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Abstract Decidualization of the human endometrium is critical for establishing pregnancy and is entailed by differentiation of endometrial stromal cells (ESCs) into decidual cells. During decidualization, the actin cytoskeleton is dynamically reorganized for the ESCs’ morphological and functional changes. Although actin dynamically alters its polymerized state upon external stimuli not only in the cytoplasm, but also in the nucleus, nuclear actin dynamics during decidualization have not been elucidated. Here, we show that nuclear actin was specifically assembled during decidualization of human ESCs. This decidualization-specific formation of nuclear actin filaments was disassembled following the withdrawal of the decidualization stimulus, suggesting its reversible process. Mechanistically, RNA-seq analyses revealed that the forced disassembly of nuclear actin resulted in the suppression of decidualization, accompanied with the abnormal upregulation of cell proliferation genes, leading to incomplete cell cycle arrest. CCAAT/enhancer-binding protein beta (C/EBPβ), an important regulator for decidualization, was responsible for downregulation of the nuclear actin exporter, thus accelerating nuclear actin accumulation and its assembly for decidualization. Taken together, we demonstrate that decidualization-specific nuclear actin assembly induces cell cycle arrest for establishing the decidualized state of ESCs. We propose that not only the cytoplasmic actin, but also nuclear actin dynamics profoundly affect decidualization process in humans for ensuring pregnancy.

Objective: Reports demonstrating the effectiveness and safety of strut-adjusted volume implants (SAVI) in Japan are limited. Therefore, this study aimed to compare the treatment outcomes of SAVI and whole-breast irradiation (WBI) at a single facility. Materials and Methods: Data were retrospectively extracted from the medical records of patients treated with SAVI or WBI following partial mastectomy (Bp). Patients undergoing Bp, sentinel lymph node biopsy, and SAVI spacer insertion followed by brachytherapy with the SAVI device were compared to those followed with WBI. Local recurrence was assessed annually by physical examination, bilateral mammography, and breast ultrasonography. Results: The SAVI and WBI groups comprised 53 and 113 patients, with a median age of 55 and 52 years, respectively; among them, 47 and 91 patients had a pathological tumor diameter ≤2 cm and six and 22 had a pathological tumor diameter >2 cm, respectively. Recurrence events, acute adverse events, and late adverse events were observed in the SAVI and WBI groups in 1 and 3 (p = 0.726), 24 and 79 (p = 0.01), and 24 and 18 patients (p = 0.00002), respectively, with median observation periods of 60.0 and 47.8 months, respectively. All adverse events were grades 1–2, with dermatitis being the most common in the acute phase. In the late phase, pigmentation was common in both groups. Conclusion: The local recurrence rate does not differ between SAVI and WBI within the relatively short-term follow-up period. Longer follow-up is required to confirm our results in the Japanese population.

Abstract Background The COVID‐19 outbreak made conventional medical care impossible, forcing changes in both healthcare providers and patients. In Japan, COVID‐19 infection began spreading in earnest in 2020 and exploded in 2021. There was concern that the medical impact of COVID‐19 in 2021 would differ from that in 2020. We aimed to clarify the impact of COVID‐19 on mortality and anastomotic leakage in laparoscopic surgery for gastric cancer and rectal cancer in Japan using the National Clinical Database (NCD). Methods We collected data from patients who underwent laparoscopic distal gastrectomy (LDG) and laparoscopic low anterior resection (LLAR) from January 2018 to December 2021 from the NCD, a web‐based surgical registration system in Japan. The number of surgical cases, monthly incidence of mortality and morbidity (anastomotic leakage), standardized mortality ratio (SMR), and standardized morbidity‐leakage ratio (SMLR [ratio of observed patients to expected patients calculated using the risk calculator established in the NCD]) were evaluated. Results The numbers of LDG and LLAR cases continued to decline in the first year of the pandemic in 2020 and were as low in 2021 as in 2020. Although the numbers of robot‐assisted LDG and LLAR cases increased, the growth rate was lower than the rate of increase prior to the pandemic. Mortality and anastomotic leakage, two of the most important complications, as assessed by SMR and SMLR, did not worsen during the pandemic in comparison to the pre‐pandemic period. Conclusions Laparoscopic surgeries were performed safely in Japan and were not affected by the COVID‐19 pandemic.

Yu Kawasaki,1 Takatoshi Kasai,2– 5 Yuko Sakurama,6 Fusae Kawana,2 Nanako Shiroshita,2 Natsue Koikawa6,7 1Department of Obstetrics and Gynecology, Juntendo University Graduate School of Medicine, Tokyo, Japan; 2Cardiovascular Respiratory Sleep Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan; 3Department of Cardiovascular Management and Remote Monitoring, Juntendo University Graduate School of Medicine, Tokyo, Japan; 4Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan; 5Sleep and Sleep-Disordered Breathing Center, Juntendo University Hospital, Tokyo, Japan; 6Japanese Center for Research on Women in Sport, Juntendo University, Tokyo, Japan; 7Graduate School of Health and Sports Science, Juntendo University, Chiba, JapanCorrespondence: Takatoshi Kasai, Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 3-1-3 Hongo, Bunkyo-ku, Tokyo, Japan, Tel +81-33813-3111, Fax +81-5689-0627, Email kasai-t@mx6.nisiq.netPurpose: Female athletes with menstrual abnormalities have poor sleep quality. However, whether female athletes with poor sleep quality based on subjective assessment have distinctive changes in objective measures of sleep in association with menses remains unclear. This study aimed to compare changes in objective sleep measurements during and following menses between collegiate female athletes with and without poor subjective sleep quality.Patients and Methods: Female collegiate athletes (age range/mean ± standard deviation: 18– 22/ 22.2± 1.1) with regular menstrual cycles were recruited. The participants underwent home electroencephalogram monitoring during the first and second nights after the onset of menses and one night between the seventh and 10th nights after menses onset (mid-follicular phase). The Pittsburgh Sleep Quality Index (PSQI) was used to assess the subjective sleep quality. Interactions between the presence of poor subjective sleep quality (ie, PSQI ≥ 6) and changes in objective measures of sleep in association with menses were analyzed.Results: Data of 45 athletes, including 13 with poor subjective sleep quality, showed that changes in arousal index in athletes with poor subjective sleep quality were distinctive from those in athletes without poor subjective sleep quality (p = 0.036 for interaction). In athletes with poor subjective sleep quality, the arousal index was significantly increased in menses (p for analysis of variance, 0.015), especially on the first night after the onset of menses compared with during the mid-follicular phase (p = 0.016).Conclusion: Collegiate female athletes with regular menstrual cycles are likely to have poor subjective sleep quality in association with more frequent arousal during the first night after the onset of menses than during the mid-follicular phase.Keywords: electroencephalogram, menstruation, Pittsburgh sleep quality index, sports, women

Abstract Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E2). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E2 + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E2 + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E2 + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

The joint effect of air pollutants at relatively low levels requires further investigation. Here, a database study was performed to evaluate the effects of exposure to mixtures of air pollutants during pregnancy, infancy, and childhood on childhood persistent asthma. We used the Japan Medical Data Center database, which provides access to family linkages and healthcare provider addresses, and included child-mother dyads in which the child was born between January 2010 and January 2017. The exposure of interest was ground-level air pollutants, and the primary outcome was childhood persistent asthma at 45 years of age, as defined based on outpatient and inpatient asthma disease codes and/or asthma medication dispensing claims. The weighted quantile sum (WQS) regression was used to evaluate the effects of air pollutant mixtures on 52,526 child-mother dyads from 1149 of 1907 municipalities (60.3 %) in Japan. The WQS regression models showed that with every 10th percentile increase in the WQS index, ground-level air pollutants during pregnancy, infancy, and childhood increased the risk of childhood persistent asthma by an odds ratio of 1.04 (95 % CI: 1.02–1.05; p

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Identifying child age of RSV infection associated with increased risk of asthma is important for developing asthma prevention strategies. Our systematic review aimed to comprehensively summarize studies of the association between age of RSV infection and childhood asthma risk. The study protocol was pre-registered, and our study report adhered to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA). Inclusion criteria were prospective and retrospective cohort studies and case-control studies which assessed the association of age of RSV infection before age 2 years and risk of childhood asthma after age two years. Relevant studies were identified through MEDLINE, Embase, Cochrane and International Clinical Trials Registry Platform (ICTRP) from study inception through May 5, 2023. Studies were evaluated with the Quality In Prognosis Studies (QUIPS) tool. From 149 studies screened, five studies (two prospective cohort studies and three retrospective cohort studies) were included in our systematic review, including 47,603 participants. Available studies only assessed age of severe RSV infection and asthma risk. The included studies used different age categories and outcome definitions, and were rated as having high risk of bias. Two studies had sample sizes of less than 300 and did not provide conclusive results related to age of RSV hospitalization and asthma risk. The other three studies reported RSV hospitalization between age 6 months and 23 months compared with age 0-6 months being associated with a higher odds ratio, hazard ratio, or incidence rate ratio of asthma diagnosis/hospitalization. Due to the heterogeneous epidemiological designs, including exposures and outcome ascertainments of the included studies, we could not perform a meta-analysis, or calculate weighted averages of the effect estimates. Our systematic review highlights a major gap in our knowledge about the relationship between age of RSV infection and asthma risk.

Abstract Background Segmental absence of intestinal musculature (SAIM) is a partial defect of the intrinsic muscular layer of the intestinal tract. In this report, we describe a case of perforation of the sigmoid colon due to SAIM accompanied by vascular Ehlers–Danlos syndrome (vEDS), which was successfully treated by surgical therapy. Case presentation A male in his 30 s was being followed up for vEDS diagnosed by genetic testing. He had undergone two major vascular surgeries, abdominal aortic artery revascularization and thoracic endovascular aortic repair for a residual dissection and enlarging abdominal aortic aneurysm. On postoperative day 11, the patient developed perforation of the sigmoid colon for which intraperitoneal lavage and drainage, Hartmann surgery, and transverse colostomy were performed. Histological findings showed no disturbance of blood flow or diverticulum but did show a defect in the intrinsic muscular layer around the perforation site, leading to the pathological diagnosis of SAIM and associated perforation of the sigmoid colon. Postoperatively, the patient had no complications and was discharged on postoperative day 18. The patient is being followed as an outpatient and has experienced no relapse. Conclusions Both SAIM and vEDS, which may be related diseases, are associated with the presence of tissue fragility and have a high potential to cause intestinal perforation Caution should be exercised during surveillance in patients with constipation and examinations that cause increased intestinal pressure.

Abstract Background “Arterialization” of the dorsal hand vein is well‐established in human medicine, but not in veterinary medicine. Objectives To compare cephalic and saphenous venous blood collected by continuously heating the paws to 37°C (“arterialization”), with arterial blood (AB) for measurement of blood gas variables in well‐perfused dogs. Animals Eight healthy dogs. Methods Experimental study. Fore and hind paws were continuously heated to 37°C to “arterialize” cephalic and saphenous venous blood. AB and “arterialized” cephalic and saphenous venous blood (ACV and ASV, respectively) were simultaneously collected from lightly anesthetized dogs with induced metabolic and respiratory acid‐base disorders. The pH, partial pressures of carbon dioxide (PCO2) and oxygen (PO2), bicarbonate concentration [HCO3−], and base excess (BE) were measured once in each state. Systolic blood pressure was maintained above 100 mm Hg. The AB, ACV, and ASV values were compared. Results The pH, [HCO3−], and BE values had no significant difference and good agreement, the PCO2 values had a strong correlation (correlation coefficient of .91‐1.00), and the PO2 values had a significant difference (P

Abstract Aim This study aimed to evaluate the impact of the coronavirus disease (COVID‐19) pandemic on elective endoscopic surgeries in Japan using the National Clinical Database. Methods We retrospectively analyzed the clinicopathological factors and surgical outcomes of laparoscopic cholecystectomy (LC), laparoscopic distal gastrectomy (LDG), and laparoscopic low anterior resection (LLAR) and compared the monthly numbers of each procedure performed in 2020 with those in 2018 and 2019. The degree of infection in prefectures was classified into low and high groups. Results In 2020, the number of LCs (except for acute cholecystitis) was 76 079 (93.0% of that in 2019), the number of LDGs was 14 271 (85.9% of that in 2019), and the number of LLARs was 19 570 (88.1% of that in 2019). Although the number of robot‐assisted LDG and LLAR cases increased in 2020, the growth rate was mild compared with that in 2019. There was little difference in the number of cases in the degree of infection in the prefectures. The numbers of LC, LDG, and LLAR cases decreased from May to June and recovered gradually. In late 2020, the proportion of T4 and N2 cases of gastric cancer and the number of T4 cases of rectal cancer increased compared with those in 2019. There was little difference between the proportions of postoperative complications and mortality in the three procedures between 2019 and 2020. Conclusion The number of endoscopic surgeries decreased in 2020 as a result of the COVID‐19 pandemic. However, the procedures were performed safely in Japan.

Evidence on the optimal antibiotic strategy for empyema is lacking. Our database study aimed to evaluate the effectiveness of empirical anti-pseudomonal antibiotics in patients with empyema. We utilised a Japanese real-world data database, focusing on patients aged ≥40 diagnosed with empyema, who underwent thoracostomy and received intravenous antibiotics either upon admission or the following day. Patients administered intravenous vasopressors were excluded. We compared thoracic surgery and death within 90 days after admission between patients treated with empirical anti-pseudomonal and non-anti-pseudomonal antibiotics. Cause-specific hazard ratios for thoracic surgery and death were estimated using Cox proportional hazards models, with adjustment for clinically important confounders. Subgroup analyses entailed the same procedures for patients exhibiting at least one risk factor for multidrug-resistant organisms. Between March 2014 and March 2023, 855 patients with empyema meeting the inclusion criteria were enrolled. Among them, 271 (31.7%) patients received anti-pseudomonal antibiotics. The Cox proportional hazards models indicated that compared to empirical non-anti-pseudomonal antibiotics, empirical anti-pseudomonal antibiotics were associated with higher HRs for thoracic surgery and death within 90 days, respectively. Thus, regardless of the risks of multidrug-resistant organisms, empirical anti-pseudomonal antibiotics did not extend the time to thoracic surgery or death within 90 days.

Abstract Aim Whether a laparoscopic procedure can contribute to the improvement of clinical outcomes in obese patients with stage II/III rectal cancer compared to an open procedure remains unclear. Objective This study evaluated the technical and oncological safety of laparoscopic surgery versus open surgery in obese patients (body mass index [BMI] ≥25 kg/m2) with rectal cancer. Patients and Methods Data were collected from patients with pathological stage II/III rectal cancer and analyzed. Operations were performed via laparoscopic or open surgery from 2009 to 2013. A comparative analysis was performed after applying propensity score matching to the two cohorts (laparoscopic group and open group). The primary endpoint was 3‐y relapse‐free survival (RFS). Results Overall, 524 eligible cases were collected from 51 institutions. Equal numbers of propensity score‐matched patients were included in the laparoscopic (n = 193) group and open (n = 193) group. Although the rate of D3 lymph node dissection did not differ between the laparoscopic group (87.0%) and the open group (88.6%), the median number of harvested lymph nodes was significantly lower in the laparoscopic group versus open group (17.5 vs 21, P = 0.0047). The median postoperative hospital stay was also significantly shorter in the laparoscopic group (14 d) vs the open group (17 d) (P = 0.0014). Three‐y RFS was not significantly different between the two groups (hazard ratio 1.2454, 95% confidence interval 0.9201–1.6884, P = 0.4689). Conclusion The short‐ and long‐term results of this large cohort study (UMIN ID: UMIN000033529) indicated that laparoscopic surgery in obese rectal cancer patients has advantageous short‐term outcomes and no disadvantageous long‐term outcomes.

Akihiro Shiroshita,1– 3 Shungo Yamamoto,4 Keisuke Anan,3,5 Hokuto Suzuki,1 Masafumi Takeshita,1 Yuki Kataoka3,6– 8 1Department of Respiratory Medicine, Ichinomiyanishi Hospital, Ichinomiya, Japan; 2Division of Epidemiology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA; 3Scientific Research Works Peer Support Group (SRWS-PSG), Osaka, Japan; 4Department of Infectious Disease, Kyoto City Hospital, Kyoto, Japan; 5Division of Respiratory Medicine, Saiseikai Kumamoto Hospital, Kumamoto, Japan; 6Department of Internal Medicine, Kyoto Min-Iren Asukai Hospital, Kyoto, 606-8226, Japan; 7Section of Clinical Epidemiology, Department of Community Medicine, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan; 8Department of Healthcare Epidemiology, Kyoto University Graduate School of Medicine/Public Health, Kyoto, 606-8501, JapanCorrespondence: Akihiro Shiroshita, Department of Respiratory Medicine, Ichinomiyanishi Hospital, 1 Kaimeihira, Ichinomiya, Aichi Prefecture, 494-0001, Japan, Tel +81-80-3807-4960, Fax +81-586-48-0055, Email akihirokun8@gmail.comPurpose: Whether the empirical use of anti-pseudomonal antibiotics actually improves patient outcomes is unclear. Hence, we aimed to determine whether empirical anti-pseudomonal antibiotics are better than anti-pseudomonal antibiotics in treating patients with recurrent lower respiratory tract infections (LRTIs).Patients and Methods: We extracted data from the Japanese nationwide database of the Real World Data Co., Ltd. Our target population was patients with LRTIs, defined as chronic obstructive pulmonary disease exacerbation and pneumonia. We included patients aged ≥ 40 years who were admitted for lower respiratory tract infections ≥ 2 times within 90 days. We excluded patients who had an event (death or transfer) within 24 h after admission. We ran a frailty model adjusted for the following confounding factors: number of recurrences, age, body mass index, activities of daily living, Hugh-Johns classification, altered mental status, oxygen use on admission, blood urea nitrogen, and systemic steroid use.Results: We included 893 patients with 1362 observations of recurrent LRTIs. There were 897 (66%) observations in the non-anti-pseudomonal antibiotic group and 465 (34%) in the anti-pseudomonal group; the numbers of in-hospital deaths were 86/897 (10%) and 63/465 (14%), respectively. Our frailty model yielded an adjusted hazard ratio (HR) (anti-pseudomonal group/non-anti-pseudomonal group) of 1.49 (95% confidence interval, 1.03– 2.14).Conclusion: The empirical use of anti-pseudomonal antibiotics was associated with a higher HR of in-hospital mortality than the use of non-anti-pseudomonal antibiotics. Physicians might need to consider limiting the prescription of anti-pseudomonal antibiotics based on background factors such as the patient’s baseline function and disease severity. Further studies are needed to evaluate the causal relationship between empirical anti-pseudomonal antibiotics and mortality, and identify specific patient population who benefit from empirical anti-pseudomonal antibiotics.Keywords: clinical epidemiology, COPD, Emphysema, infection and inflammation, pneumonia

Abstract Background Situs inversus totalis (SIT) is a relatively rare condition, in which the thoracic and abdominal organs are reversed or mirrored from their normal positions. Here, we reported a case of a patient with SIT and advanced gastric cancer with lymph node metastasis who underwent laparoscopic distal gastrectomy (LDG). Case presentation A 67-year-old man with SIT was clinically diagnosed with T3N2M0 advanced gastric cancer located in the middle gastric body. Three-dimensional reconstruction of computed tomography angiogram revealed that the common hepatic artery originated from the superior mesenteric artery. The patient underwent LDG with D2 lymph node dissection and Roux-en-Y reconstruction. The postoperative course was uneventful. Conclusion This case report showed that LDG could be safely performed on a patient even under complex conditions, such as advanced gastric cancer with lymph node metastasis with SIT and vascular anomalies.

Abstract We performed a systematic review and meta‐analysis to re‐evaluate the effectiveness of angiotensin‐converting enzyme inhibitors (ACE‐I) in the reduction of pneumonia risk. We searched relevant publications in five databases. All studies included patients older than 18 years, who had used ACE‐I as an intervention, and had assessed pneumonia. Seven RCTs (n = 8704) and 38 observational studies (n = 1,705,030) were included. The overall risk of bias was high. ACE‐I‐treated patients were associated with a slightly lower risk of pneumonia, both from pooled estimates of RCTs [pooled odds ratio (OR), 0.75; 95% confidence interval (CI), 0.62–0.90; low certainty of evidence] and observational studies (pooled OR, 0.85; 95% CI, 0.78–0.92; very low certainty of evidence). Considering the small effect size of ACE‐I in preventing pneumonia and the low quality of the evidence, routine use of ACE‐I for pneumonia prevention is not recommended.

Abstract Background Although many quality indicator (QI) sets have been developed for acute cardiovascular diseases, a comprehensive summary is lacking. In this scoping review we aimed to summarize the available evidence on the QI sets for acute cardiovascular diseases, and assess the QI set development process. We followed the Joanna Briggs Institute framework and the PRISMA extension for scoping reviews. Methods We conducted a systematic search of MEDLINE, EMBASE, and major international guidelines on QIs for acute major cardiovascular diseases. The study included articles published after 2000. Results Among the 3112 articles screened, 18 were included in this scoping review. Among the 18 articles included, 12 were on acute coronary syndrome (ACS), five on acute heart failure (AHF), and two on acute aortic dissection (AAD); one article included QIs for both ACS and AHF. Only four of these studies conducted a systematic search with a search strategy. From the 18 articles, 268 QIs containing duplication between articles were identified (191 QIs were for ACS, 57 were for AHF, and 20 were for AAD) and QI measurements varied across articles. Conclusions This scoping review provides a comprehensive list of QIs for acute cardiovascular diseases. Our results may be helpful to clinicians and organizations seeking to develop QIs for acute cardiovascular care in the future.

Summary: Preculture is indispensable for achieving highly efficient non-homologous end joining (NHEJ)-based genome editing. Here, we present a protocol for optimizing genome editing conditions for murine hematopoietic stem cells (HSCs) and evaluating their function following NHEJ-based genome editing. We describe steps for sgRNA preparation, cell sorting, preculture, and electroporation. We then detail post-editing culture and transplanting of bone marrow. This protocol can be used to study genes related to HSC quiescence.For complete details on the use and execution of this protocol, please refer to Shiroshita et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.