent described that the term was related merely to body weight, and 7% described that the term was related to overall health. The descriptive statistics of the survey sample are shown in Almost all survey respondents (99%) agreed that being overweight may lead to negative consequences for dogs. In the open-ended follow-up question, the respondents were asked to give a few examples of negative consequences, and 1061 free-text answerers were recorded. The answers were grouped according to type of problem described, and the two largest groups were “joint problems” (46%) and “heart- and coronary diseases” (23%). The least frequent groups were “a shortened life span” (4%) and “a reduced quality of life” (1%) .p < 0.0001), where the normal-weight dog was the dog that was most commonly underestimated, followed by the slightly overweight dog, while the obese dog was the dog that was most commonly accurately assessed, when compared to the exact in vivo assessment of the same dogs performed by the primary investigator (which was considered the gold standard method in the survey sample) , and about half of the respondents could not identify a slightly overweight (BCS = 6) or an overweight dog (BCS = 7). On the contrary, as many as 71% could correctly identify an obese dog (BCS = 8) with an exact score or higher . Among a sample) .p = 0.0014). In the age group of 61+ years, the dog with a BCS of 5 was given a mean score of 3.5, and 46% assessed the normal-weight dog as being underweight (BCS = 1–3). For the obese dog (BCS = 8), the different age groups did not differ in proportion for equivalent assessment or underassessment (p = 0.15). The respondents with no previous knowledge of the BCS scale showed a significantly lower proportion (61%) in terms of equivalent BCS assessment for the normal-weight dog (BCS = 5) compared to the respondents with previous knowledge (77%) (p < 0.0001). For the obese dog (BCS = 8), previous knowledge of the BCS scale did not affect the proportion of equivalent assessment or underassessment (p = 0.15). Within all age groups, having “no previous knowledge” of the BCS scale was numerically more frequent than having “previous knowledge” (p = 0.09).For the survey respondents, both “age group” and “previous knowledge” of the BCS scale had an effect on the accuracy of owner perceptions in the indirect assessment based on photos. The respondents aged 61+ years showed a significantly lower proportion (51%) in terms of equivalent BCS assessment for the normal-weight dog (BCS = 5) compared to the other age groups (64–75%)  showed systematic patterns in their assessment of their dogs, which were defined as constant under-assessment, equivalent assessment, or over-assessment.The gender distribution is presented in p < 0.0001) . Owners with no previous experience of dog ownership were few and were almost exclusively from the youngest age group, and all owners aged 38–55 years had previously owned at least one and up to 12 dogs  of the observations, overestimated their dogs in 4/82 (5%) of the observations, and performed equivalent assessment in 49/82 (60%) of the observations. Of the underestimated dogs, 21/29 (72%) were overweight (BCS = 6–7), 6/29 (21%) were of normal weight (BCS = 4–5), and 2/29 (7%) were underweight (BCS = 3). Owner perceptions, in terms of the mean BCS based on predefined oral descriptions and no further education, were significantly lower (4.6 ± 1.0) than the BCS assessed by the veterinary health care personnel (5.2 ± 1.1) in the overall comparison ( 0.0001) . An anal 0.0001) a and own 0.0001) b were eq 12 dogs .p < 0.0001) compared to their previous assessment (p ≥ 0.89)  . After tp < 0.0001), and increasing age of the owners showed a weak positive correlation  with increasing number of owned dogs.The tabulation of the two explanatory factors, “owner age groups” and “number of previously owned dogs”, is shown in p = 0.02, R-squared = 0.12). Female dogs had a (mean ± SD) BCS of 5.4 ± 1.0, which did not differ from the BCS of male dogs of 4.9 ±1.1 (p = 0.07). Both male and female neutered dogs had a significantly higher (mean ±SD) BCS of 5.7 ± 0.9 compared to intact dogs with a BCS of 5.0 ±1.1 (p = 0.006).Of all participating dogs, 30/82 (37%) were assessed as being overweight  and overweight ), 57% was assessed as being of normal weight (BCS = 4–5), and 6% was assessed as being underweight (BCS = 2–3) by the veterinary health care personnel. There was a weak positive association between dog age and BCS, where the BCS of participating dogs increased with increasing dog age  compared with the other dogs (black). All dogs shown in the photos in the survey were, however, Labrador Retriever dogs of the type for shows, were assessed in vivo by the same primary investigator, and were photographed in a standardized setting. The lighter color could perhaps have contributed to the obese state being more obviously spotted. However, coat color has previously been shown to have no effect on the assessment of BCS when comparing visual assessment using photos to in vivo assessment of the same dogs .The method of visual BCS assessment using photos has been shown to be moderately to highly correlated with in vivo assessment ,41, but In the indirect assessment using photos, dog owners who had no previous knowledge of the BCS scale and/or were of older age were less likely to perform assessment that was equivalent to the primary investigator, especially in the assessment of the normal-weight dog, where the results differed significantly. In fact, 46 percent of the respondents aged 61+ years in the survey assessed the normal-weight dog (BCS = 5) as being underweight (BCS = 1–3), thus underestimating the dog with an ideal body condition by two steps or more on the scale, which is a clinically significant amount.In the clinical study sample, overweight dogs were about twice as likely to be underestimated compared to normal-weight/underweight dogs in the pre-educational assessment, a finding that is consistent with other studies investigating the accuracy of BCS assessment among owners evaluating their own dogs ,23,37,38The fact that certain owner-related factors can affect the precision of owner BCS assessments has been confirmed by another study, showing that type of dogs (sport or pet dogs)  may affeThe group of owners with no previous experience of dog ownership was small and did not overlap with the group of owners aged 38–55 years, in which all owners had previously owned at least one dog. The oldest participants in the clinical sample differed in their perceptions compared to the veterinary health care personnel before the standardized education. However, owner age as an explanatory factor for accuracy in assessments could possibly be a marker for some other factors not detectable by the current study design. Importantly, it should be emphasized that all dog owners responded to the standardized education given, regardless of age and previous dog ownership experience. Hence, a recommendation is that veterinary health care personnel should take the time during veterinary visits to provide standardized education to their clients.Although exact owner ages were grouped slightly differently between the two study samples, the age groups might be considered comparable as they represent similar life stages of the participants. Even though many dog owners participated in this study as a whole, and participants in the clinical study sample were from three different regions of Sweden, the results do not necessarily represent perceptions of Swedish dog owners at a national level due to the use of convenience sampling based on voluntary participation and non-randomized selection. Another limitation of the study is the unintentional overrepresentation of women, health care professionals, and students from animal-related programs in both study samples. The results were not analyzed with regard to owner gender and education, even though one previous study of owner perceptions of canine body composition has shown that women underestimated body composition to a lower degree than men , a factoThe results from the two study populations evaluating owner perceptions via indirect assessment based on photos and direct assessment of own dogs based on oral instructions suggested they were influenced by some similar factors, i.e., body condition status of the dogs and owner age. This study shows no overlap between the survey and the clinical samples. In future studies, owner perceptions of what an ideal body condition looks like, as evaluated in dogs owned by others and/or from photos, should be investigated and compared to the accuracy of BCS assessment of a dog owned by the participant in the same study population of dog owners. This will lead to a better understanding of the impact of individual perceptions on the accuracy of BCS assessment.The canine overweight prevalence in the clinical sample was 37%, and of those dogs, about two-thirds were slightly overweight, one-third were overweight, and no dog was obese. A prevalence of nearly 40% is slightly higher than previously reported in Sweden ,7 but inIn this study, canine BCS increased with the age of the dogs and neutered dogs had a significantly higher BCS than intact dogs. The fact that these intrinsic dog factors affect BCS has previously been verified in other studies ,45,46,47Both study samples underestimated body condition at assessment when they had no previous education; however, after the standardized practical BCS education given to the dog owners in the clinical sample, these owners performed the BCS assessments equivalent to the veterinary health care personnel overall, regardless of owner age and/or number of previously owned dogs. The standardization of the education is unique to the authors’ knowledge and likely contributed to the significantly improved agreement between the assessments from the dog owners and the veterinary health care personnel. This suggests that a systematic education could be vital for owners’ ability to correctly assess their dogs’ body condition.Other studies have reported no or only little improvement in the accuracy of BCS assessment after using the BCS scale and/or after some training directed to dog owners ,21,22,23We suggest that the concept of “weight blindness”, defined here as an underassessment of normal-weight dogs and an inability to identify overweight dogs, could be introduced as a novel concept. Weight blindness captures the bias underlying the underestimation of canine body condition and is likely to have a negative impact on canine overweight prevalence as owners will not address an unknown problem. In accordance with other studies ,41, we sThe results from the two different study samples indicate that owner perceptions of an “ideal body condition” in dogs are sliding and that the ability to identify overweight in one’s own dogs might be limited. We propose a concept called “weight blindness”, which could be successfully reversed with standardized practical education on how to use the 9-point BCS scale. When addressing canine overweight, it should be recognized that not only the body condition of dogs but also owner-related factors might influence owners’ perceptions of different canine body compositions and the accuracy of BCS assessment when there is no previous education. Future studies should preferably investigate perceptions among owner populations based on a randomized selection of participants. Dogs owned by others and/or perceptions based on photos, as well as assessments of one’s own dogs, in the same study population of owners could be used to generate a better understanding of the impact of individual perceptions on the accuracy of BCS assessment.


The number of grains per panicle is an important factor in determining rice yield. The DST-OsCKX2 module has been demonstrated to regulate panicle development in rice by controlling cytokinin content. However, to date, how the function of DST-OsCKX2 module is regulated during panicle development remains obscure.ABNORMAL PANICLE 1 (ABP1), a severely allele of FRIZZY PANICLE (FZP), exhibits abnormal spikelets morphology. We show that FZP can repress the expression of DST via directly binding to its promotor. Consistently, the expression level of OsCKX2 increased and the cytokinin content decreased in the fzp mutant, suggesting that the FZP acts upstream of the DST-OsCKX2 to maintain cytokinin homeostasis in the inflorescence meristem.In this study, the FZP plays an important role in regulating spikelet development and grain number through mediating cytokinin metabolism.Our results indicate that The online version contains supplementary material available at 10.1186/s12870-023-04671-4. Oryza sativa) is one of the most important cereal crops in the world, providing the main energy for more than half of the world's population [Rice , encodes cytokinin oxidase/dehydrogenase 2 (OsCKX2), which can catalyze the degradation of active cytokinins [OsCKX2 expression has been successfully used in rice breeding practice to increase seed yield [CKX2 through directly binding to its promoter [Cytokinin is a specific hormone that affects plant growth and development, and plays an important role in regulating shoot apical meristem (SAM) in plants , 20–22. tokinins . Decreastokinins . Increased yield . Transcrpromoter . ERECTA1promoter . A recenpromoter . HoweverFRIZZY PANICLE (FZP), is involved in the transition from spikelet to floral meristem identity, which contains an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain transcription factor. FZP is an ortholog of the maize transcription factor BD1 [FZP at the transcriptional stage [FZP translational repression by interacting with CUREs in the 3’ UTR of FZP mRNA, leading to changes in the number of secondary branches and the grain number per panicle [The spikelet identity gene, ctor BD1 , 31, 32.al stage . Deletioal stage . In anot panicle . HoweverABNORMAL PANICLE 1 (ABP1) gene, which regulates spikelets development and grain yield in rice. Map-based cloning revealed that ABP1 was identical to FRIZZY PANICLE (FZP). Molecular evidence shows that FZP directly binds to the motif AGCCGCC in the DST promoter that represses its expression. Consequently, the expression level of OsCKX2 was upregulated and the level of cytokinin decreased in the inflorescence meristem of fzp mutant, which reduces the SAM activity and the grain number of per panicle. Our findings reveal that FZP regulates grain number by negatively controlling the function of DST-OsCKX2 module, which provides novel insight into the regulation of cytokinin homeostasis in rice.In this study, we identified an abnormal panicle 1 (abp1) from an EMS-treated mutant pool of the elite indica rice variety YK17 (Zhongjiazao17). The abp1 mutant was similar to the wild type in plant morphology  were constructed. The gene was mapped in the chromosome 7 primarily, and delimited to a 995 kb region between SSR markers MM0148 and MM3834, which harbors a Frizzy panicle  gene  experiments was in the young panicle (≤ 0.5 cm) of fzp and wild-type. A total 578 differentially expressed genes (DEGs) were identified, including 161 downregulated and 417 upregulated in fzp  assay, the result showed that FZP could bind to the promotor of DST  assay was conducted by using proDST: LUC as a reporter. In rice protoplasts, effectors 35S: FZP significantly repressed the transcription of the reporter compared with the negative control  controls panicle development by regulating the activities of florescence meristem activity, mutation in TAW1 shows the differentiation of spikelet meristem was delayed, the differentiation time of branch meristem was prolonged, and significantly increases the number of secondary branches and spikelets in rice [RCN1and RCN2 (RICE CENTRORADIALIS) delayed the transition from branch meristem to spikelets meristem, resulting in an increased number of branches and the grains number of per panicle [FZP is involved in the differentiation of spikelets meristem and prevent the formation of axillary meristem, has become a marker gene for the development of spikelet meristem in grasses, several FZP alleles have been cloned, Our results showed that ABP1 is a severely active allele of FZP, grain number per main panicle of abp1 was reduced by 66% compared to wild type, the floral primordia was not differentiated in abp1 panicle but replaced by higher-order branch primordia, and the grains were also smaller than the normal plants  in maize belonged to AP2/ERF family, specifies determinate fates by suppressing indeterminate growth within the spikelets meristem, and which is required for timely conversion of spikelets meristem to floral meristems timely [MULTI-FLORET SPIKELET1 (MFS1) belong to AP2/ERF family, plays an important role in the regulation of spikelets meristem determinacy and floral organ identity, mutation in MFS1 resulted in an extra hull-like organ and an elongated rachilla [FZP also encodes an AP2/ERF-type transcription factor, ERF domain can be divided into three classes according to the amino acid characteristics, FZP belongs to the class II, and transient analysis shows that the class II members usually have transcriptional repressor activity [DST which controlling the number of grains per panicle was significantly up-regulated in fzp by analysis the transcriptome data, and the promoter of DST also contained the cis-acting GCC-box , a highly conserved DNA domain, are a class of plant-specific transcription factors . Previouelopment . A few es timely . Rice MUrachilla . FZP alsactivity . Howeverbox Fig. . We revents Fig. . Our resOsCKX4 antisense inhibition could increase the panicle size and spikelets number of plants without affecting other agronomic traits [OsCKX11 plays an important role in delaying leaf senescence, increasing seed number, and coordinating source and sink, osckx11 showed significantly increased branching, tillering, and seed number compared to WT [OsCKX family were also checked, the OsCKX3, 4, 8 and 9 were downregulated, which were considered to be the reason why the DHZ content was unchanged in the mutant , an important plant hormone, was first discovered in tobacco tissue culture, CTK are widely involved in regulating plant growth, development and response to the environment, such as promoting cell division, removing apical dominance and shortening the transition time from vegetative growth to reproductive growth . The shoc traits . OsCKX11ed to WT . In thisnt Fig. S. The DSTole Fig. F.DST and regulates spikelet development by maintaining cytokinin homeostasis in inflorescence meristem via DST-CKX2 module. Our results indicate that FZP plays a critical role in regulating grain number through mediating cytokinin metabolism.Collectively, our findings indicate that FZP represses the transcription of fzp mutant was identified from EMS-treated seeds of the elite indica rice (Oryza sativa) variety YK17 (Zhongjiazao 17), the mutant stably inherited for multiple generations was applied for further experiments. The F2 mapping populations was derived from a cross between fzp mutant and tropical japonica rice variety D50. All plants were grown under natural conditions in the experimental fields of Zhejiang Academy of Agricultural Science in Hangzhou, China.The fzp were fixed in 2.5% glutaraldehyde solution at 4 °C overnight, and the samples were rinsed three times with 0.1 M, pH7.0 phosphate buffer for 15 min each time. Then the samples were fixed in 1% osmic acid solution for 2 h, rinsed the samples three times with 0.1 M, pH7.0 phosphate buffer for three times. The samples were dehydrated with ethanol solutions of gradient concentrations, and then treated with 100% ethanol twice, 20 min each time. Drying process was performed in a Hitachi HCP-2  critical point dryer. The samples were subsequently sputter-coated with gold and analyzed using the Hitachi SU-8010 scanning electron microscope .For scanning electron microscopy analysis, the tissue samples collected from YK17 and 2 plants, we delimited the gene to 995 kb region between MM0148 and MM3834. For the complementation of fzp, the FZP genomic DNA fragment containing 2.6-kb upstream sequences and 1.2-kb downstream sequence was inserted into the pCAMBIA2300 vector using kpnI and xbaI sites. The resulting plasmid was introduced into fzp mutant to generate transgenic plants [To determine the mutated gene, both parents and 20 individuals with the abnormal panicle phenotype were selected for initial linkage analysis. More than 200 simple sequences repeated (SSR) markers evenly distributed on 12 chromosomes of rice were used for preliminary mapping. The mutant control gene was initially located on chromosome 7. Using another 192 Fc plants . All prifzp using Trizol reagent (Life Technologies). The method of RNA extraction from developing seeds was according to a modified SDS-Trizol method [UBQ gene (LOC_Os03g13170) was used as internal control. The relative mRNA level of tested genes was normalized to UBQ and calculated by the 2–DDCT method [Total RNA was extracted from different plant tissues except developing seeds of wild-type and l method . First Sl method . ExperimT method . Primersfzp and wild-type were collected, RNA extraction as previously described. Illumina Hiseq platform was used for sequencing in Novogene (China). Differentially expressed genes (DEGs) were identified as genes with absolute value of log2Fold changed ≥ 1 and q-value < 0.005 using DEGseq. For GO and KEGG analysis, Goseq and KOBAS (2.0) were used. The threshold of corrected P-value < 0.05 was used to identify significantly enriched GO terms and KEGG pathways.Young panicle samples (≤ 0.5 cm) of ◦C in the dark, the GFP signals were observed under a Zeiss LSM510 laser-scanning confocal microscopy . The primers of this experiment were listed in Supplemental Table SThe full-length coding sequence of FZP without stop codon from YK17 was amplified by PCR. Then the PCR product was cloned into pAN580 vector to get the FZP-GFP construct . The fusFZP was amplified and inserted into pB42AD, the DST promoter sequence was inserted into pLacZ2u to generate ProDST: LacZ2u. The plasmids were co-transformed into the yeast strain EGY48, the empty pB42AD in combination with the LacZ reporter constructs were used as negative controls [For yeast one-hybrid assay, the CDS of controls . The traE. coli DE3 cells and purified using MBPSep Dextrin 6FF Chromatography Column  based on the manufacturer’s instructions. For EMSA assays, the probes of DST (62 nt in length) containing a motif AGCCGCC was commercially synthesized and labeled with an EMSA probe biotin labeling kit . Unlabeled DNA oligos were used as competitors. Reaction system was performed in a 10μL reaction volume which containing 2μL 5 × EMSA/Gel-shift binding buffer, 5 nmol purified recombinant protein and 2 nmol biotin-labeled probe. EMSA assays were performed with the chemiluminescent EMSA kit .Full length CDS of FZP was cloned into the Pmal-c2x to fuse with MBP tag, the recombinant protein was then transformed into 35S: NONE as an effector. Total 10 μg of plasmid  was used for the transformation in rice protoplast. Protoplast extraction and transformation were performed as previously described [For dual-LUC assay, the 1.2 kb promoter sequence of DST was amplified and cloned into vector 190LUC as a reporter, and the full-length coding sequences of FZP was inserted into the control vector pescribed . All theFor endogenous cytokinin level assay, 1 g young panicles (≤ 0.5 cm) were collected and pooled for measurements. Three biological replications were performed. Quantification of endogenous cytokinin was performed as previously described .Additional file 1. Additional file 2. Additional file 3. Additional file 4.


Reherniation and reoperation rates of 4.5%–36% are reported in canine patients treated for intervertebral disc herniation (IVDH). Decision‐making for surgical reintervention can prove challenging, especially since common postoperative changes are poorly described on MRI. The purpose of this single‐center, retrospective, descriptive study was to describe the MRI characteristics of the surgical site in dogs treated for thoracolumbar IVDH and presenting for ongoing or recurrent neurological signs. Twenty‐one patients were included for a total of 42 MRI studies. Chondrodystrophic breeds, specifically Dachshunds, were overrepresented. Mean number of days between surgery and second MRI was 335 (range 2–1367). Metallic susceptibility artifacts were seen in seven of 21 cases (33%), but these were limited in extent, spanning on average 1.3 vertebral bodies. In 11 cases, spinal cord compression suspected to be clinically significant was found at the surgical site; the extradural compressive material consisted of intervertebral disc material only, or a combination of intervertebral disc material and hematoma or inflammatory changes in 10 cases, and a displaced articular process and fibrous tissue in one case. The latter is a newly described complication of mini‐hemilaminectomies. Paravertebral soft tissue changes and vertebral new bone formation varied according to the postoperative stage at which the patients were imaged. The results of this study supported the use of MRI as a diagnostic modality for spinal imaging following IVDH surgery, and showed that the presence of extradural disc material at a spinal surgical site is common along with various vertebral and paravertebral changes. ECVNEuropean College of Veterinary NeurologyIVDintervertebral discIVDHintervertebral disc herniationIVDMintervertebral disc material1Recurrence of clinical signs affects an estimated 4.5–12.3%Residual spinal cord compression is commonly reported following surgery, with variable rates depending on the technique performed.The objectives of this study were therefore to search for a population of dogs having undergone surgery for thoracolumbar IVDH as well as a postoperative MRI, and (1) assess the frequency and diagnostic impact of susceptibility artifacts on postoperative MRI studies; and (2) describe the changes seen at the surgical site on MRIs performed to investigate ongoing or recurrent neurological signs.22.1This was a single‐center, retrospective, descriptive study. Records of canine patients seen at the Queen's Veterinary School Hospital (QVSH) between January 2009 and September 2020 were screened. Informed consent was obtained from all owners at the time of admission, and the authors obtained ethical approval from the Cambridge University Ethics & Welfare Committee reference CR553.Criteria for case inclusion were (1) a diagnosis of thoracolumbar IVDH based on MRI findings and surgical confirmation; (2) a postoperative follow‐up MRI study was performed due to clinical need ; and (3) all MRI studies included at least T2‐weighted (T2W) and T1‐weighted (T1W) sagittal and transverse sequences of the surgical site. A radiology resident (A.‐L.P.) and a European College of Veterinary Diagnostic Imaging (ECVDI)‐certified veterinary radiologist (M.‐A.G.) were responsible for final decisions for case inclusion or exclusion, as well as for consensus reviewal of the images.2.2Patients’ records were reviewed by a veterinary radiology resident (A.‐L.P.). The following data were recorded: breed (classified as chondrodystrophic or non‐chondrodystrophic according to available lists of breeds associated with the FGF4‐12 and FGF4‐18 mutationsThe magnetic resonance (MR) images were retrieved and reviewed by the veterinary radiology resident (A.‐L. P.) and an ECVDI‐certified veterinary radiologist (M.‐A. G), using an open source 64‐bit medical image viewer . At the time of image review, the two assessors were blinded to patient data and previous imaging reports. The MR images were assessed for susceptibility artifacts compatible with the presence of metallic material (referred to as susceptibility artifacts for the remainder of the study), presence and characteristics of extradural material, and changes to the vertebrae, spinal cord, and paravertebral soft tissues. On the first MRI study of each patient, the degree of degeneration of the herniated IVD was classified as completely degenerate, partially degenerate, or non‐degenerate, based on an entirely lost, decreased, or intact T2W hyperintensity of the nucleus pulposus, respectively, and this is illustrated in Figure 33.1Clinical and imaging details for each case are provided in Supporting Information 3.2Duration of neurological clinical signs prior to first presentation ranged between 24 h and over 180 days (median 6 days). The most common clinical signs included back pain, lethargy, reluctance to walk, paraparesis, paraplegia, and ataxia.The affected spinal segment was the thoracolumbar junction (T10‐L3) in 18 patients , and the lumbar spine (L3‐L7) in 3 patients .® Surgairtome Two® and OralMax™ Pneumatic System, ConMed, Utica, New York) and rongeurs.All patients underwent spinal surgery within 24 hours of their first MRI. In total, 14  dogs underwent a hemilaminectomy and seven  dogs undervent a mini‐hemilaminectomy. All surgeries were carried out by a European College of Veterinary Neurology (ECVN) resident, under the supervision of an ECVN‐boarded specialist. Several residents and a total of 10 ECVN specialists were involved, in many different combinations. Although the surgical approaches were grossly standardized for both hemilaminectomies and mini‐hemilaminectomies, technical specifications such as the number of muscle detachments and extent of surgical window in the bone were not available from the surgical reports. None of the surgeons involved used subcutaneous fat grafts. To create the vertebral bony window, all surgeons used a high‐speed drill . One postoperative MRI was performed using a 1.5 Tesla MR magnet . Besides the required T2W and T1W sagittal and transverse sequences focused on the surgical site, additional sequences were also reviewed when available including T1W dorsal, three‐dimensional hybrid contrast enhancement, and dorsal Short Tau Inversion Recovery. The sequences and settings varied between patients (Supporting Information 3.4Pertinent imaging findings of sampled dogs are summarized in Supporting Information 3.5The mean number of days between surgery and second MRI study was 335 (range 2–1367 days) days. Eight patients were included in the early postoperative period , and 13 patients in the late postoperative period .3.6Susceptibility artifacts were present in seven cases 7/21, 33%), of which three  cases were classified as moderate and four  cases as marked. An example of each magnitude of signal distortion is available in Figure 1, 33%, oDue to interference from susceptibility artifact at the surgical site, certain patients had to be excluded from the assessed population for the following imaging criteria located at the surgical site: degree of compression of the spinal cord, length of intramedullary T2W hyperintensity, tethering of the spinal cord, bony changes, reduction in volume of the epaxial muscles, change in intensity of the ipsilateral epaxial muscles, and presence of collections of fluid in the paravertebral soft tissues. In some patients with moderate susceptibility artifact, the disruption in signal was lateralized enough to assess the adjacent vertebral canal accurately, and this was taken into account when excluding cases for certain imaging features. Eventually, exclusions from specific imaging features applied to one patient from the early postoperative group (case 20) and four patients from the late postoperative group . Purely imaging‐based results are therefore presented out of a total of 7 patients for the early postoperative group, out of 9 patients for the late postoperative group, and out of a total of 16 when both groups are combined.3.7Among the eight patients of the early postoperative group, only one  patient recovered well from the surgery before relapsing (case 13), with the others showing poor post‐surgical improvement or even deterioration of their neurological signs. Five  of these patients had evidence of ongoing or recurrent compression of the spinal cord at the surgical site , including case 13, with only one  patient having neurological signs attributed to a disc extrusion distant from the surgical site, and one  patient showing no evidence of spinal cord compression. In one  patient, the MRI was inconclusive due to susceptibility artifact and no second surgery was performed, precluding an accurate diagnosis. In this patient, the surgical site was presumed to be the site of the focal myelopathy responsible for the neurological signs in the absence of other visible lesions in the remainder of the studied spinal segment. In the 5 patients with identified compressive extradural material at the surgical site, this was causing marked, moderate or mild compression of the spinal cord in one, three, and one patients, respectively. Four patients from this group underwent surgical revision of the surgical site and all but one showed improvement of their neurological status after the second surgery.Of the 13 patients that belonged to the late postoperative group, four patients had a poorly diagnostic MRI study at the level of the surgical site due to susceptibility artifact. In this group, 10  patients made a full recovery following surgery and three  patients only made a partial recovery. These three patients showed mild chronic neurological deficits that eventually acutely deteriorated; one had no evidence of ongoing compression at the surgical site and a compressive distant IVDH, 1 had evidence of mild compression at the surgical site only, and one had a compressive distant IVDH with a poorly characterizable surgical site due to localized susceptibility artifact. Of the 10 patients that had recovered fully from the surgery, three had imaging evidence that compression of the spinal cord at the surgical site was the cause of the presenting neurological signs and an additional patient had suspected spinal cord compression at the surgical site in the absence of other visible compressive lesions of this spinal segment. This last patient (case 16) underwent surgical revision of the previous mini‐hemilaminectomy site which resulted in removal of a displaced articular facet and fibrous material from the vertebral canal. Of the three patients that recovered fully from initial surgery, then represented in the late postoperative period with clinically significant spinal cord compression at the surgical site by extradural IVDM , two had concurrent IVD fenestration at the time of initial surgery. Finally, six patients were thought to have relapsed due to IVDH at a different disc space, of which two were free of compression at the surgical site, two showed evidence of extradural compression at both the surgical site and a distant disc space, and two had too much susceptibility artifact to characterize the surgical site fully on MRI. The degree of spinal cord compression at the surgical site was deemed to be moderate in one patient, and mild in five patients. Five patients underwent spinal surgery to remove extradural material at a site distant from the previous surgical site, with only one patient undergoing revision of its previous surgical site (case 16).When assessing specifically the central T2W hyperintensity of the intervertebral discs that were not herniated on the first MRI study but herniated on the second study, six were classified as completely degenerate, two as partially degenerate, and one as nondegenerate on the initial MRI.3.8The changes to the vertebrae could not be accurately described in the presence of adjacent susceptibility artifact, and therefore the aforementioned 5 postoperative MRI studies were excluded from the following results.Bony defects were clearly visible without evidence of new bone formation in 10 patients 10/16, 63%); this included all patients  from the early postoperative period, and three patients  from the late postoperative period, up to 839 days after surgery , and hyperplastic vertebral new bone formation was identified in three of 16 patients (19%). These six patients all belonged to the late postoperative group.There was evidence of displacement of a cranial articular facet of L1 following a left‐sided T13‐L1 mini‐hemilaminectomy in two patients imaged within a week of surgery. One of them showed a clearly visible non‐compressive displaced articular facet on MRI Figure , while t3.9On 13 of the first MRI studies , the spinal cord showed a focal area of T2W hyperintensity that remained T1W isointense, overlying the diseased IVD space. This spanned a length of less than two vertebral bodies in nine cases (43%) and over two vertebral bodies in four cases (19%). Of these four cases, two made a full recovery after the initial surgery, one remained mildly ataxic for 27 months postsurgery, and one developed myelomalacia within days of surgery, highly suspected from the clinical evolution and second MRI and confirmed at postmortem examination.On the second MRI studies, after exclusion of the five cases with susceptibility artifacts, this focal T2W hyperintense region of the spinal cord had increased in length , decreased in length , or remained absent .The increase in length was superior to two vertebral bodies in three cases , equivalent to 1 to 2 vertebral bodies in length in two cases , or inferior to one vertebral body in two cases .Of the nine cases in which either the spinal cord was within normal limits, or the T2W hyperintensity of the spinal cord decreased, one belonged to the early postoperative group and eight to the late postoperative group. Five of these patients had a good clinical outcome, and four were lost to follow‐up.Of the 16 follow‐up MRI studies that were not affected by susceptibility artifacts, tethering of the spinal cord to the surgical site was present in four 4/16, 25%) studies, all belonging to the late postoperative group. Concurrent attenuation of the CSF and fat columns ipsilateral to the tethering was visible, along with widening of the contralateral CSF column. This is illustrated in Figure % studies3.10Unilateral changes in the paravertebral soft tissues were also appreciated in the 16 postoperative MRI studies that were not affected by susceptibility artifacts, both in the early (7 studies) and in the late (9 studies) postoperative groups.Common changes seen in the patients imaged within 14 days of surgery included increased volume of the epaxial muscles over the surgical site, associated with a diffuse to patchy T2W hyperintensity that remained T1W isointense , focal collections of T2W markedly hyperintense, T1W mildly hyperintense to muscle material in the paravertebral soft tissues , and multifocal subcutaneous small rounded signal voids . These changes were never seen in patients imaged later than 14 days after surgery.From 14 days onward, five of nine 56%) patients showed a focal reduction in the volume of the epaxial muscles ipsilateral to the surgery, and these muscles had a similar increased signal intensity on both T2W and T1W sequences when compared to the preoperative studies. A tract of T2W and T1W hyperintense tissue could be identified in two  patients of this group along the spinous process, in the area of the surgical approach. This is also illustrated in Figure 6% patienFinally, a common finding that could be appreciated in all 21 postoperative MRI studies despite the presence of susceptibility artifacts was the presence of thin linear T2W/T1W hypointense subcutaneous tracts in the area of the surgical approach, found in all patients in the early postoperative group (100%), and eight of 13 patients in the late postoperative group (62%).4Findings from this study supported using follow‐up MRI of dogs treated for IVDH for characterizing the changes at the surgical site, with only a few occurrences of MRI studies being rendered poorly diagnostic due to susceptibility artifacts. Several of these postoperative changes could also be related to the stage of the postoperative period, with some being more or less common depending on the time elapsed since surgery. Due to the presence of pain or neurological deficits in the patients included in this study, the clinical impact of these changes diagnosed on MRI could not be ascertained. The population included in the present study showed characteristics that were similar to those from other publications on canine IVDH, with chondrodystrophic breeds and more particularly Dachshunds being overrepresented, and most patients being middle‐aged (median age of 6 years 2 months old).In 16 of 21 patients (76%) included in this study, metallic susceptibility artifacts were either not present or did not hinder the evaluation of the vertebral canal. This does not support the findings of an earlier study, that speculated that susceptibility artifacts would be common postoperatively and therefore MRI may not be an adequate imaging modality for patients that had undergone spinal surgery.Extradural IVDM at the surgical site was described in 11 patients (5 from the early postoperative group and 6 from the late postoperative group), which represents 52% of the study population. It remains unclear whether this was residual from surgery or newly herniated IVDM. The imaging evidence of persistent extradural compression of the spinal cord alongside the history of clinical deterioration or failure to improve prompted surgical reintervention in all patients which showed compression of the spinal cord at the surgical site during the early postoperative period.The clinical significance of the presence of spinal compression at the surgical site was considered in light of the presence or absence of other spinal lesions. Compression at the surgical site was suspected to be the cause of the neurological signs in 11 of the included patients, in the absence of another site of significant spinal cord compression. Following conclusions from previous studies,Nine patients showed a disc hernia distinct from the initial surgical site on their second MRI. When reviewing the central T2W hyperintensity of these intervertebral discs on the first MRI study of each patient, most  showed some degree of loss of this hyperintensity, indicating degeneration. This is consistent with the pathophysiology of IVD disease and previous publications.Bony defects were more clearly discerned in the early postoperative period, where all patients imaged within 2 weeks of surgery showed a clearly identifiable gap in the vertebral bone corresponding to the surgical window. Marked vertebral new bone formation was found in three MRI studies , all in the late postoperative period. Interestingly, different degrees of bony defects and new bone formation could be seen in patients imaged at similar postoperative stages, showing that the amount of new bone formation did not necessarily relate to the amount of time elapsed since surgery. From a clinical point of view, the presence of a large amount of new bone formation may complicate the surgical approach if surgical reintervention is required—this was not the case for the included patients.When considering the difference in length of the T2W hyperintense region of the spinal cord between the first and second MRI studies, the five patients in which it increased in length by more than one vertebral body had a poor clinical outcome, whereas the other seven patients for which follow‐up was available were associated with good outcomes. Hypotheses as to the cause of this hyperintensity include contusion, edema, and gliosis, depending on the time frame since the insult to the spinal cord occurred—the mechanism that causes this hyperintensity to increase or decrease in length over time is however unknown.Tethering of the cord to the surgical site was found in a quarter of patients with postoperative MRIs free of susceptibility artifacts. All of these four patients belonged to the late postoperative group. It is unclear why some patients developed this feature compared to others, and it is also difficult to estimate the clinical impact of this as at least two of these patients had a good neurological outcome.Unilateral changes in the epaxial muscles were common, and their nature varied depending on the time elapsed since surgery. In all patients imaged within 2 weeks of surgery, the epaxial muscles over the surgical site were mildly swollen, with a change in signal intensity consistent with inflammation or edema. Collections of fluid in the soft tissues adjacent to the surgical site, thought to be seroma or hemorrhagic fluid, were also common in the 2 weeks postsurgery (86% of cases). Finally, in five patients, multifocal small subcutaneous signal voids were present and thought to be due to subcutaneous emphysema and/or suture material. Soft tissue changes were evaluated subjectively and there was no appreciable difference between the patients that underwent hemilaminectomy and those that underwent mini‐hemilaminectomy.After the initial postoperative two weeks, just over half of patients (56%) showed a reduced volume of the operated epaxial muscles compared to the contralateral musculature, occasionally associated with an increase in signal intensity compatible with fatty infiltration. Although no muscle biopsies were performed, possible explanations for this include loss of innervation and/or loss of blood supply, causing muscular atrophy.The T2W and T1W hyperintense tract visible in two patients of the late postoperative group along the spinous process of the operated vertebra were hypothesized to be fatty infiltration in the area of dissection of the epaxial muscles from their bony attachments.Finally, approximately three quarters of the included population (76%) showed a linear tract in the subcutaneous fatty tissues in the area of the surgical approach, of a signal intensity suggestive of fibrosis or scar tissue.Limitations of this study include first of all its retrospective nature, which introduces variables such as non‐standardized MRI sequences, and record‐keeping. The authors however implemented strict inclusion criteria to mitigate this. Several different combinations of residents and specialists were involved in the surgeries, and although surgical approaches for both hemilaminectomies and mini‐hemilaminectomies are standardized to some degree, certain aspects of these techniques will inherently vary with the surgeon involved. Surgical reports did not offer details such as the length of the incision, the number of muscle detachments, and the size of the bony window. All the data were also collected from a single institution, and may therefore be biased by a number of factors such as the clinicians involved, the surgical material used, and the imaging acquisition methods. Case numbers were also small, although still comparable to other studies on the subject. Because several patients were treated conservatively following their second MRI, surgical confirmation was often not available for the findings of the follow‐up MRIs. All the patients included in the present study underwent a second MRI due to ongoing or relapsed neurological signs; it is therefore uncertain whether the changes described would be found in patients that recover uneventfully, and to what degree they contributed to the recurrence of neurological signs in our patients. Assessment of bony changes would also potentially have been more accurate on CT than MRI, and therefore further studies using CT to evaluate bony healing and remodeling following spinal surgery may be of interest. Finally, it is also important to consider that the low diagnostic impact of the susceptibility artifacts in this study may only be valid for low‐field MRI, as it is less sensitive to local magnetic field inhomogeneities compared to high‐field MRI.In conclusion, findings from this sample of dogs indicated that, contrary to what was suggested in a previous study, metallic susceptibility artifacts are uncommon in postoperative low‐field MRI studies, and that the disruption caused to the diagnostic quality of the MR images is focally limited to the surgical site. MRI can therefore be considered an adequate modality for patients that require postsurgical spinal imaging, although further studies may be required to extrapolate this finding to high‐field magnets. Additionally, this study has also shown that the appearance of spinal surgical sites on MRI is variable, with certain findings increasing or decreasing in prevalence according to the stage of the postoperative period. Additionally, ongoing or recurrent compression of the spinal cord at the surgical site is common, even in patients that have made a full recovery and present with a late recurrence of clinical signs.Category 1(a)Conception and Design: Peschard, Freeman, Genain(b)Acquisition of Data: Peschard(c)Analysis and Interpretation of Data: Peschard, Freeman, GenainCategory 2(a)Drafting the article: Peschard(b)Revising Article for Intellectual Content: Peschard, Freeman, GenainCategory 3(a)Final Approval of the Completed Article: Peschard, Freeman, GenainCategory 4(a)Agreement to be accountable for all aspects of the work ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: Peschard, Freeman, GenainThe authors have declared no conflict of interest.Preliminary results were presented at a QVSH meeting on Friday 5th of February 2021. The abstract was presented at the pre‐BSAVA EAVDI meeting on Wednesday 24th of March 2021.An EQUATOR network checklist was not used.Supplement 1: Details for individual cases.Click here for additional data file.Supplement 2: Technical parameters used for MRI studies  and number of studies for each MRI sequence used.Click here for additional data file.Supplement 3: Frequencies of the main imaging findings for included dogs, according to the stage of the postoperative period at which the second MRI was performed.Click here for additional data file.