An inducible germ cell ablation chicken model for high-grade germline chimeras.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Laser speckle flowgraphy has comparable accuracy to indocyanine green fluorescence angiography in assessing bowel blood perfusion.

PURPOSE: To investigate the accuracy of laser speckle flowgraphy (LSFG), a noninvasive method for the quantitative evaluation of blood flow using mean blur rate (MBR) as a blood flow parameter in the assessment of bowel blood perfusion compared to indocyanine green fluorescence angiography (ICG-FA). METHODS: We enrolled 46 patients who underwent left-sided colorectal surgery. LSFG and ICG-FA were applied to assess blood bowel perfusion, with MBR and luminance as parameters, respectively. In both measurement methods, the position where the parameter suddenly decreased was defined as the blood flow boundary line. Subsequently, the blood flow boundaries created after processing the blood vessels flowing into the intestinal tract were determined using LSFG and ICG-FA, and concordance between the two was examined. Blood flow boundaries were visually identified using color tone changes on a color map created based on MBR in LSFG and using differences in luminance in ICG-FA. The distances between the transection line and blood flow boundaries determined using each method were compared. RESULTS: The location of blood flow boundaries matched in 65% (30/46) of cases. Although locations differed in the remaining 35% (16/46), all were located on the anal side near the transection line, and the difference was not clinically significant. The average distances between the transection line and blood flow boundary were 2.76 (SD = 3.25) and 3.71 (SD = 4.26) mm, respectively. There was no statistically significant difference between the two groups (p = 0.38). CONCLUSION: LSFG was shown to have comparable accuracy to ICG-FA, and may be useful for evaluating bowel perfusion.

Differentiating gallbladder cancer from polyps using non-enhanced magnetic resonance imaging.

Purpose: This retrospective cohort study assessed the efficiency of non-enhanced magnetic resonance imaging (MRI) for differentiating gallbladder cancer (GBC) from gallbladder polyps (GBPs) measuring ≥ 10 mm. Material and methods: Patients diagnosed with GBCs or GBPs ≥ 10 mm and GBC ≤ T2 stage were eligible for inclusion. Two independent blinded readers assessed the continuity of the mucosal and muscular layers (CMML; present or absent) and normalised signal intensity ratio (NIR) on the apparent diffusion coefficient map (NIR-ADC), T1-weighted image (NIR-T1WI), and T2-weighted half-Fourier acquisition single-shot turbo spin-echo image. Univariate and multivariate logistic regression analyses and interobserver agreement analyses were performed to detect predictive variables differentiating GBCs from GBPs. Receiver operating characteristic (ROC) analysis was performed to evaluate diagnostic performance. A reproducibility test was performed to verify the predictive variables. Results: Multivariate analysis showed significant differences in CMML, NIR-ADC, and NIR-T1WI (p < 0.001). The positive predictive value (PPV) and specificity of the absence of CMML were approximately 100%. The CMML showed the best specificity, accuracy, and PPV in the reproducibility study. The sensitivity of CMML alone was approximately 50%, whereas it increased to approximately 70% when combined with NIR-ADC. The diagnostic performance of the combination, including sensitivity, was almost like that of tumour size. The combined tumour size and CMML assessment showed higher diagnostic performance than tumour size alone. Conclusions: The absence of CMML and NIR-ADC ≤ 1.86 helped in differentiating GBCs from GBPs. Evaluation of the absence of CMML and measurement of tumour size could better aid in determining between the two than measurement of tumour size alone.

VGLL3 confers slow-twitch muscle differentiation via PGC-1α expression in C2C12 myocytes.

Vestigial-like family member 3 (VGLL3) is a cofactor for the TEA-domain transcription factor (TEAD) family. Although VGLL3 influences myogenic differentiation, its involvement in slow- and fast-twitch fiber specification remains unknown. In this study, we established a cell line stably overexpressing VGLL3 and analyzed effects of VGLL3 on the myogenic differentiation of murine myoblast C2C12 cells. We found that VGLL3 expression promotes slow-twitch muscle differentiation. Mechanistically, VGLL3 expression induced the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master transcriptional regulator of slow-twitch muscle development. We also found that VGLL3 proteins are degraded by the proteasome, which causes switching of TEAD cofactors from VGLL3 to Yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ). These results suggest that the balance between the two kinds of TEAD cofactors VGLL3 and YAP/TAZ controls muscle fiber-type specification.

Optimal threshold score of aortic valve calcification for identification of significant aortic stenosis on non-electrocardiographic-gated computed tomography.

OBJECTIVES: This study evaluated the association between aortic valve calcification (AVC) and aortic stenosis (AS) by scoring the AVC to determine the threshold scores for significant AS on non-electrocardiographic (ECG)-gated computed tomography (CT). METHODS: We retrospectively analyzed the AVC scores of 5385 patients on non-contrast non-ECG-gated CT, who underwent transthoracic echocardiography (TTE) from March 1, 2013, to December 26, 2019, at our institution. Multivariable logistic regression models were used to identify potential risk factors for significant AS. The thresholds for significant AS were computed using receiver operator characteristic (ROC) curves, based on the AVC scores after propensity score matching. RESULTS: A significant association was found between AS and age (p < 0.001; odds ratio [OR], 1.04; 95% confidence interval [CI], 1.02-1.06), female sex (p < 0.001; OR, 4.5; 95% CI, 2.75-7.36), bicuspid aortic valve (p < 0.001; OR, 23.2; 95% CI, 7.35-72.9), and AVC score (AVC score/100) (p < 0.001; OR, 1.82; 95% CI, 1.71-1.95). All sex-specific AVC thresholds for significant AS (moderate and over AS severity, moderate and over AS severity without discordance, discordant severe AS, and concordant severe AS) showed high sensitivity and specificity (AUC, 0.939-0.968; sensitivity, 84.6-96%; specificity, 84.2-97.1%). CONCLUSIONS: We determined the optimal AVC threshold scores for significant AS, which may aid in diagnosing significant asymptomatic AS on incidental detection of AVC through non-ECG-gated CT for non-cardiac indications. KEY POINTS: • Increased frequency of non-electrocardiographic (ECG)-gated computed tomography (CT) for non-cardiac indications has led to the increased incidental identification of aortic valve calcification (AVC). • It is important to identify patients with significant aortic stenosis (AS) who require additional echocardiographic assessment on incidental detection of AVC via non-ECG-gated CT. • We determined the AVC thresholds with high sensitivity and specificity to identify significant AS on non-ECG-gated CT, which could lead to early diagnosis of asymptomatic significant AS and improved prognosis.

Polymerization Properties of n-Butyl Cyanoacrylate Mixed with Ethiodized Oil in the Lymphatic System: In Vivo Investigation in a Rabbit Model.

PURPOSE: To evaluate the polymerization properties of a mixture of n-butyl cyanoacrylate (nBCA) and ethiodized oil in the lymphatic system using an animal model. MATERIALS AND METHODS: Nineteen male Japanese White rabbits underwent 28 lymphatic embolization procedures under fluoroscopic guidance using manually injected mixtures of nBCA and ethiodized oil at ratios of 1:2 (nBCA density of 33%), 1:4 (20%), 1:6 (14%), and 1:8 (11%) via the popliteal lymph node. The time required for polymerization and the distance traveled by the mixture were evaluated and compared among the groups using the Kruskal-Wallis test. Histopathologic intergroup comparisons and time-course changes were also evaluated using embolized lymph nodes. RESULTS: Among 23 successful procedures, the mean polymerization times were 14 ± 3, 88 ± 93, 331 ± 292, and 932 seconds ± 540 and the mean distances traveled were 13 ± 10, 31 ± 44, 85 ± 89, and 108 mm ± 35 in the 33% (n = 5), 20% (n = 6), 14% (n = 6), and 11% (n = 6) groups, respectively. The 11% group demonstrated a significantly longer polymerization time than the 33%, 20%, and 14% groups and distance traveled than the 33% group. Pathologically, the embolized lymph nodes showed inflammatory changes and massive necrosis regardless of the nBCA density. CONCLUSIONS: Polymerization times and distances traveled were increased when nBCA was diluted with increasing quantitites of ethiodized oil in this rabbit model of lymphatic embolization. These relationships should be considered when dilution is prescribed for clinical use.

Long-term results of Chiari pelvic osteotomy on the preservation of hip function with mean follow-up of more than 30 years and its prognostic factors.

BACKGROUND: Clinical outcomes of Chiari pelvic osteotomy for acetabular dysplasia, including conversion to total hip arthroplasty (THA), have not been adequately explored. The purpose of this study was to examine the long-term results and clinical outcomes of Chiari pelvic osteotomy as the primary outcome and to analyze its prognostic factors as the second outcome. METHODS: This study was a multicenter, retrospective cohort study. Ninety-seven patients underwent Chiari pelvic osteotomy at three hospitals between March 1975 and October 1997. The long-term clinical outcomes of Chiari pelvic osteotomy, including conversion to THA and hip pain, were analyzed using the Kaplan-Meier method. In addition, the prognostic factors for conversion to THA after Chiari pelvic osteotomy were evaluated with clinical variables and radiographic parameters. RESULTS: The study included 51 hips in 45 patients (4 men and 41 women) with long-term follow-up. The survival rates assessed by Kaplan-Meier analysis with conversion to THA as an endpoint, were 90.2% (95% confidence interval (CI) 82.0-98.4%) at 20 years and 73.5% (95% CI 61.1-86.0%) at 30 years. In contrast, the Kaplan-Meier survival rates with the Japanese Orthopaedic Association hip score for pain ≤20 as an endpoint, were 86.3% (95% CI 76.8-95.7%) at 20 years and 65.6% (95% CI 52.3-79.0%) at 30 years. Only older age at osteotomy was the significantly poor prognostic factor for conversion to THA, with a hazard ratio of 1.11/year, 95% CI 1.06 to 1.18, (p < 0.01). CONCLUSION: Chiari pelvic osteotomy may still be a good alternative to bony reconstructive surgery for acetabular dysplasia especially in young patients. Only older age at the osteotomy was related to the poor prognosis of preserving hip function.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

BACKGROUND: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. RESULTS: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. CONCLUSIONS: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

Real-world safety and effectiveness of omalizumab in Japanese patients with chronic spontaneous urticaria: A post-marketing surveillance study.

BACKGROUND: The safety and efficacy of omalizumab in chronic spontaneous urticaria (CSU) patients has been established, but real-world long-term data remain scarce, especially in Japan. METHODS: 52-week, open-label, single-arm, observational study evaluated the safety and effectiveness of first-time omalizumab in Japanese CSU patients responding inadequately to conventional therapies. RESULTS: Overall, 235 of 280 patients completed the study. Most patients were aged ≥ 18 and < 65 years; adolescents (≥ 12 and ≤ 18 years) accounted for 9.6% of the total population. The mean ± standard deviation (SD) duration of CSU at baseline was 1.6 ± 3.1 years; 46.1% of patients had had CSU for < 6 months. At baseline, the mean ± SD of Urticaria Control Test (UCT) score, Weekly Urticaria Activity Score (UAS7), and Dermatology Life Quality Index (DLQI) were 5.1 ± 3.2, 25.2 ± 11.9, and 8.4 ± 5.9, respectively. The mean ± SD duration of the observation period was 330.3 ± 86.2 days. Relapse was reported in 65 patients, 51, 9, and 5 of whom required retreatment with omalizumab 1, 2, and ≥ 3 times, respectively. The incidence of adverse events (AEs), serious AEs, and adverse drug reactions (ADRs) was reported in 11.8%, 1.4%, and 3.9% of patients, respectively. The most common AEs were urticaria (1.8%) and eczema (1.1%). No adolescents experienced ADRs. A cumulative of 92.8% of patients responded in the Physician's Global Impression of Change, with 81.3%, 75.0%, and 95.1% of patients achieving UCT ≥ 12, UAS7 ≤ 6, and DLQI ≤ 5 up to Week 52, respectively. CONCLUSIONS: This study supports the safety and effectiveness of omalizumab in CSU patients who responded inadequately to conventional therapies in real-world clinical practice in Japan.

[Laparoscopic Colectomy for a Patient with Transverse Colon Cancer in Situs Inversus Totalis].

A 74-year-old man with situs inversus totalis visited our hospital for a positive fecal occult blood. He was diagnosed with transverse colon cancer by total colonoscopy. We performed laparoscopic partial colectomy. He was discharged on the 8th postoperative day, without postoperative complications. Histopathological examination revealed well differentiated adenocarcinoma, pT1aN0M0, pStage Ⅰ. Preoperative assessment of the anatomical position and vascular malformations, using 3- dimensional computed tomography, was essential for our safe surgical conduct.

[Treatment Strategy for Familial Adenomatous Polyposis Presenting with Obstructive Colorectal Cancer and Concurrent Liver Metastasis].

A 27-year-old man was referred to our hospital for a detailed examination of abdominal distention, bloody stool, anorectal pain, and weight loss. A colonoscopy revealed a circumferential type 2 tumor at 9 cm from the anal verge which was diagnosed as an adenocarcinoma based on biopsy. Contrast-enhanced CT of the abdomen showed an elevated perineal lipid concentration in the rectum(Ra)which was suspicious for clinical T4a stage, and simultaneous S7/8 liver metastasis. We strongly suspected familial adenomatous polyposis(FAP)because his mother had a past history of total proctocolectomy for FAP. We decided to first create a loop stoma at the transverse colon for the obstructive rectal cancer, and then administer neoadjuvant chemotherapy(mFOLFOX6 plus panitumumab). We performed total proctocolectomy with permanent stoma and S8 ventral resection for the liver metastasis after 5 courses of mFOLFOX6 plus panitumumab. As for clinicopathological findings, round 50 polyps were identified in the colon and rectum, and rectal cancer invaded into the muscularis propria. Finally, the patient was diagnosed as a clinically attenuated FAP with ypT2 rectal cancer.

[Successful Continuation of Treatment in Advanced Colorectal Cancer by Switching to a Prior Biologic Agent after Emergence of Bevacizumab Allergy to a Biosimilar Drug-A Case Report].

Biosimilar(BS)drugs have recently been introduced owing to concerns with healthcare economics. In this report, we present a case in which a patient discontinued bevacizumab treatment following an allergic reaction to a BS formulation of bevacizumab but was able to safely continue treatment by switching to an original bevacizumab formulation in the late-line setting. The patient was a 66-year-old man diagnosed with unresectable colorectal cancer with synchronous multiple liver metastases. After primary tumor resection, chemotherapy including the original bevacizumab formulation was initiated. Allergic reactions to the BS formulation of bevacizumab occurred during the second-line treatment; however, in the late-line setting, switching back to the original bevacizumab formulation enabled the safe continuation of therapy. Overall, our case study suggests that switching of biologic agents may contribute to the ongoing management of chemotherapy.

Immediate effects of verbal instructions with internal focus of attention and external focus of attention on forward reach movement while standing.

[Purpose] This study aimed to examine the immediate effects of verbal instructions with an internal and external focus of attention on forward reach movement while standing. [Participants and Methods] Thirty-seven healthy young males performed reach movement in three conditions: control, internal focus of attention, and external focus of attention. The measurements recorded were the movement distance of the third metacarpal bone (reach distance), the distance of the center of pressure, and the movement angles between the acromion and malleolus lateralis and between the acromion and trochanter major. [Results] Compared to the control condition, the internal focus of attention condition had a lower reach distance, angles between the acromion and malleolus lateralis and between the acromion and trochanter major, and center of pressure distance. In contrast, compared to the control condition, the external focus of attention condition showed higher reach distance and angles between the acromion and malleolus lateralis and between the acromion and trochanter major. The change rate of reach distance in the internal and external focus of attention conditions correlated significantly with the change rates of the angles between the acromion and malleolus lateralis and between the acromion and trochanter major. [Conclusion] Verbal instructions with attentional focus resulted in the simultaneous adjustment of the positional relationship between trunk and hip and immediately affected the reach distance. Our findings suggest that verbal instruction with attentional focus is an important factor affecting reach movement.

Disseminated Mycolicibacter arupensis and Mycobacterium avium co-infection in a patient with anti-interferon-γ neutralizing autoantibody-associated immunodeficiency syndrome.

BACKGROUND: Disseminated infections of Mycolicibacter arupensis, a slowly growing nontuberculous mycobacteria (NTM) which causes synovitis, osteomyelitis, or pulmonary infections have rarely been reported. We report a case of disseminated M. arupensis and Mycobacterium avium co-infection in a patient with anti-interferon (IFN)-γ neutralizing autoantibody-associated immunodeficiency syndrome. CASE PRESENTATION: A 68-year-old Japanese male without human immunodeficiency virus infection was referred with complaints of persistent low-grade fever, arthralgia of the upper limbs, and weight loss of 10 kg. Cervical and mediastinal lymphadenopathies as well as a nodular opacity in the right lung were detected, and biopsy specimens of the cervical lymph node yielded M. arupensis without evidence of malignant cells. M. arupensis was also detected in sputum and peripheral blood. Computed tomography (CT) revealed deterioration of the right supraclavicular lymphadenopathy with internal necrosis and multiple low-density splenic lesions. Bone marrow and aspirates from the cervical lymph node collected at initiation of treatment yielded M. avium. The presence of anti-IFN-γ neutralizing autoantibodies was detected, leading to a diagnosis of co-infection of M. arupensis and M. avium with anti-IFN-γ neutralizing autoantibody-associated immunodeficiency syndrome. Post initiation of treatment with clarithromycin, ethambutol, and rifabutin, his fever declined, and his polyarthritis resolved. He developed disseminated varicella zoster during treatment; however, a follow-up CT scan six months after treatment revealed improvement of the lymphadenopathies, consolidation in the right lung, and splenic lesions. CONCLUSION: This is the first report of disseminated M. arupensis and M. avium co-infection in a patient with anti-IFN-γ neutralizing autoantibody-associated immunodeficiency syndrome.

Transanal down-to-up dissection of the distal rectum as a viable approach to achieve total mesorectal excision in laparoscopic sphincter-preserving surgery for rectal cancer near the anus: a study of short- and long-term outcomes of 123 consecutive patients from a single Japanese institution.

BACKGROUND: In rectal cancer (RC) surgery, the complexity of total mesorectal excision (TME) in laparoscopic sphincter-preserving surgery (lap-SPS) for RC near the anus has been a critical issue. Recently, technical assistance via the anus for complete TME has been receiving attention. This study aimed at clarifying the transanal down-to-up dissection viability for achieving TME in lap-SPS for RC near the anus. METHODS: We evaluated surgical and oncological outcomes of a total of 123 consecutive patients undergoing either a transanal rectal dissection (TARD) under direct vision mobilizing the most difficult portion of TME via the anus or the transanal TME by using an endoscopic system (TaTME) for achieving TME in lap-SPS for RC near the anus between January 2006 and February 2021. RESULTS: A total of 123 consecutive patients (83 men) with a median age of 66 years (range 33-86 years) were included. TARD and TaTME were performed for 50 (40.7%) and for 73 (59.3%) patients, respectively. Preoperative treatment was performed for 40 (32.5%) patients, resulting in a complete pathological response in 5 (12.5%) patients. Intersphincteric resection was performed significantly more in the TARD group (p<0.001). Although the TaTME group needed a longer operative time at the transanal portion (p<0.001), the median blood loss was lower (p<0.001). Postoperative complications with the Clavien-Dindo classification grade ≧2 developed in 52 (42.3%) patients. Urinary dysfunction and stoma-related complications were found most frequently. More patients needing medication for urinary dysfunction were found in the TARD group, but a significant difference was not observed (10.0% vs. 6.8%, p=0.526). The quality of TME was good for almost all patients. Recurrence developed in 18 (14.6%) patients. The 5-year overall survival (OS) and relapse-free survival (RFS) rates in 123 patients were 95.8% and 88.8%, respectively. The 5-year OS and RFS between the two groups were comparable. CONCLUSIONS: Our data suggested that a transanal down-to-up dissection of the distal rectum might be a viable approach in lap-SPS for RC near the anus. Further studies are needed to examine the differences between TARD and TaTME.

The dynamic spatial and temporal relationships between the phalanx-forming region and the interdigits determine digit identity in the chick limb autopod.

BACKGROUND: Interdigits (IDs) determine digit identity in chick limbs. They are located between the digital rays and act as secondary signaling centers downstream of sonic hedgehog to provide positional information for determining digit identity in the phalanx-forming region (PFR). We examined the dynamic developmental mechanism by which PFR cells obtain positional information from IDs to determine the identity of individual digits in the chick hindlimb. RESULTS: We identified the specific region of the IDs responsible for determining digit identity and showed that PFR cells actively receive positional information only from the posteriorly, and not the anteriorly, located IDs. We also demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4. Finally, we found that both ID4 and digital ray 4 are necessary for determining digit 4 identity. CONCLUSIONS: The digital rays are naïve during the initial stages of their development, at which time digit identity is not determined. To determine digit identity, each PFR cell shows a unidirectional response to obtain positional information specifically from the IDs located posterior to the PFR, regardless of the signal strength from the anteriorly located IDs.

DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost (Xenotoca eiseni).

Nutrient transfer from mother to embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni (family Goodeidae), the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trophotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.