Background: We analyzed the predictive value of the tap test (TT) on the outcome of cerebrospinal fluid (CSF) shunting in patients with idiopathic normal pressure hydrocephalus (iNPH) and cognitive impairment up to 12 months postoperatively.Methods: We analyzed the data of two prospective multicenter studies on ventriculoperitoneal shunt (VPS) and lumboperitoneal shunt (LPS) use in iNPH patients. We selected patients with Mini-Mental State Examination (MMSE) scores ≤ 26 points as study subjects. We used a multivariate logistic regression model to obtain the optimal threshold of MMSE scores after TT to predict the score improvement at 12 months following shunting and that helped to control for confounding factors such as age and MMSE scores before TT. We used logistic regression models to identify variables with age-adjusted odds ratio (A-OR) and multivariate-adjusted OR (M-OR).Results: For an improvement of ≥3 points in the MMSE score cutoff 7 days following TT in VPS and LPS cohort studies, the MMSE scores improved by 6 points after 12 months. The VPS cohort had sensitivity, specificity, and area under the curve (AUC) of 69.2, 73.7, and 0.771%, respectively; however, for the LPS cohort, they were 86.2, 90.9, and 0.906%, respectively. For MMSE scores that improved by ≥3 points in patients after the TT, the possibility of an improvement by 6 points at 12 months following CSF shunt had A-OR 7.77 and M-OR 6.3 times for the VPS, and A-OR 62.3 and M-OR 59.6 times for the LPS cohort.Conclusion: CSF shunting contributes to improved cognitive function in iNPH patients. Furthermore, MMSE score evaluation at the TT can sensitively predict improvement in postoperative MMSE scores following LPS intervention.Clinical Trial Registration: SINPHONI-1 , first posted: September 22, 2005.SINPHONI-2 [University Hospital Medical Information Network (UMIN) Clinical Trials no. UMIN000002730], the posted: February 1, 2010. Idiopathic normal pressure hydrocephalus (iNPH) is a syndrome that occurs in older adults presenting with enlarged cerebral ventricles during diagnostic imaging and neurological symptoms, including gait disturbance with trunk balance disorder, cognitive impairment, and urinary incontinence –3. A cerThe rate of cognitive impairment in iNPH patients varies widely . A decliIn a multicenter, prospective cohort study of iNPH on neurological improvements (SINPHONI)-1, shunting was routinely performed on all enrolled patients, without judging the indications of surgery according to the CSF tap test (TT), which involves the removal of 30–50 ml of CSF , 21. PatWe aimed to analyze the predictive value of the TT for the effects of CSF shunting on patients with iNPH and cognitive impairment up to 12 months postoperatively, from the multicenter collaborative studies SINPHONI-1 and SINPHONI-2, involving two different shunting techniques.ClinicalTrials.gov, no. NCT00221091) and SINPHONI-2 [University Hospital Medical Information Network (UMIN) Clinical Trials no. UMIN000002730] patient cohorts. The recruitment and methodology of these studies have been described previously , 75 (72–78) years] who underwent a TT and VPS and (ii) SINPHONI-2 group . Patients in the latter group included a postponed group  who underwent shunting after 3 months of waiting and an immediate group  who underwent LPS at an early stage  and an MMSE score ≤26 points as study subjects. A total of 156 subjects were classified as follows : (i) SINly stage . There wn = 71; SINPHONI-2, n = 69).We evaluated the MMSE scores before TT, 7 days after TT, and 12 months postoperatively . Cognition was evaluated using MMSE scores 7 days following TT , 26, as We evaluated the degree of improvement by dividing the group of patients with cognitive impairment by three points on the MMSE score and dividing them by the severity of their illness. In the first study, the severity was classified into four groups using the MMSE scores before surgery as follows: grade (G)1, 24–26 points, MCI; G2, 21–23 points, mild dementia; G3, 18–20 points, moderate dementia; and G4, 0–17 points, severe dementia. MMSE scores alone were used as an indicator of the outcome. Patients with iNPH and MMSE scores that improved to ≥27 points following the TT or postoperatively were classified as G0. The degree of change in MMSE scores after the TT and 12 months following the shunt intervention were classified as one of the following four levels: excellent improvement (two ranks up from the severity group classification at entry), improvement (one rank up), no change, and worse (one or more ranks down).Other cognitive function tests such as frontal assessment battery (FAB) scores, symbol search subtest of the Wechsler Adult Intelligence Scale—Third Edition (WAIS-III), Trail Making Test (TMT)-A (sec.), and Zarit Caregiver Burden Interview (ZBI) were assessed before and 12 months following shunting , 2.In the second study, we analyzed the optimal threshold, sensitivity, and specificity for MMSE score change after the TT for predicting MMSE score improvements at 12 months following shunting for SINPHONI-1 and SINPHONI-2.To assess the predicted outcomes 12 months after the shunt surgery, we applied two definitions of MMSE score improvement: (i) ≥3 points or (ii) ≥6 points.We used a per protocol set for all efficacy analyses, which made the data set compatible with those of SINPHONI-1 and SINPHONI-2. We performed the Wilcoxon's signed-rank test to analyze the median values and interquartile range  for age, MMSE, FAB, WAIS-III, TMT-A, and ZBI. The relationship of MMSE scores before TT to after TT and after CSF shunt was examined using Spearman's rank correlation coefficients.We plotted the receiver-operating characteristic curve (ROC) of CSF TT for predicting the shunt effectiveness. Furthermore, we examined the association of improvement in MMSE scores following TT and after shunting to elucidate the reasons behind differences in accuracy of TT between SINPHONI-1 and SINPHONI-2. We used logistic regression models to search for variables with age-adjusted odds ratio (A-OR) and multivariate-adjusted OR (M-OR). A multivariate logistic regression model was used to control for confounding factors, such as age and MMSE scores before TT.p) values of Fisher's exact test were calculated. Statistical significance was considered at a p-value < 0.05. All missing data were treated as deficit data that did not affect other variables. All statistical analyses were performed using SPSS version 25  for Windows and R software ; http://www.Rproject.org).The odds ratios, 95% confidence intervals (CIs), and the probability , the patients were enrolled between November 2004 and November 2006. The Translational Research Informatics Center  together with the steering committee, monitored all clinical data, imaging data, data related to safety issues, and protocol compliance via a web-based case report system. In SINPHONI-2 , patients were recruited from 20 Japanese institutes and hospitals between March 2010 and October 2011, and was designed by all the SINPHONI-2 investigators including a biostatistician and Independent Data and Safety Monitoring Committee members, in conformity with the Guidelines for Good Clinical Practice and the Declaration of Helsinki of the World Medical Association. The institutional review boards for the SINPHONI-1 and−2 studies approved the study protocol. All patients or their representatives provided written informed consent. All clinical and radiological data were prospectively recorded in an independent protocol compliance center via a web-based case report system. An authorization has been obtained for information disclosure (consent to disclose) for publication in a journal, in derivative works by the AAN, or on a journal's website.In SINPHONI-1 (n = 28), G2 (n = 11), G3 (n = 12), and G4 (n = 29)] and LPS  cohorts, respectively. The median MMSE score before TT  in SINPHONI-1 (VPS cohort) and SINPHONI-2 (LPS cohort) were 20 (15–24) points and 20 (16–23) points, respectively  . Twenty-We performed ROC analysis to evaluate the optimal threshold, sensitivity, and specificity of MMSE scores at the TT to predict improvement at 12 months following shunting. Improvements in MMSE score cutoff values of three points at TT for both VPS and LPS cohorts predicted a likely improvement by six points after treatment. Following shunt intervention, the VPS cohort had a sensitivity, specificity, and AUC of 69.2, 73.7, and 0.771%, respectively, after 12 months. However, the LPS cohort had a sensitivity, specificity, and AUC of 86.2, 90.9, and 0.906%, respectively. Regarding the predicted improvement of three points before TT to 12 months postoperatively, the VPS cohort revealed a sensitivity, specificity, and AUC of 90.3, 40.0, and 0.624%, respectively, for an MMSE score with no change at 1 week after the TT. In contrast, the LPS cohort revealed a sensitivity, specificity, and AUC of 57.1, 85.3, and 0.789%, respectively .For MMSE scores that improved by ≥3 points after TT, the possibility of an improvement by 6 points after 12 months following the intervention was A-OR 7.77 (95% CI: 2.22–27.2) and M-OR 6.30 (1.74–22.8) times higher in the VPS cohort. Conversely, the possibility was A-OR 62.3 (6.96–557.5) and M-OR 59.6 (6.64–535.6) times higher in the LPS cohort .For no change in MMSE scores by the TT, the possibility of an improvement by three points by 12 months following the intervention was A-OR 1.06 (0.26–4.39) and M-OR 1.18 (0.27–5.09) in the VPS cohort, compared with A-OR 7.51 (2.33–24.2) and M-OR 7.54 (2.91–24.8) in the LPS cohort. For cutoff values (MMSE score 0 improvement), the prediction was effective only for the LPS cohort .The iNPH syndrome has emerged as a more treatable gait disorder than a treatable form of dementia . There aHerein, 57.8% of iNPH patients with cognitive impairment demonstrated “cognitive improvement” (improvement by one or more grades) at 12 months after shunt placement. However, there was a difference in the improvement between G2 (70%), G3 (72%) on one side, and G1 (54%), G4 (48%). Patients with moderate cognitive decline (G2 and G3), whose preoperative MMSE scores ranged from 18 to 23 points, showed a significant improvement in cognitive function after shunt intervention. Following shunt placement, about half of the patients in the G1 group with MCI revealed improved cognition compared with the normal range. This was partially because elderly people are the target base of iNPH and partly because of a ceiling effect. Also, improvement was less in patients with severe cognitive disturbance (G4 group) who had MMSE scores ≤17 . The finCognitive function changes appear later compared with other iNPH symptoms, such as gait disturbance . ConsideOn the other hand, we can speculate that in VPS, cognitive function improvement was not reflected by the TT result for patients with iNPH where the CSF flow to the lumbar subarachnoid space might be affected, e.g., due to spondylosis, but shunt effectiveness can still be obtained. These trends, such as the LPS being more reflective of TT results, are similar to previous reports assessing gait disturbance after TT . We suppIn an earlier report, Miyajima et al. reported that there was no significant difference between the two methods, using data from SINPHONI-1 and SINPHONI-2 trials . No randThis study has some limitations. The number of participants who were recruited was small. We conducted a multicenter cooperative study and entrusted patient selection to each facility. Despite the inclusion of patients meeting the clinical criteria, surgeons may have excluded cases complicated by neurodegenerative diseases, such as Alzheimer's disease or dementia with Lewy bodies, and a considerable number of them are expected to have iNPH, causing a potential selection bias.Furthermore, in this study, short-term improvement in cognitive function was achieved with shunting. However, elucidating cognitive changes over a longer-term course or in cases of iNPH with comorbid diseases is a challenge for iNPH treatment selection. It has been reported that CSF biomarkers are useful to differentiate patients with iNPH with comorbid neurodegenerative diseases and that patients with Alzheimer's disease are less likely to have their cognitive function improved –44. We bCurrently, all iNPH severity classifications are based on preoperative symptoms. The necessity of revising severity classifications that predict the degree of improvement from shunting for intervention selection is another challenge of the future.In conclusion, CSF shunting contributes to an improvement in cognitive function for patients with iNPH. Furthermore, MMSE score evaluation at the TT can sensitively predict an improvement in postoperative MMSE scores after LPS intervention.The data that support the findings of this study are available on reasonable request from the corresponding author, MN. The data are not publicly available since they include information that could compromise the privacy of the research participants.The studies involving human participants were reviewed and approved by the Ethics Committee of Juntendo University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Masaaki Hashimoto, MD, PhD ; Hideki Origasa, MD, PhD ; Haruko Yamamoto, MD, PhD ; Hajime Arai, MD, PhD ; Koreaki Mori, MD, PhD ; Shigenobu Nakamura, MD, PhD ; Tamotsu Miki, MD, PhD ; Kazunari Ishii, MD, PhD ; Hiroji Miyake, MD, PhD ; Nobumasa Kuwana, MD, PhD ; Naoyuki Samejima, MD, PhD ; Daisuke Kita, MD, PhD ; Takahiko Tokuda, MD, PhD ; Mitsuhito Mase ; Satoru Mori, MD, PhD ; Yoshinaga Kajimoto, MD, PhD ; Teiji Nakayama, MD, PhD ; Osamu Hirai, MD, PhD ; Masatoshi Takeda, MD, PhD ; Chia-Cheng Chang, MD, PhD ; Isao Date, MD, PhD ; Masahiro Kameda, MD, PhD ; Takaharu Okada, MD, PhD ; Junichiro Hamada, MD, PhD ; Mitsuya Watanabe, MD, PhD ; Mitsunobu Kaijima, MD, PhD ; Souichi Sunada, MD, PhD ; Yoshihumi Hirata, MD, PhD .MI and EM conceived and coordinated the study and chaired the steering committee. MN prepared the first draft of the manuscript. All authors were members of the steering committee and designed and wrote the study protocol with input from all listed members of the study advisory board, involved in the interpretation and/or presentation of the data, reviewed and revised the initial draft and subsequent versions of the report, and approved the submitted version, listed investigators contributed to the patient enrollment, the steering committee, independent data, and safety monitoring committee monitored the study.This work was supported by the Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research  and the Japan Agency for Medical Research and Development (grant number AMED JP20dm0207073).This investigator-initiated study was supported in part by Johnson & Johnson K.K. and Nihon Medi-Physics Co. Ltd. This work was also supported in part by the Ministry of Health, Labor, and Welfare of Japan , and by Grants-in-Aid for Scientific Research  from the Japan Society for the Promotion of Science. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. MN has received speakers' honoraria from Johnson & Johnson K.K., Janssen Pharmaceutical K.K., and Medtronic, Inc. SY received speaker honoraria from Johnson & Johnson, K.K. and Medtronic, Inc. MM has received speaker's honoraria from Johnson & Johnson K.K., Nihon Medi-Physics Co. Ltd., Medtronic, Inc., Eisai Inc., and Daiichi-Sankyo Co., Ltd. HK has received speakers' honoraria from Johnson & Johnson K.K., Nihon Medi Physics Co. Ltd., Medtronic, Inc., Ono Pharmaceutical Co., Ltd, Pfizer Inc., Eisai Inc., Daiichi-Sankyo Co., Ltd., Novartis Pharma K.K., Janssen Pharmaceutical K.K., and Takeda Pharmaceutical Co., Ltd. EM has received research grants from Eisai Co. Ltd. and Fuji Film RI, consulting fees from Lundbeck, and speaker's honoraria from Johnson & Johnson K.K., Nihon Medi-Physics Co. Ltd., Medtronic, Inc., Eisai, Co., Ltd., Daiichi Sankyo Co., Ltd., Janssen Pharmaceutical K.K., Novartis Pharma K.K., and Ono Pharmaceutical Co. Ltd. MI received speaker honoraria from Johnson & Johnson, K.K. and Medtronic, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca Ca2+ influx across the sarcolemma activates Ca2+-sensitive ryanodine receptors in the SR promoting a synchronous cell-wide Ca2+ release via the phenomenon of calcium-induced calcium release (CICR), ultimately leading to myocyte contraction. Many studies have demonstrated TT loss in ventricular myocytes during heart failure (HF) in small animal models such as mice (Transverse tubules (TTs) are membranous invaginations primarily associated with mammalian ventricular myocytes. These inward projections of the external sarcolemmal membrane bring voltage-gated calcium channels on the surface membrane into proximity with the sarcoplasmic reticulum (SR), the main intracellular Ca as mice  and rats as mice  as well  as mice , pigs  of amphiphysin-II (BIN1) may play a more central role in TT formation in skeletal and heart muscle. Isoform 8 of BIN1 has the ability to form TTs in skeletal muscle cells  and muta2) interaction with Bin1 in TT formation and maintenance is established skeletal muscle but unclear in the heart. Skeletal BIN1 contains a unique sequence domain of 15 amino-acids called Exon10  and age-matched Wistar Kyoto and spontaneously hypertensive rats (SHR) ranging between 2 and 20 months of age. Rats were injected with 750 units of heparin 5 min prior to anesthesia induced by an injection mixture (80 mg/kg:10 mg/kg) of ketamine and xylazine. When the rats were fully anesthetized, the heart was quickly excised and processed for myocyte isolation, protein extraction or tissue fixation.Normal human tissue samples were obtained from The Gift of Hope of Illinois. All samples were obtained from healthy organ donors whose hearts were considered unsuitable for transplantation. Upon harvest, these hearts were immediately placed in cold cardioplegia solution. Tissue samples were then perfused with 2% paraformaldehyde for subsequent processing. The protocol for tissue use was reviewed by the Institutional Review Board of Northwestern University (exemption 4) and informed consent was obtained from families of organ donors before tissue was collected for research purposes.All reasonable requests for original data analyses will be fulfilled upon written request to the corresponding author in support of transparency and reproducibility requirements. Full details of all methods are provided in the online 2+ channel protein present at a high density in TTs, indicated that isoforms of cardiac Bin1 (cBIN1) co-localized with Cav1.2 along the TTs in both normal rat (Wistar Kyoto or WKY rats) and human ventricular tissue P2] [PIP3], but not with phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidic acid (PA).To investigate the interaction between cBIN1 and PIP2, we cloned two splice variants of mouse cBIN1 (#1 and #4) as well as human skeletal muscle BIN1 (isoform 8) in pET6xHN. These cBIN1 splice variants exclude mouse exon11 (previously exon10) and either include 13 and 17 (full-length variant #4) or exclude these two exons , an enzyme required for PIP2 synthesis (2 regeneration and thus reduces its membrane levels. Endothelin-1 (Et-1) is the endogenous activator of a receptor-mediated signaling cascade involving a Gαq activation of several isoforms of phospholipase C  by which PIP2 is hydrolyzed to diacylglycerol and inositol-3-phosphate (DAG and IP3). Finally, m-3M3FBS is a direct activator of PLCβ (and subsequent PIP2 hydrolysis).To determine if depletion of PIPynthesis . InhibitTo demonstrate that TT loss mediated by these pharmacological interventions was indeed a result of PIP2 depletion/reduction, PIP2 levels were measured following each of these treatments. First HL1 cells were transfected with a PLCδ1-PH-GFP construct that acts indirectly as a PIP2 reporter. PLCδ1-PH-GFP binds with sarcolemmal PIP2 but is released in the cytosol when PIP2 levels decline. We monitored PH-GFP translocation after treating Bin1- cells with either 15 μM wortmannin (2 h), 200 nmol/L Et-1 (for 48 h), 30 μm m-3M3FBS  or the combination of Et-1 and m-3M3FBS (for 45 min) . In the p < 0.05), Et-1 (p < 0.05), m-3M3FBS (p < 0.05), and the combination of Et-1 and m-3M3FBS (p < 0.05)  . Our resIn order to extend our observation that PIP2 plays a key role in tubule disruption in a cell culture system to adult ventricular myocytes, we investigated the effects of pharmacological depletion of PIP2 on TTs in isolated adult rat ventricular myocytes (ARVMs). We have previously described our measurements of TT organization, measured as organization index (OI), in adult myocytes using di-4-ANNEPS staining .2+ release properties in adult myocytes following pharmacologically-induced PIP2 depletion. We and others have demonstrated that progressive heart failure is characterized by TT loss and profound disruption of sarcoplasmic reticulum Ca2+ release and reuptake  and rapid  pacing rates in the control myocyte, Ca2+ release was rapid and uniform along the entire cell length and the decay of the transient was also rapid at both rates  show dramatic slowing in release and recovery properties as OI decreases. There is no obvious departure of any cell group from the overall relationship, suggesting a commonality in mechanism, in this case the decrease in OI. These data suggest that the relationship between OI and each characteristic of Ca2+ release or removal occurs along a continuum that is largely independent from the specific treatment involved so that the slowing of release and of removal is reliant on the reduction in OI and not on how it was achieved. This is best demonstrated in the graphs in the right panels in which the linear fit for each overall relationship is shown. Each graph shows a significant reliance of Ca2+ release and reuptake on OI that is not related to the specific treatment by which reduced OI was induced. We have previously shown nearly identical results in individual myocytes from aged SHRs  is located in the cytoplasm unlike membrane-bound PLCβ1b , 2011. THypertension and other cardiovascular diseases that often develop into HF are known to increase levels of endothelin-1  and angiv1.2 localization to cardiac TTs, thus promoting specific microtubule-induced trafficking of new Cav1.2 molecules to the T-tubular membranes , both of which subsequently were found to abolish TTs in adult ventricular myocytes. The translocation of the membrane PIP2-binding reporter PLCδ1-PH-GFP from sarcolemma to cytosol following these pharmacological treatments suggested that tubule loss was a result of sarcolemma PIP2 depletion. These results suggest that not only does BIN1 have the potential to promote tubulation but it also requires the presence of membrane PIP2 to do so.We postulated that the N-BAR domain alone, without the Exon 10/11 sequence such as in cBIN1 isoforms, can still bind PIP2. In fact, the concept of structurally related F-BAR domains capable of binding phosphoinositol was studied previously by In order to extend our observations in the HL-1 cell culture system to native cardiac myocyte structure and function, we repeated the PIP2 depletion experiments in native ventricular myocytes and found a similar dramatic reduction of TT organization following pharmacological PIP2 depletion. PIP2 depletion induced by either PI4K inhibition or PLCβ1 activation had virtually identical effects to disrupt TT organization to that reported for HF myocytes. As postulated earlier, PLCβ1 activation, as a member of putative BIN1, PIP2, and PLCβ1 complex located in the TTs, may lower endogenous PIP2 membrane levels in a manner similar to what we observed in culture. Thus, PIP2 depletion via PLCβ1 hydrolysis may eventually disrupt BIN1 targeting to plasma membranes where PIP2 ordinarily helps to localize this protein causing the cBIN1/PIP2 complex to lose its ability to maintain TTs and inducing TT remodeling in HF.2+ release that contribute to poor contractile performance at the subcellular and cellular levels and ultimately to systolic dysfunction in the heart. Slow rise times and times to peak in myocytes after PIP2 depletion is consistent with the finding that PIP2 loss disrupts TTs thereby reducing Ca2+ influx through L-type Ca2+ channels and impairing CICR from SR. This point is important since cellular regions lacking TTs then have large distances across which remote TT Ca2+ influx must diffuse to reach the SR membrane  in order to induce CICR compared to areas with normal TTs. Thus, instead of a simultaneous activation of contraction throughout the cell, individual cell regions without local TTs will do so with a delay resulting in a dyssynchronous contraction, reducing contractile efficiency. Another feature of altered Ca2+ cycling is slow reuptake  PIP2 binds to Bin1, which has been a highly controversial issue in the heart, (2) Bin1 isoforms are capable of inducing tubule formation, and (3) PIP2 depletion causes loss of TTs in the heart in a manner that mimics the TT remodeling that occurs in HF. On the other hand, we have not provided definitive proof that the PIP2 depletion is directly responsible for TT loss. Our current methodologies do not permit us to measure this interaction directly but our results provide critical new evidence that PIP2 depletion occurs in HF and that pharmacological PIP2 depletion is associated with the loss of TTs in cardiac myocytes. The hope is that, in the future, we will be able to observe this relationship with higher precision in order to provide definitive proof of this reliance of TT remodeling on PIP2 regulation during the development of cardiac disease.2+ channels are present on TTs, which in turn are critical for the synchronous release of Ca2+ in myocytes and normal EC coupling in heart. Our observation that both delayed Ca2+ release and reuptake are closely related to TT organization due to PIP2 depletion strongly suggests that the loss of PIP2 may be the mechanism for TT disruption during disease development and the resulting defects in Ca2+ cycling and progressive decline in myocardial mechanics. Our study provides important new insights into the mechanistic understanding of TT organization and its implications in Ca2+ dynamics in normal and diseased states.Our data suggest that BIN1 localization at the sarcolemma is required for TT formation and that PIP2 functions as a membrane-targeting and stabilization element for the formation of TTs. These observations are crucial to our understanding of TT remodeling in HF since reduced sarcolemmal PIP2 levels would then be expected to reduce BIN1 targeting to the sarcolemma and hence tubulation. This finding suggests a novel mechanism for TT formation and maintenance, both normal and abnormal. Approximately 80% or more of all L-type CaBased on our results, it might be possible to interfere with degenerative TT remodeling by targeting pathways leading to PIP2 loss. A better mechanistic understanding of the roles of BIN1 and PIP2 in TT remodeling may be important to the development of novel therapeutic interventions aimed at the restoration of this putative phospholipid-protein interactive complex both for the prevention of and the reversal of HF once it has developed.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Northwestern University Institutional Review Board, Northwestern University, Evanston, IL. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee, Northwestern University, Evanston, IL.JZ, NS, JW, and JJ designed and performed experiments, analyzed the data, wrote parts of the manuscript, and approved the final version. CM, WM, and LG designed and performed the experiments, analyzed the data, and approved the final version. MF, WL, PK, SV, RK, and GA designed the experiments and approved the final version. EN and MM analyzed the data and approved the final version. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Predicting which cryptic-donors may be activated by a splicing variant in patient DNA is notoriously difficult. Through analysis of 5145 cryptic-donors , we define an empirical method predicting cryptic-donor activation with 87% sensitivity and 95% specificity. Strength  and proximity to the annotated-donor appear important determinants of cryptic-donor activation. However, other factors such as splicing regulatory elements, which are difficult to identify, play an important role and are likely responsible for current prediction inaccuracies. We find that the most frequently recurring natural mis-splicing events at each exon-intron junction, summarised over 40,233 RNA-sequencing samples (40K-RNA), predict with accuracy which cryptic-donor will be activated in rare disease. 40K-RNA provides an accurate, evidence-based method to predict variant-activated cryptic-donors in genetic disorders, assisting pathology consideration of possible consequences of a variant for the encoded protein and RNA diagnostic testing strategies. Genetic variants affecting the consensus splicing motifs can alter binding of spliceosomal components and induce mis-splicing. Here, the authors develop a method, showing that ranking the most common recurring mis-splicing events in public RNA-Seq data can predict the activation of cryptic-donors. Splicing variants can simultaneously induce different mis-splicing outcomes, including skipping of one or more exons, activation of a cryptic splice-site(s), and/or retention of one or more introns1. Whether induced mis-splicing disrupts the reading frame or affects a region of known functional  importance, has significant diagnostic implications. Therefore, knowing the specific mis-splicing outcome of genetic variant is necessary to conclusively link it to a disease. While the accuracy of in silico algorithms in predicting whether a variant will cause mis-splicing has been comprehensively assessed9, there is currently no reliable means to predict which mis-splicing event(s) may occur in response to a variant that activates mis-splicing. As a result of this and other factors, the vast majority of splice site variants are classified as variants of uncertain significance (VUS); a non-actionable diagnostic endpoint in genomic medicine10.Genetic variants affecting the conserved sequences of the consensus splicing motifs can alter binding of spliceosomal components and induce mis-splicing of precursor messenger RNA (pre-mRNA)11 and algorithms within Alamut Visual® 13, to predict splicing outcomes arising from genetic variants identified in 74 families with monogenetic conditions subject to RNA diagnostic studies 14. Algorithmic predictions of the strengths of activated cryptic splice sites were highly discordant, especially for cryptic donors. SAI’s deep learning showed the greatest accuracy in predicting activated cryptic splice-site(s) , whereas historical algorithms within Alamut Visual® resulted in 34–69% false negatives14.We recently evaluated the accuracy and concordance of SpliceAI (SAI)In this study we focus on determining empirical features that inform prediction of variant-associated spliceosomal selection of a cryptic-donor, in preference to the annotated-donor and other nearby decoy-donors (any GT or GC not used by the spliceosome). Through analysis of 4811 variants in 3399 genes, we show that while splice-site strength and proximity to the annotated-donor strongly influence spliceosomal selection of a cryptic-donor, these factors alone are not sufficient for accurate prediction. Importantly, we show that the most common mis-splicing events seen at each exon-intron junction across 40,233 publicly available RNA-seq samples compiled within the 40K-RNA database, predict with accuracy which cryptic-donor will be activated in rare disease.16  to the eighth nucleotide of the intron , as constraint on sequence diversity eases beyond this point   Fig. . AM-varinor Fig. , and AM-nor Fig. . 89% of nor Fig. .n = 2252)  Cryptic-Modified (CM): a genetic variant modifies a cryptic-donor and does not affect the annotated-donor (52) Fig. . CM-varinor Fig. , with 32nor Fig. .n = 373)  of the cryptic-donor  Annotated-Modified/Cryptic-Modified (AM/CM): a genetic variant that simultaneously modifies the annotated-donor and nearby cryptic-donor (73) Fig. . AM/CM-vnor Fig. , with 31nor Fig. . AM/CM-vnor Fig. .Box 1 GlossaryAnnotated-donor: A donor in an ensembl-annotated transcript.Decoy-donor: Any essential donor dinucleotide (GT/GC) that is not an annotated-donor.Cryptic-donor: A decoy-donor shown to be activated (i.e. used by the spliceosome) by a genetic variant.Annotated-Modified (AM): A genetic variant modifies the annotated-donor resulting in activation one or more unmodified cryptic-donors.Cryptic-Modified (CM): A genetic variant modifies a cryptic-donor and does not affect the annotated-donor.Annotated-Modified/Cryptic-Modified (AM/CM): A genetic variant that simultaneously modifies the annotated-donor and nearby cryptic-donor.Donor Frequency (DF): A measure of donor strength based on how many annotated-donors in the human genome have the exact same sequence.Competitive decoy-donor: A decoy-donor with a DF score at least 10% the score of the nearby annotated-donor.40K-RNA: An aggregated database of splice-junctions detected across 40,233 publicly available RNA-seq samples.-4 or D+5 position .99% of cryptic-donors activated by AM-variants and 71% of cryptic-donors activated by CM-variants, lie within 250 nt of the annotated-donor 12 and SpliceAI (SAI)11 as well as our own method termed Donor Frequency (DF)  among all annotated-donors, converted to a cumulative frequency distribution. For example, if an E-3 to D+6 sequence has a raw frequency of 222, this combination of nine bases occurs at the analogous position for 222 annotated-donors, corresponding to the 35th percentile of a cumulative frequency distribution across the human genome  or variant (VAR) donor sequence.We examined the ability of four algorithmic measures of splice-site strength to predict cryptic-donor activation Fig. . We compThe four algorithms use different methods to measure the intrinsic strength of a given donor splice-site. In the following discussion we use the term stronger and weaker to denote a donor that has a higher or weaker score, respectively, according to that algorithm. Comparisons such as weaker by >50% denote that the donor score has been reduced by more than half by the variant.VAR  or strengthening of the cryptic-donor (67–98% of variants). However, only 35–70% of activated cryptic-donors are stronger than the annotated-donor+3 position are enriched in G dinucleotides whereas introns with A (or T) at the D+3 position are enriched in T dinucleotides  depleted more than in-frame decoy-donors (orange), while showing negligible depletion in the intron Fig. . GC deco18 and Intropolis19 (40K-RNA). Unannotated splice-junctions (representing stochastic mis-splicing), seen rarely in RNA-seq samples aggregated across a population, constitute empirical evidence that both splicing reactions can be executed using a decoy-donor. Therefore, we mined 40K-RNA for splice-junctions representing the use of cryptic-donors within 250 nt of any annotated-donor, and ranked them according to the number of samples they were present in (see methods). Overall, ~7% of all unannotated decoy-donors are in fact present as rare, stochastic mis-splicing events in 40K-RNA.We next assessed what proportion of decoy-donors in the genome are used, albeit rarely, via unannotated splice-junctions detected across 40,233 publicly available RNA-seq samples from GTExThe proportion of exonic GT decoy-donors present in 40K-RNA  dramatically increases with proximity to the annotated-donor, with intronic decoys showing only a modest change Fig. . This miThe ability of DF to measure donor strength is evidenced by Fig. 22.The fact that intronic decoy-donors are less depleted and less likely to be seen in 40K-RNA Fig.  was initWe defined competitive decoy-donors as those with a DF of at least 10% that of the associated annotated-donor see Fig. . In the GAA (NM_000152.3:C.2646 + 2 T > A) identified in an individual affected with glycogen storage disease type II23 that induces splicing to an exonic cryptic-donor 21 nt upstream of the annotated-donor. NNS, MES, and DF rank the decoy-donor at +57 as the strongest donor - however this donor is enveloped within a G-repeat rich region which may mask it, and accordingly is not present in 40K-RNA. SAI instead predicts use of the cryptic-donor at −21. Notably, this cryptic-donor is present in 137 samples in 40K-RNA, providing empirical evidence that despite its weak primary motif, it can be used by the spliceosome.Figure 24 . 90% of AM-variant activated cryptic-donors are present in 40K-RNA, while 91% of unused decoy-donors are absent. Therefore, 40K-RNA provides potent predictive information with respect to both true positives (cryptic-donors) and true negatives (decoy-donors). Notably, while cryptic-donors were observed in multiple independent samples across 40K-RNA, they were typically very low frequency splice-junctions  as our predicted cryptic-donors as this maximised sensitivity (87%) without compromising specificity (95%) Fig. . Use of We define SAI prediction of cryptic-donor activation as a donor-gain Δ−score of 0.1 or greater, which accurately predicts 75% of cryptic-donors and inaccurately predicts only 1% of decoy-donors Fig. . SAI shoUse of 40K-RNA has caveats for CM-variants and AM/CM-variants, and cannot be used for variants that create a GT (or GC) motif. However, for the subset of CM-variants and AM/CM-variants where the variant modifies the extended splice site region of an extant GT/C decoy-donor . Accordingly, even if a GC decoy-donor is not present in 40K-RNA, conversion by a variant to a GT donor presents high risk for cryptic-activation. SAI performed well for CM-variants and AM/CM-variants, correctly predicting 96% of variants that created an essential donor motif and 90% which modified an existing essential motif  and of equal importance to strategise functional testing for RNA diagnostics14. While activation of a cryptic-donor 6 nucleotides away will remove or insert two amino-acids, activation of a cryptic-donor 4 nucleotides away will induce a frameshift, with vastly different implications for pathology interpretation.The ultimate goal of splicing predictions is to determine if and how a genetic variant will induce mis-splicing of pre-mRNA. Even for essential splice-site variants that almost invariably cause mis-splicing, consideration of probable consequences of the variant is critical for pathology application of the ACMG-AMP code PVS1We learned five key lessons from our analyses of 4811 cryptic-donor variants in 3399 genes: (1) Decoy-donors that show evidence of natural stochastic use by the spliceosome in population-based RNA-Seq data  have the greatest probability of activation as cryptic-donors. (2) Decoy-donors closer to the annotated splice site are inherently more likely to be used by the spliceosome, likely due to the presence of all required sequence features that are facilitating use of the annotated donor.(3) Cryptic-donors do not necessarily need to be stronger than the annotated-donor to present substantive risk for mis-splicing, with decoy-donors only 10% of the strength of the annotated-donor able to compete for spliceosomal binding. (4) Intronic G-repeats can diminish the likelihood of spliceosomal recognition and use of intronic decoy splice sites. (5) SAI’s deep-learning appreciates the broader sequence context determining spliceosomal usability of a cryptic-donor, though less accurately predicts activation of more distal cryptic-donors (>100 nt from the annotated-donor).SAI’s deep learning presents a major improvement in predicting cryptic-donor activation. However, use of SAI in a pathology context is limited by the challenge of deriving a clinically-meaningful interpretation of a number on a 0–1 scale, without independently verifiable evidence. In contrast, 40K-RNA provides an accurate, evidence-based means to rank cryptic-donors likely to be activated by genetic variants.26 used deep sequencing of twelve major cancer susceptibility genes to catalogue all alternative and aberrantly spliced transcripts. They found variant-activated cryptic splicing was often seen at much lower levels in disease controls, suggesting that the dominant transcript in rare disease may be seen as a stochastic mis-splicing event in other samples. We use this insight, mining the breadth of publicly available RNA-seq data across numerous tissues to comprehensively catalogue stochastic cryptic splicing events across all genes.Brandão et al.The heightened sensitivity and empirical nature of using 40K-RNA is of vital importance for pathology assessment of variants affecting the essential donor splice-site, as not considering a likely cryptic-donor activated can lead to profound complications in variant interpretation. Prospectively, the sensitivity of 40K-RNA can be enhanced by ultra-deep sequencing. It is also easy to envisage extending the method to predict other mis-splicing events such as exon skipping, and mis-splicing events at the acceptor splice site. 40K-RNA can reliably identify distal cryptic-donors with high likelihood of activation, which may not be identified by SAI. Conversely, SAI can reliably identify cryptic donors with high likelihood of activation not detected in 40K-RNA, due to low read depth of the target gene.27 (https://kidsneuro.shinyapps.io/splicevault-40k/). Our research establishes that for AM-variants, if a cryptic-donor is activated, in 87% of cases it will be among the top 4 events. We hope this evidence-based method may improve clinical interpretability of donor variants.In conclusion, we define an accurate, evidence-based method to predict cryptic-donor activation in the context of a variant affecting the annotated-donor, based on stochastic mis-splicing events observed in 40,233 publicly available RNA-seq samples (40K-RNA). We provide a web resource that reports and ranks the most commonly (mis)used cryptic donors proximal to every ensembl annotated-donor15 and supplemented by curation from published literature of 75 additional variants29 (2) 4372 variants derived from RNA-seq studies: Variant-associated aberrant cryptic-donor activation detected from RNA-seq data identified by SAVnet in somatic tumor samples (n = 3259)16 and 1113 variants identified in GTEx samples by spliceAI and verified using RNA-seq data11. The following inclusion criteria applied: (1) Variants had to occur within E-4-D+8 of the annotated or the cryptic-donor, otherwise they were excluded as outside the bounds of this analysis. (2) annotated cryptic-donors were within the same exon/intron as the variant . (3) The annotated cryptic-donor VAR sequence had to have an essential GT/GC dinucleotide at D+1/D+2, to minimise misannotated variants being included.Variants were derived from several sources: (1) 439 variants curated from literature, predominantly comprised of 364 variants in DBASS5-4-D+8 of the annotated-donor, it was an AM-variant, if it occurred within E-4-D+8 of the cryptic-donor it was a CM-variant, and if it occurred within E-4-D+8 of both the annotated- and cryptic-donor it was an AM/CM-variant. For 37/373 of AM/CM-variants, an additional unmodified cryptic-donor was activated, in addition to the cryptic-donor modified by the variant- these were excluded from analyses.We annotated variants with categories we defined– if the variant occurred within E30 was used to extract (up to) 500 nt of genomic sequence preceding and succeeding the annotated-donor (GRCh37). For each variant in the cryptic-donor database, we extracted up to 250 exonic nucleotides in the 5′ direction , and up to 250 intronic nucleotides in the 3′ direction, in the same fashion . We also identified any other essential donor dinucleotides  which were present in the sequence and extracted the E-4-D+8 sequence surrounding them. These sequences we define as decoy-donor- sequences containing the essential donor dinucleotides  but which weren’t utilised by the spliceosome as a result of the variant 31. Importantly, without additional filtering, no cryptic-donors in the database violated this rule.From the (up to) 500 nt of sequence we pulled Eant Fig. , lower. 13 scores were retrieved using the perl script provided at http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html. MES scores below 0 were standardised to 0 as predicted non-functional splice sites (2) NNSplice (NNS)12 scores were retrieved using the online portal (https://www.fruitfly.org/seq_tools/splice.html), set to human, with default settings  (3) SpliceAI (SAI)11 scores were retrieved using a script adapted from the SAI GitHub repository (https://github.com/Illumina/SpliceAI) which allows spliceAI to score custom sequences. We rounded the scores to three decimal places, and scores at 0 when rounded as such  were termed non-functional splice site predictions. (4) Donor Frequency (DF) calculates the median frequency among four 9 nt windows of sequence spanning the donor  MaxEntScan (MES)27, with first and last introns and single exon transcripts removed. We filtered the set so that junctions were within the open reading frame for the given gene, so we knew that cryptic splicing here would affect the protein. We also removed exons with alternative 5′ or 3′ ends already annotated in different transcripts. We used these criteria to form a set of 142,014 canonical exon-intron junctions that are not alternatively spliced (or at least not annotated as such). We extracted sequences surrounding annotated-donors and extracted all decoy-donors just as for the cryptic database (see methods section creating a database of cryptic-donor variants).Our set of naturally occurring human decoy-donors were derived from the set of all canonical Ensembl transcripts (Release 75)17 that controls for dinucleotide frequencies . We performed the shuffle 15 times and took the average count of decoy-donors at each nucleotide position as our expected count at this position. The observed count of decoy-donors was then divided by the expected count at each position.Decoy-donor depletion was calculated using a method we adapted from a previous study19 and GTEx18. Intropolis is a set of ~42 M splice-junctions found across 21,504 human RNA-seq samples from the Sequence Read Archive (SRA). Samples were aligned using Nellore et al. annotation-agnostic aligner Rail-RNA33. Intropolis was downloaded from its dedicated github repository (https://github.com/nellore/intropolis). Per sample splice-junction files were obtained from GTEx (phs000424.v8.p2 [https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000424.v8.p2]). Using Datamash34, splice-junction read counts were summarised across all samples for each unique splice-junction and translated from GRCh38 to GRCh37 using liftOver35.We had two sources of data for 40K-RNA- RNA-seq data from IntropolisFor each set of splice-junctions (Intropolis and GTEx), we cross-referenced and located junctions within ensembl transcripts. We filtered to cryptic-donor events by scanning for any unannotated donors used between the 5′ end of the exon and the 3′ end of the intron for that respective exon-intron junction, where the junction also spliced to the next annotated acceptor. Events from the two sources were merged, sample counts were tallied across the two datasets, and splice-junctions present in at least 3 samples and representing cryptic-donor use within 250 nt of any annotated-donor were retained.36.For Fig. 11. That is, the importance score of each nucleotide was calculated as:sactual is the score calculated on the genuine sequence, and sA, for example, is the score calculated when an A is substituted at this position.For Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


At present, artificial intelligence (AI) has already been applied in cardiovascular imaging  and it has been propelled to the forefront of cardiovascular medical imaging research. In this review, we presented the current status of artificial intelligence applied to image analysis of coronary atherosclerotic plaques, covering multiple areas from plaque component analysis  to risk prediction. Additionally, we discuss the current evidence, strengths, limitations, and future directions for AI in cardiac imaging of atherosclerotic plaques, as well as lessons that can be learned from other areas. The continuous development of computer science and technology may further promote the development of this field. Although modern medical care has increasingly advanced, cardiovascular disease (CVDs) that has an increasing incidence worldwide still poses a serious threat to the quality of human life and health. According to the latest report, CVDs remains the main cause of premature death in most countries, especially low- and middle-income countries , which sWith the continuous development of imaging technology and the popularization of imaging examination, massive image datasets have been generated. Meanwhile, big data are a major driver in the development of precision medicine clinicians and researchers alike have more opportunities than ever before to engage in the development and evaluation of novel image analysis algorithms, with the ultimate goal of creating new tools to optimize patient care ,5. ArtifThe aim of this paper was to focus on research that applied AI for coronary atherosclerotic plaques so as to summarize imaging methods  and different fields of coronary atherosclerotic plaques . Finally, we pointed out some current existing problems and future directions.Previous articles have described in detail AI algorithms for cardiovascular imaging ,9,10,11.Coronary atherosclerosis is a common physiological disorder characterized by the formation of fatty streaks proliferation of intimal smooth muscle cells, which eventually leads to coronary artery stenosis . AtherosDifferent components of coronary atherosclerotic plaques correspond to different mechanisms, leading to different outcomes . TherefoIn the field of noninvasive examinations, Zreik  and Rajep = 0.001) [Masuda and colleagues proposed a model combining ML with a histogram to detect the characteristics of coronary atherosclerotic plaques  in CCTA. The model shows a significantly higher area under the curve than the traditional method  . Yamak e= 0.001) . In the field of invasive examinations, Kim  and SheeThere were many studies directed at OCT. Shalev  and Xu  used a sAfter years of research, a variety of medical imaging techniques have now been used to analyze atherosclerotic plaques. These techniques can detect anatomical and functional abnormalities caused by atherosclerosis, provide detailed information about plaque composition, and even evaluate the risk of atherosclerotic plaques. These methods provide a reference for measuring the severity of coronary atherosclerosis in daily clinical practice and cardiovascular research and have an important role in the diagnosis and treatment of related patients . Mainstream noninvasive measurements of coronary atherosclerotic plaques include CT and MRI. The CT can be used to characterize luminal stenosis, assess the component load of plaques and vascular remodeling. As a noninvasive test, it can detect asymptomatic patients with high-risk plaques and stratify the risk of cardiovascular disease . At the Mainstream invasive measurements of coronary atherosclerotic plaques include OCT and IVUS, which are intravascular techniques that provide a cross-sectional view of the coronary artery. IVUS has special advantages in detecting vulnerable plaques as it can clearly distinguish the properties and composition of different plaques . Yet, IVExisting imaging tools can analyze coronary atherosclerotic plaques based on their morphology and structure, but modern precision medicine requires a more detailed analysis of plaque. A large amount of data in the image is inevitably overlooked due to the limitations of the naked eye. Additionally, the methods mentioned above produce large amounts of image data. Working long hours increases the possibility of missed diagnosis or misdiagnosis risks made by radiologist due to the subtle variations in the image that can be easily ignored. Therefore, new imaging diagnosis approaches are urgently required to improve diagnosis efficiency and accuracy by using existing medical imaging data with the ultimate goal of Precision Medicine. Vulnerable plaque rupture is the most common cause of acute coronary syndrome (ACS), which is the most dangerous type of CAD . PatholoNumerous studies have focused on the field of CCTA. Kolossvary et al. extracted 4400 radiological features from CCTA images of 60 patients by using radiomics and found that 916 features (20.6%) were associated with napkin-ring sign (NRS), of which 440 (9.9%) multiple radiographic features  were more sensitive to high-risk plaques than plaque volume and other conventional quantitative parameters . Then, tMadani formulated the training model to predict the maximum von Mises stress, which could indicate the risk of plaque rupture, and provide new ideas for the detection of high-risk plaques in the clinical field .Bae  and Jun Concerning OCT, Wang et al.  proposedp values < 0.01), indicating noninvasive hemodynamic assessment enhanced the identification of high-risk plaques that subsequently caused ACS. This study suggests that the integration of noninvasive hemodynamic assessment would enhance the prediction ability for ACS risk and may help provide optimal treatment for those high-risk patients.Fractional flow reserve (FFR) derived from coronary CTA(CT-FFR) is a promising noninvasive maker of coronary physiology and identification of high-risk plaques. Lee, J.M.  investigSince the recent machine learning algorithm with pixel-level coarse coronary segmentation was insufficient for surface model reconstruction, a new CT-FFR technique with a “Coarse-to-Fine Subpixel” algorithm for lumen contour was proposed to achieve more precise reconstructions. This technique computed subpixel level lumen contour generating the artery centerline after the first coarse coronary segmentation on a pixel level. The new technology would lead to more precise lumen boundary and vessel reconstructions and provide a high diagnostic performance in identifying hemodynamically significant stenosis, “gray zone” lesions, high-risk plaques, and severely calcified lesions.At present, the gold standard for the diagnosis of myocardial specific ischemia is the fractional flow reserve (FFR), which can guide interventional therapy and improve the prognosis of patients with CAD . The stuThe risk assessment of cardiovascular disease depends on a variety of factors, such as sex, age, weight, smoking, drinking, and so on . MoreoveIn the field of IVUS, Araki presented a model to assess the risk of coronary heart disease by combining the IVUS grayscale plaque morphology and carotid B-mode ultrasound carotid intima-media thickness (cIMT) based on SMV, which is a marker of subclinical atherosclerosis . The teaConsidering the studies of risk predicting focuses on CCTA in the field of noninvasive examination, van Assen  used ML There are still limitations in this field. In the research of ML used in coronary atherosclerotic plaques analysis, more prominent problems are the following two points: first, in almost all studies, data derived from a single research center or an old public dataset make it difficult to cover patients with different conditions and scanning parameters, making the training model difficult to satisfy the complex scenarios of clinical. Larger, rich public datasets should be established in the future for higher-quality research. Second, most of the research in this field takes the diagnostic opinion of artificial experts as the standard, lacking validation of the gold standard of pathology; therefore, the individual bias of experts may affect the accuracy of the final model. Future research on artificial intelligence for coronary atherosclerotic plaque analysis should be based on more big data; additionally, multicenter research is necessary to provide better algorithmic models.In summary, artificial intelligence has the potential to expand and improve medical technologies for better patient care, by reducing the analysis time and provide automated recommendations to physicians regarding diagnosis and downstream treatment decision making. A proposed workflow for the incorporation of machine learning and deep learning analysis of imaging modalities in clinical practice. The workflow brings in a promising algorithm, based on a recurrent convolutional neural network, for automatic detection and characterization of coronary artery plaque, as well as detection and characterization of the anatomical significance of coronary artery stenosis. The areas of AI-based cardiovascular imaging covered range from imaging analysis  to diagnostic imaging, including identifying plaques, assessing plaque vulnerability, myocardial hemodynamic evaluation, such as deep learning-based CT-FFR, and carrying out risk prognosis assessments. Specifically, the ability of the AI algorithms to make more accurate diagnoses is useful for physicians to detect diseases earlier in their course to plan for the right treatment action . With th


Purpose: To report an unusual presentation with kyrieleis like plaques in a patient with ocular Behcet’s disease.Case presentation: A 28-year-old young male presented with blurring of vision in the left eye, fundus examination revealing focal segmental intra-arterial plaques involving all branches of retinal artery characteristics of kyrieleis-like plaques with no features of retinitis in retinal periphery and mild vitritis. All routine investigations were normal, the patient testing positive for HLA-B51 marker, diagnosed as a case of ocular Bechet’s disease and managed with oral steroids and immunosuppressive agents. Results: The patient had good recovery of vision with substantial resolution of intra-arterial plaques.Conclusion: Ocular Bechet’s disease can present with uncommon features of kyrieleis like plaques. Ophthalmologists need to be vigilant for the presence of such potential ocular manifestations as the likely initial presentation in order to obtain early diagnosis and initiate timely management.  The exact pathogenesis of Kyrieleis plaques is still not known, but it has been suggested that they may represent an immunological response to an infectious agent resulting in the deposition of immune cells within or adjacent to the vessel wall [2]. Kyrieleis plaques were originally described in association with ocular tuberculosis and are most commonly seen in patients of toxoplasmic retinochoroiditis. The similar plaques have also been reported with Rickettsia conorii, Mycobacterium tuberculosis, Syphilis, and Herpes Simplex infections . It has also been hypothesized to result from extravasation of blood lipids into the arterial walls following retinal arterial damage. Wise et al. suggested that these plaques resulted from arteriosclerosis, but few others found them to be a result of the migration of exudates from active choroiditis to periarterial sheaths [4]. These intra-arterial plaques may heal on treatment with corticosteroids or antibiotics depending on aetiology [5]. It results mainly from deposition of immune cells within or adjacent to the vessel wall [6]. It typically presents as bilateral panuveitis with predominant posterior segment manifestations in the form of vitritis, retinitis, vasculitis and papillitis with potentially blinding complications [7]. Kyrieleis like plaques has not been documented as a feature of ocular Behcet’s disease. In the present case, isolated uniocular presentation with kyrieleis like plaques was seen without any significant anterior segment inflammation, which responded considerably to high dose oral steroids and immunomodulator drugs. Behcet’s disease is typically characterized by a triad of oral ulcers, genital ulcers, and ocular lesions [Our case emphasized that Bechet’s disease can present with atypical ocular features like intraarterial plaques. Therefore, the presence of kyrieleis like plaques in an individual with signs of uveitis, should prompt an ophthalmologist for early systemic investigation, to diagnose and treat underlying aetiology with good visual recovery. Conflict of Interest statementThe author(s) declares that they have no potential conflicts of interest.Informed Consent and Human and Animal Rights statementInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the review board of Military Hospital, Kirkee, Pune, India.AcknowledgementsNil.Sources of FundingNone to declare.DisclosuresNone.


Idiopathic normal pressure hydrocephalus (INPH) is a neurodegenerative disorder characterized by excess cerebrospinal fluid (CSF) in the ventricles, which can be diagnosed by invasive CSF drainage test and treated by shunt placement. Here, we aim to investigate the diagnostic and prognostic power of systematic volumetric analysis based on brain structural MRI for INPH.We performed a retrospective study with a cohort of 104 probable INPH patients who underwent CSF drainage tests and another cohort of 41 INPH patients who had shunt placement. High-resolution T1-weighted images of the patients were segmented using an automated pipeline into 283 structures that are grouped into different granularity levels for volumetric analysis. Volumes at multi-granularity levels were used in a recursive feature elimination model to classify CSF drainage responders and non-responders. We then used pre-surgical brain volumes to predict Tinetti and MMSE scores after shunting, based on the least absolute shrinkage and selection operator.r = 0.80 for Tinetti and r = 0.88 for MMSE. The anatomical features that played important roles in the diagnostic and prognostic tasks were also illustrated.The classification accuracy of differentiating the CSF drainage responders and non-responders increased as the granularity increased. The highest diagnostic accuracy was achieved at the finest segmentation with a sensitivity/specificity/precision/accuracy of 0.89/0.91/0.84/0.90 and an area under the curve of 0.94. The predicted post-surgical neurological scores showed high correlations with the ground truth, with We demonstrated that volumetric analysis with fine segmentation could reliably differentiate CSF drainage responders from other INPH-like patients, and it could accurately predict the neurological outcomes after shunting.• We performed a fully automated segmentation of brain MRI at multiple granularity levels for systematic volumetric analysis of idiopathic normal pressure hydrocephalus (INPH) patients.• We were able to differentiate patients that responded to CSF drainage test with an accuracy of 0.90 and area under the curve of 0.94 in a cohort of 104 probable INPH patients, as well as to predict the post-shunt gait and cognitive scores with a coefficient of 0.80 for Tinetti and 0.88 for MMSE.• Feature analysis showed the inferior lateral ventricle, bilateral hippocampus, and orbital cortex are positive indicators of CSF drainage responders, whereas the posterior deep white matter and parietal subcortical white matter were negative predictors.The online version contains supplementary material available at 10.1007/s00330-020-07531-z. Idiopathic normal pressure hydrocephalus (INPH) is characterized by the clinical triad of dementia, gait dysfunction, and urinary incontinence due to excess cerebrospinal fluid (CSF) buildup in the brain. In the absence of any proven medical therapy, surgical placement of a shunt to drain excess CSF is shown to be an effective treatment , 2, althMRI features of INPH involve ventricular enlargement, steep callosal angle, Sylvian fissure expansion, disproportionately enlarged subarachnoid space hydrocephalus (DESH), and cerebral atrophy . These fThe previous volumetric studies of INPH used coarse tissue segmentation of the GM, WM, and CSF, or examined single brain region based on certain hypotheses, but did not systematically evaluate the whole-brain structural volumes and their distribution. In this study, we aim to evaluate the full potential of volumetric analysis in the diagnosis and prognosis of INPH, using an automatic whole-brain segmentation pipeline  that pari) a diagnosis of probable INPH based on routine radiological reports, characteristic gait, and cognitive symptoms; (ii) completion of gait assessments before and within 2 h after the CSF drainage test via LVLP; and (iii) completion of MRI scans between 1 and 3 months prior to the test. Patients with obstructive hydrocephalus, congenital hydrocephalus, or secondary hydrocephalus were excluded. One hundred four patients were selected according to these criteria, with their demographic and clinical information summarized in Table Probable INPH were included to test the diagnostic accuracy of separating responders to CSF drainage from non-responders. Inclusion criteria were as follows:  tests before shunting and within 1 year after shunting and had MRI scans between 0 and 10 months prior to shunting. The exact interval between the tests and shunt surgery and between surgery and MRI scans are provided in Table Patient data were retrospectively collected between the years of 2009 and 2016. All research protocols were approved by the local Institutional Review Board. Written informed consent was obtained from all patients in this study. Two patient cohorts were included in this study:A physical therapist (A.M.) administered the Tinetti Performance Oriented Mobility Assessment  and TUG All patients had 3D high-resolution T1-weighted images scanned on a 3-T Trio or Verio scanner (Siemens). Images were acquired using an MPRAGE sequence with the following imaging parameters: field of view of 240 × 240 mm, in-plane resolution of 0.76–0.94 mm, slice thickness of 0.9–1.2 mm, sagittal orientation, echo time of 2.1–3.25 ms, repetition time of 1800–2110 ms, and flip angle of 8–9°, parallel acceleration factor of 2, and a scan time of approximately 5 min.www.mricloud.org). Details of the segmentation algorithm were provided in [The high-resolution T1-weighted images were segmented using a multi-atlas algorithm  via a clvided in . We usedvided in  that wasvided in . Five grvided in .Fig. 1Muscikit-learn.org). Age and gender were also included in the model as additional features. A linear support vector machine (SVM) was incorporated into the RFE as the classifier. Class-weighting [The discriminative features (ROI volumes) for differentiating responders to CSF drainage were selected using a recursive feature elimination (RFE) method  using theighting  was usedwww.r-project.org). The ROI volumes at levels 1–4, along with the age, gender, test score before shunting, the time between shunt surgery and post-surgical test, and time between the pre-surgical MRI and shunt surgery, were used as predictors. The regularization factor in LASSO was kept at 0.5 for training. ROI volumes at level 5 were not used because the number of features (n = 283) was too large for this cohort (n = 37for MMSE and 47 for Tinetti). We used leave-one-out cross-validation to predict the outcomes of individual patients, and correlated the predicted scores with the ground truth.We used the least absolute shrinkage and selection operator (LASSO)  method tt test with unequal variance for Evan’s index and MMSE before and after CSF drainage, and chi-square test for gender. The Wilcoxon matched-pairs signed-rank test was used to compare the test scores before and after CSF drainage or shunt surgery in cohort 2. The statistical tests were performed in R.Differences of the clinical characteristics between the responders and non-responders in cohort 1 were tested using the Wilcoxon rank-sum test for data not satisfying normal distribution , unpaired p < 0.013). Ninety-four percent of the responders and 91.3% of the non-responders had Evan’s index above the threshold of 0.3, which is one of the diagnostic criteria for INPH [The demographic and clinical characteristics were equivalent between the responder and non-responder groups Table , except for INPH . Using EFigure The ROIs selected by RFE and their weights were extracted to characterize the discriminative features in classification. The weight maps in Fig. r of 0.36  between the LASSO predicted Tinetti scores and the clinically measured ground truth were 0.55, 0.56, 0.76, and 0.80 at levels 1, 2, 3, and 4, respectively . The predicted MMSE after shunting was strongly correlated with the ground truth with r’s of 0.85, 0.86, 0.87, and 0.88 at levels 1, 2, 3, and 4, respectively . The predicted scores at level 4 were shown in Fig. r = 0.48 for the Tinetti and r = 0.80 for MMSE).The pre-shunt Tinetti showed a moderate correlation with the post-shunt Tinetti with an r values at levels 1–4 were 0.53, 0.53, 0.77, and 0.75 for Tinetti and 0.61, 0.75, 0.69, and 0.83 for MMSE, suggesting that the structural volumes alone were also predictive of the post-shunt outcome but combining pre-shunt information could further enhance the performance. Evan’s index–based prediction accuracy was only 0.42 and 0.46 for the Tinetti and MMSE, correspondingly, without the pre-shunt scores.We also tested the predictive performance of the LASSO models without the pre-shunt scores. The The weights of the predictors in the LASSO model were examined for their contributions in the prediction. The features selected in predicting Tinetti primarily involved the ventricle and sulci, such as the right parietal and frontal sulci and bilateral inferior LV Fig. . Noticear = 0.80–0.93).This study investigated the role of systematic volumetric analysis for the diagnosis of INPH shunt candidates as assessed by their response to CSF drainage and prediction of neurological outcomes after shunt placement. We achieved a high diagnostic accuracy of 0.94 in discriminating CSF drainage responders from non-responders in a cohort of probable INPH patients. The volumetric analysis also showed promising performance in predicting the gait and cognitive outcomes after shunt placement, with high correlations with the ground truth  supports the feasibility of using pre-surgical MRI to estimate patient outcomes. The features associated with the prediction showed distinctive patterns for Tinetti and MMSE. While the ventricle and sulci played essential roles in predicting Tinetti, the cortical gyri and WM regions were selected for MMSE prediction, indicating the gait and cognitive outcomes may be associated with different brain regions. This finding agreed with previous findings that CSF-based anatomical features were more effective in predicting gait outcomes than predicting cognitive outcomes [The use of brain MRI in predicting neurological outcome in post-surgical INPH was largely limited to qualitative or semi-quantitative assessments, such as the DESH scale , 19, 21.= - 0.79  and that= - 0.79 ; Ahmed e= - 0.79 . We showoutcomes , 19, 21.There are several limitations in the current study. Due to the limited patient number in cohort 2, we could not use the volumetric analysis at the finest segmentation as the number of volumetric features far exceeded the sample size. A larger cohort would allow a more flexible analysis and higher prediction accuracy. Also, the current study only retrospectively included patients from a single site with a homogenized imaging protocol. Future prospective multi-center studies are needed to test the generalizability and accuracy of the proposed method. Moreover, the current study only concerned brain volumes, and morphological features, such as the callosal convexity and DESH, were not assessed or compared. Future work that incorporates the volumetric and shape information into the feature analysis may further improve the prediction accuracy. Also, a combination of the morphological analysis with advanced imaging techniques, such as phase-contrast-based CSF flow imaging , diffusiIn summary, we demonstrated that systematic volumetric analysis with fine segmentation could reliably differentiate responders to CSF drainage test and predict the neurological outcomes after shunt placement in INPH patients. The high-performance diagnostic tool with cloud-based multi-granularity segmentation could be readily integrated into clinical routine.ESM 1(DOCX 23 kb)


The spermatogonial stem cell (SSC) is the only adult stem cell in males to transmit genetic information to offspring. SSC transplantation (SSCT) is a laboratory technique to regenerate spermatogenesis in recipient males, thus can be used as a novel breeding tool to benefit animal production. Although successful SSCT in rodent models has been established, progress in realizing SSCT in large animals has been limited. Here we discuss what we learned in this area from past experiments and highlight possible directions in developing effective SSCT protocol in large animals.Spermatogonial stem cell transplantation (SSCT) can restore male fertility through transfer of germline between donor and recipient males. From an agricultural perspective, SSCT could be an important next-generation reproductive and breeding tool in livestock production. Current SSCT approaches in large animals remain inefficient and many technical details need further investigation. This paper reviews the current knowledge on SSCT in large animals, addressing (1) donor spermatogonial stem cell (SSC) preparation, (2) recipient male treatment, and (3) SSC injection, homing, and detection. The major studies showing unequivocal evidence of donor SSC-derived spermatogenesis in large animals (mainly in livestock for breeding purpose) are summarized to discuss the current status of the field and future directions. Spermatogenesis, a highly complex and tissue-coordinated process, maintains sperm production throughout the lifetime of male mammals. The high efficiency of the spermatogenesis system depends on the continuous proliferation and differentiation of spermatogonial stem cells (SSCs) ,2,3. SSCFor the first time in 1994, Brinster and his colleagues transplanted testicular cell suspensions containing SSCs from fertile donor mice into the seminiferous tubules of the testes of infertile recipient mice. They observed complete donor cell-derived spermatogenesis, which resulted in the formation of mature sperm, and obtained the offspring of donor spermatogonia through natural mating ,9. This The purpose of this review is to provide the latest information in the field of SSCT study in large animal models, focusing on the use of agricultural animals as a breeding tool for surrogacy purposes. We provide an overview of current progress in SSC biology, recipient preparation, and cell transfer methods, and summarize the key attempts and outcomes of SSCT in various large animal models.Bos taurus bulls and 9–9.5 months for Bos indicus bulls [SSCs derive from gonocytes, which come from primordial germ cells (PGCs) in early embryos ,38,39. Dus bulls ,45. The us bulls .single spermatogonia (As), Apaired spermatogonia (Apr) (chains of 2 cells connected by an intercellular bridge), and Aaligned spermatogonia   generated by successive mitotic divisions. The least differentiated As spermatogonia are generally considered to be SSCs, while Apr are considered to be progenitor spermatogonia [al cells transition without cell division to A1 differentiating spermatogonia [1, A2, A3, A4, In, and B spermatogonia [1, A2, A3, In, B1, and B2 spermatogonia [The stem cell pool in testis is maintained and expanded through self-renewing divisions of SSCs and a series of mitotic divisions that facilitate the transition of SSCs to spermatogonia with different states. The spermatogenic process is generally similar in almost all mammals . In the atogonia . Broadlyatogonia ,49, and atogonia . In cattatogonia . Afterwa6/g depending on species [6/g DSP [6/g DSP [Spermatogenesis in mammals occurs in a finely regulated and highly productive manner. The frequency of the spermatogenic cycle is around 10 days and the whole of spermatogenesis is estimated to consume 1–2.5 months (approximately 4.5 cycles) in the mammalian species that were already investigated . Daily s species ,50. In p[6/g DSP ,51,52. HLike other adult stem cells, SSCs are very sparse in number. There are only about 35,000 SSCs per testis in adult mice, accounting for about 0.03% of the total number of germ cells in testis . In neonDolichos biflorus agglutinin, to specifically enrich pig primitive germ cells, and cultured purified gonocytes in medium containing 10% FBS for a short time [While researchers had success with these long-term in vitro cultures in rodent models, several teams only achieved short-term culture of large animal SSCs under similar culture conditions. Goel et al. used a lectin, ort time ,75. Howeort time . Thereafort time . Recentlort time . So far,ort time ,74,75,76ort time , and goaort time , but wheAnother key issue that restricts the effective application of SSCT in large animals is the preparation of ideal recipients that can support re-established donor-derived spermatogenesis following cell transfer. The ideal recipients used in SSCT should have endogenous spermatogenesis ablation or suppression while retaining complete somatic cell structure and function . EndogenThe chemotherapeutic agent busulfan can effectively ablate endogenous germline cells to support SSCT in mouse models. However, its effectiveness in preparing large animal recipients is limited. Busulfan usually cannot ablate the germ cell pool completely, and different degrees of recovery of endogenous germ cells are almost inevitable. This could preclude the homing of transplanted SSCs. Busulfan is also systemically toxic, causing bone marrow suppression and anemia . A speciThe latest gene editing technology provides new options for eliminating endogenous SSCs in animals. Using gene editing technology to knock out (or inactivate) a certain gene, which only expresses in male germ cells or plays a key role in their survival and development can selectively eliminate endogenous SSCs without affecting the integrity of somatic support cell structure and function, thus providing sufficient niches for colonization and differentiation of transplanted SSCs ,90. The Three routes are usually used to inject donor cells into recipient testes. SSCs can either be injected into the efferent ducts connecting the rete tubules and epididymis, the rete tubules formed by the convergent ends of all seminiferous tubes, or the seminiferous tubes themselves ,92. In mThe homing ability of stem cells allows them to migrate and colonize existing niches. SSC homing refers to a process in which the transplanted SSCs pass through the blood-testis barrier (BTB) formed between two supporting cells in the opposite direction toward open niches on the basement membrane . Homing Application of SSCT in livestock as a breeding tool requires allogeneic or homologous transplantation. This requires consideration of the genetic and immune compatibility between recipients and cell donors. On the one hand, the success of SSCT seems to depend on the availability of recipients that are either genetically compatible with the donor animal, or have innate immune deficiency or receive immunosuppressive treatment ,104. On Escherichia coli β-galactosidase gene (lacZ) or enhanced green fluorescent protein (EGFP)) to label donor cells. Generally, researchers use lentiviral or adeno-associated viral (AAV) vectors to deliver reporter genes to donor SSCs for transplantation. Using this method, recipient ejaculate harboring donor-derived transgenic sperm can be detected after SSCT in pigs [Tracing the colonized SSCs and re-established spermatogenesis that results from SSCT is generally challenging; most previous attempts in large animals involved the administration of cytotoxic treatments to recipients, resulting in residual endogenous spermatogenesis. Some studies used fluorescent dyes ) to monitor the location of the donor cells and the duration of their persistence in the seminiferous tubules of recipient testes ,21,22,24 in pigs ,14, goat in pigs ,109,110, in pigs , and mon in pigs . Further in pigs ,14, goat in pigs , as well in pigs  and tree in pigs , validat in pigs ,112. Lik in pigs ,32,33. TOverall, the current experimental approaches to donor SSC-derived spermatogenesis remain inefficient; low proportions of donor genotypes are readily identifiable in recipients in the existing literature, especially in large animals. We here draw a brief schematic diagram outlining the typical procedure of SSCT in laboratory mouse models and large animals . The proIn this section, we summarize the clear evidence from previous experiments that donor-derived spermatogenesis occurs following SSCT in large animals. The studies that only included single lines of evidence, such as repopulation of transplanted donor cells identified by fluorescent dye labeling or histological assessment, or genotyping of ejaculate by microsatellite markers or transgenic DNA, are not included here. In addition, SSCT in monkey models is also excluded, as these models are usually developed as a strategy to address human infertility caused by chemotherapy or radiotherapy. In these approaches, testicular tissue cryopreservation and autologous transplantation are required, which differs from SSCT application in livestock for breeding purposes. The technical details of these reports are listed in In the first example referred to in Honaramooz et al. also demonstrated success in goats, transplanting the mixed testicular cells isolated from transgenic goat testes expressing human alpha-1 antitrypsin into untreated immature wild-type recipient goats, using the rete testis injection method. The presence of transgene was detected in the ejaculate of 2 recipients, and transgenic offspring were obtained through natural mating . This stSimilar strategies were also employed in sheep. Herrid et al. transplanted isolated sheep testicular cell suspension into recipient testes (from the same or different breeds) treated with 9 or 15 Gy irradiation. The semen collected starting from 6–10 months after transplantation showed donor-derived DNA in some ram recipients. Production of donor-derived sperm was detected beginning at 15 weeks after SSCT. Animals receiving 15 Gy irradiation generated more significant donor-derived sperm than 9 Gy and non-irradiated groups. The proportion of donor sperm in 15 Gy irradiation recipients was 9.7% on average, with the highest percentage (30%) identified at 40 weeks. Subsequently, the semen of transplanted recipients was used to perform AI on 151 ewes whose estrus cycles were synchronized. Microsatellite marker analysis of the newborn lambs showed that 4 donor-derived lambs (7.6% of progeny) resulted from a recipient in Merino-to-Merino transplantation, and 6 lambs (14.6% of progeny) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram . These cGermline ablation through genetic modification substantially advances SSCT progress in large animals. Genetic sterility can completely and inheritably remove endogenous germ cells without impairing somatic supporting cells and testicular function. The sterile condition in animals is stable without recovery of endogenous spermatogenesis over time. Demonstrating this, Park et al. generated NANOS2 knockout pigs by directly injecting the CRISPR reagent into the cytoplasm of pig embryos to produce inactivating mutations in the NANOS2 coding region. The resultant NANOS2 knockout pigs mimicked the phenotype of knockout mice, showing a loss of SSCs while retaining intact seminiferous tubules. Serum testosterone secretion level was not different from that of normal pigs . The resModern livestock production pursues high-efficiency propagation of elite animals with desirable genetics. To this end, alternative and novel reproductive techniques applicable to livestock have been subjects of ongoing study for multiple generations, from natural breeding to AI, SCNT, and then SSC manipulation. The SSCT-based “surrogate sires” concept proposed by some active investigators in this field can rapidly and selectively proliferate and disseminate gametes from desirable sires (without having to use the sires themselves), significantly accelerating the progress of livestock breeding and production ,114,115.In light of the recent success of NANOS2-knockout large animal models in SSCT application, cultivating such genetically sterile recipient males could be a simple and uniform strategy to facilitate effective SSCT in large animals. Many other genes associated with germline maintenance and development can be explored and modified to create ideal recipient male models. In addition, an efficient genetic modification technique in SSCs should be investigated to support generation of SSC-derived transgenic or gene-edited large animals, overcoming the low efficiency, and high cost of embryo-based genome modification approaches including pronuclear injection and SCNT.