High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae.

BACKGROUND: The current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections. METHODS: We developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using "eNTRy rules" criteria associated with increased likelihood of intracellular accumulation in Escherichia coli. FINDINGS: We identified 72 compounds with confirmed activity against K. pneumoniae and/or drug-resistant A. baumannii. Two new chemical series with activity against XDR A. baumannii were identified meeting our criteria of potency (EC50 ≤50 µM) and absence of cytotoxicity (HepG2 CC50 ≥100 µM and red blood cell lysis HC50 ≥100 µM). The activity of close analogues of the two chemical series was also determined against A. baumannii clinical isolates. INTERPRETATION: This work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K. pneumoniae and A. baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects. FUNDING: BMBF and GARDP.

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells.

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.

Development of fluorogenic substrates for colorectal tumor-related neuropeptidases for activity-based diagnosis.

The M3 metalloproteases, neurolysin and THOP1, are neuropeptidases that are expressed in various tissues and metabolize neuropeptides, such as neurotensin. The biological roles of these enzymes are not well characterized, partially because the chemical tools to analyse their activities are not well developed. Here, we developed a fluorogenic substrate probe for neurolysin and thimet oligopeptidase 1 (THOP1), which enabled the analysis of enzymatic activity changes in tissue and plasma samples. In particular, the probe was useful for studying enzyme activities in a single-molecule enzyme assay platform, which can detect enzyme activity with high sensitivity. We detected the activity of neurolysin in plasma samples and revealed higher enzyme activity in the blood samples of patients with colorectal tumor. The result indicated that single-molecule neurolysin activity is a promising candidate for a blood biomarker for colorectal cancer diagnosis.

Fatigue on Waking, Insomnia, and Workplace Relationship Problems May Help to Detect Suicidal Ideation among New Middle-Aged Primary Care Patients: A 6-Month Prospective Study in Japan.

Signs of suicidal depression often go undetected in primary care settings. This study explored predictive factors for depression with suicidal ideation (DSI) among middle-aged primary care patients at 6 months after an initial clinic visit. New patients aged 35-64 years were recruited from internal medicine clinics in Japan. Baseline characteristics were elicited using self-administered and physician questionnaires. DSI was evaluated using the Zung Self-Rating Depression Scale and the Profile of Mood States at enrollment and 6 months later. Multiple logistic regression analysis was conducted to calculate adjusted odds ratios for DSI. Sensitivity, specificity, and likelihood ratios for associated factors were calculated. Among 387 patients, 13 (3.4%) were assessed as having DSI at 6 months. Adjusted for sex, age, and related factors, significant odds ratios for DSI were observed for "fatigue on waking ≥1/month" (7.90, 95% confidence intervals: 1.06-58.7), "fatigue on waking ≥1/week" (6.79, 1.02-45.1), "poor sleep status" (8.19, 1.05-63.8), and "relationship problems in the workplace" (4.24, 1.00-17.9). Fatigue on waking, sleep status, and workplace relationship problems may help predict DSI in primary care. Because the sample size in this investigation was small, further studies with larger samples are needed to confirm our findings.

Prognostic Factors of Cervical Adenocarcinoma With Positive Pelvic Lymph Node Metastases Without Preoperative Lymph Node Enlargement Treated With Radical Hysterectomy.

BACKGROUND/AIM: Pelvic lymph node (LN) metastases are found histopathologically after radical hysterectomy (RH) in some cases of cervical adenocarcinoma with no enlarged LNs on preoperatively. The aim was to clarify whether LN metastasis is a prognostic factor in these patients, and whether any postoperative therapy is advisable. PATIENTS AND METHODS: Sixty-one patients with stage I-II cervical adenocarcinoma [International Federation of Obstetrics and Gynecology (FIGO) 2008] with no enlarged pelvic LNs on preoperative imaging evaluation who underwent RH at our institution were retrospectively examined for clinicopathological, treatment, and prognosis-related factors. RESULTS: The median age was 47 years. FIGO stages were IB1 in 44 patients, IB2 in 10, IIA in 4, and IIB in 3. Seventeen patients (27.9%) had positive pelvic LNs. The multivariate analysis for both overall (OS) and disease-free (DFS) survival showed only pelvic LN metastasis was an independent prognostic factor. In a multivariate analysis of LN-positive cases, multiple LN metastases was an independent prognostic factor for poorer DFS. OS rates were 100%, 83.3%, and 30.0%, and DFS rates were 85.5%, 83.3%, and 12.5% for patients with no LN metastasis, single metastasis, and multiple metastases, respectively, showing a significant difference. Eight recurrences were observed in 10 patients with multiple node-positive disease, and six (75%) had an intrapelvic recurrence. CONCLUSION: Among patients who underwent RH for cervical adenocarcinoma with no preoperative enlarged LN and positive pelvic LNs confirmed postoperatively, multiple positive pelvic LNs are an independent poor prognostic factor. Because pelvic recurrence is common, concurrent chemoradiotherapy, possibly using paclitaxel and cisplatin, should be considered as adjuvant therapy.

Dietary High-Dose Biotin Intake Activates Fat Oxidation and Hepatic Carnitine Palmitoyltransferase in Rat.

This study investigated the effects of dietary high-dose biotin intake on fat oxidation in rats using respiratory gas analysis, and evaluated fatty-acid oxidation-related enzyme activities and gene expressions in the liver. Five-week-old male Sprague-Dawley rats were fed a control diet and three biotin-supplemented diets (additive biotin concentration: 0.05%, 0.10%, and 0.20% of diet) for 3 wk. In 2 wk, fat oxidation in the 0.20% biotin-supplemented diet group was higher than that in the 0.05% biotin-supplemented diet group; however, the energy expenditure and carbohydrate oxidation were unchanged between the dietary groups. At the end of 3 wk, body weight and epididymal white adipose tissue weight reduced in the 0.20% biotin diet group, and hepatic triglyceride levels tended to decrease. Additionally, increased plasma adiponectin concentration and hepatic mitochondrial carnitine palmitoyltransferase activity as well as decreased hepatic acetyl-CoA carboxylase 2 gene expression were observed in the 0.20% biotin-supplemented diet group compared with those in the control group. These results provide strong evidence that dietary high-dose biotin intake activated fat oxidation due to the increase in hepatic ß-oxidation, which may contribute to the decrease in hepatic triglyceride concentration and white adipose tissue weight.

Cumulative risk of developing a new symptom in patients with primary biliary cholangitis and its impact on prognosis.

Background and Aim: Symptoms of primary biliary cholangitis (PBC) frequently impair one's quality of life (QOL). Nonetheless, with improved treatment, the prognosis of PBC also improves. QOL plays an important role in patients with PBC. In this study, we aimed to reevaluate the transition of new symptom development in PBC and its predictive factors. Methods: This retrospective multicenter study enrolled 382 patients with PBC for symptom analysis. The impact of a newly developed symptom on PBC prognosis was investigated by Kaplan-Meier analysis with propensity score matching and logistic progression analysis. Results: The cumulative risk of developing a new symptom after 10 and 20 years of follow-up was 7.6 and 28.2%, and specifically that of pruritus, which was the most common symptom, was 6.7 and 23.3%, respectively. In Cox hazard risk analysis, serum Alb level (hazard ratio [HR], 1.097; 95% confidence interval [CI], 1.033-1.165; P = 0.002), the serum D-Bil level (HR, 6.262; 95% CI, 2.522-15.553, P < 0.001), and Paris II criteria (HR, 0.435; 95% CI, 0.183-1.036; P = 0.037) were significant independent predictors of a new symptom. Kaplan-Meier analysis showed that the overall survival and liver-related death were not significant between patients with and without a new symptom. Conclusion: The cumulative risk of new symptom development is roughly 30% 20 years after diagnosis and could be predicted by factors including serum albumin levels, serum D-Bil level, and Paris II criteria.

Implementation of a health education program in Asia, comparing Thailand, Indonesia, and Japan.

BACKGROUND: The global community is faced with aging societies, which will result in increased health care costs. we have been introducing our International Organization for Standardization (ISO)-certified health education system in Thailand and Indonesia. OBJECTIVE: The purpose of this study was to collect data on the effects of this ISO-certified health education system, to extend the healthy life expectancy and to study the feasibility of implementing this program and in new social contexts. METHODS: We implemented the health education program recruiting 43, 114 and 119 participants, respectively, in Japan, Thailand and Indonesia. The participants' conditions before and after the program were determined through anthropometry, physical fitness tests, blood chemistry tests, brain function tests and pedometry. RESULTS: Regarding pedometry, the Japanese participants took the highest number of daily steps on average, followed by the Indonesian and Thai participants. In the 10-m obstacle walk, the Thai and Indonesian participants had significantly increased times. Furthermore, the differentiation reaction time, reverse differentiation reaction time, total number of "misses", total number of "mistakes" and total number of errors significantly improved. CONCLUSION: It is possible to implement a program of an ISO-certified health education system, but how to develop exercise habits is important.

Efficient Induction of Primate iPS Cells Using a Combination of RNA Transfection and Chemical Compounds.

Non-human primate induced pluripotent cells (iPS cells) are useful for preclinical studies of iPS cell-based therapies and the research of primate developments. Since the initial report of iPS cells in 2006, various iPS cell induction methods have been reported. Here, we describe an efficient method for inducing iPS cells using a combination of RNA transfection and chemical compounds without using transgenes. Many kinds of marmoset cells, including difficult-to-reprogram cells, can be converted into iPS cells using this combinatorial method. Furthermore, this method can be applied to other primates, including humans.

Effects of Inadequate Folate Intake on the Onset and Progression of Hypertensive Vascular Injury.

We investigated the effects of inadequate folate intake on the onset and progression of hypertensive organ injury. In the present study, 5-wk-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed with a normal-folate (control; 160-170 µg of folate/100 g diet) or low-folate (8-10 µg of folate/100 g diet) diet until they reached 25 wk of age. After the animals reached 10 wk of age, the bodyweight of the rats in the low-folate group was lower than that of the rats in the control group. Regarding blood pressure, both groups had severe hypertension of ≥230 mmHg at 12 wk of age that was not significantly different between the groups. At 16 wk of age, the low-folate group had a low number of blood cell types. The folate levels in the serum, liver, and kidneys of these rats were significantly lower (p<0.01) and the serum homocysteine level in the low-folate group was significantly higher than in the controls. The low-folate group had a significantly lower testicular weight than the control group (p<0.05) and arterial hypertrophy, spermatogenesis arrest, and interstitial connective tissue hyperplasia were observed. However, there was no clear difference in lesions in other organs. These results indicated that under low folate status, SHRSP causes hematopoietic disorders and exacerbates hypertensive vascular injury at various degrees by organ type.

Establishment of an integrated automated embryonic manipulation system for producing genetically modified mice.

Genetically modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Here we present a fully automated system for microinjection into the pronucleus to facilitate genetic modification. We first developed software that automatically controls the microinjection system hardware. The software permits automatic rotation of the zygote to move the pronucleus to the injection pipette insertion position. We also developed software that recognizes the pronucleus in 3-dimensional coordinates so that the injection pipette can be automatically inserted into the pronucleus, and achieved a 94% insertion rate by linking the 2 pieces of software. Next, we determined the optimal solution injection conditions (30 hPa, 0.8-2.0 s) by examining the survival rate of injected zygotes. Finally, we produced transgenic (traditional DNA injection and piggyBac Transposon system) and knock-in (genomic editing) mice using our newly developed Integrated Automated Embryo Manipulation System (IAEMS). We propose that the IAEMS will simplify highly reproducible pronuclear stage zygote microinjection procedures.

Sigmoid Volvulus in Pregnancy Assessed by Contrast-Enhanced Computed Tomography Scanning.

Sigmoid volvulus requires urgent treatment, and it is particularly rare among pregnant women without a history of laparotomy. A delay in diagnosis may lead to serious consequences for the mother and fetus, and a rapid diagnosis and treatment in this setting is essential. The patient was a 19-year-old primiparous woman. She complained of repeated exacerbations and remissions of abrupt lower abdominal pain for the past 2 days and was transported to our hospital at 33 weeks of gestation. Ultrasonography revealed no placental thickening, and maternal bowel dilation was difficult to identify. Plain abdominal X-ray showed a dilated colon on the left side of the abdomen. Contrast-enhanced CT scan of the abdomen revealed a volvulus on the dorsal side of the uterus. The proximal end of the transverse to sigmoid colon was markedly dilated, and the distal end was collapsed. The elevated lactate level on blood gas analysis suggested intestinal ischemia. She was suspected of having a sigmoid volvulus at 33 weeks and 3 days of gestation. We decided to perform a cesarean section to secure the operative field for an intestinal resection following delivery. A male weighing 1840 g with Apgar scores 8/8 was delivered. The sigmoid colon was approximately 80 cm in length. A 360-degree clockwise rotation of was observed with a very distended but viable sigmoid loop. Following reduction of the volvulus, the sigmoid colon was fixed to the left side of the peritoneum. The mother had an uneventful postoperative course, and the infant was discharged without any sequelae. This case demonstrates two important lessons. First, sigmoid volvulus can occur in pregnant women even if they never had a laparotomy. Second, abdominal contrast-enhanced CT is useful for rapid diagnostic and treatment decisions relative to this pathology.

Changes in disease characteristics of primary biliary cholangitis: An observational retrospective study from 1982 to 2016.

AIM: Disease characteristics of primary biliary cholangitis have changed recently. However, detailed studies on the subject have been limited. Therefore, we aimed to clarify disease characteristics of patients with recent primary biliary cholangitis using the cohort from Niigata University and 21 affiliated hospitals. METHODS: Overall, 508 patients were enrolled in this study from 1982 to 2016, divided into three cohorts according to their year of diagnosis: ≤1999, 2000-2009 and ≥2010. We compared differences in clinical characteristics, response to ursodeoxycholic acid and prognosis. RESULTS: The male-to-female ratio increased incrementally from 1:16.4 (≤1999) to 1:3.8 (≥2010) (P < 0.001). In women, the median age at diagnosis increased incrementally from 54.0 years (≤1999) to 60.5 years (≥2010) (P < 0.001) and serum albumin decreased gradually (P = 0.001), which might have affected the increase in the Fibrosis-4 Index and albumin-bilirubin score. The ursodeoxycholic acid response rate according to the Barcelona criteria increased incrementally from 26.7% (≤1999) to 78.4% (≥2010) (P < 0.010), and those according to other criteria (Paris-I, Rotterdam and Toronto) were approximately ≥80% in all cohorts. Ten-year survival rate in the ≤1999 and 2000-2009 cohorts were 98.6% and 95.6%, respectively. These earlier cohorts were also characterized by a higher rate of asymptomatic state and mild histology (83.5% [≤1999] and 84.7% [2000-2009], and 93.6% [≤1999] and 91.1% [2000-2009]). CONCLUSIONS: Patients with primary biliary cholangitis were characterized by older age at diagnosis and an increase in male to female ratio as well as higher response rates of ursodeoxycholic acid and longer survival, resulting from the early recognition of primary biliary cholangitis.

Separation-Based Enzymomics Assay for the Discovery of Altered Peptide-Metabolizing Enzymatic Activities in Biosamples.

We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.

Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.

Short- and Long-term Outcomes of Minimally Invasive Versus Open Multivisceral Resection for Locally Advanced Colorectal Cancer.

BACKGROUND: Colorectal cancer invading the adjacent organs/structures is detected in 5% to 20% of all surgical interventions performed for the management of colorectal cancer. OBJECTIVE: Our purpose is to verify the safety and feasibility of laparoscopic surgery for the treatment of locally advanced colorectal cancer invading the adjacent organs. DESIGN: This is a retrospective study. SETTINGS: The study was conducted at a single institution in Japan. PATIENTS: We compared the morbidity, appropriate oncological resection, and disease-free survival of laparoscopic and open multivisceral resection in patients with colorectal carcinoma in the period between 2007 and 2015. MAIN OUTCOME MEASURES: The primary outcome measures were curative resection rate, morbidity rate, and recurrence of laparoscopic and open multivisceral resection in patients with colorectal cancer. RESULTS: Thirty-one patients received laparoscopic surgery, and 50 received open surgery. The amount of blood loss was smaller in the laparoscopic group than in the open group (60 vs 595 mL, p < 0.01). Curative surgery was performed in 46 patients of the open group (92.0%) and in 30 patients of the laparoscopic group (96.8%). Days until oral intake (5 vs 7 days, p < 0.01) and postoperative hospital stay (14 vs 19 days, p < 0.01) were shorter in the laparoscopic group. Overall morbidity was not different between the groups (22.5% vs 40.0%). Three-year disease-free survival rates were 62.7% in the open group and 56.7% in the laparoscopic group (p = 0.5776). LIMITATION: This study was a retrospective small study conducted at a single institute. CONCLUSION: Laparoscopic multivisceral resection may be a safe, less invasive alternative to open surgery, with less blood loss and shorter hospital stay, and was not inferior to open surgery based on long-term oncological end points. See Video Abstract at http://links.lww.com/DCR/A785.

Fusion Kinases Identified by Genomic Analyses of Sporadic Microsatellite Instability-High Colorectal Cancers.

PURPOSE: Colorectal cancers with microsatellite instability-high (MSI-H) status, due to mismatch repair deficiency, are associated with poor patient outcomes after relapse. We aimed to identify novel therapeutic targets for them. EXPERIMENTAL DESIGN: We performed MSI analyses of over 2,800 surgically resected colorectal tumors obtained from consecutive patients treated in Japan from 1998 through June 2016. Whole-exome sequencing, transcriptome sequencing, and methylation analyses were performed on 149 of 162 tumors showing MSI in BAT25 and BAT26 loci. We analyzed patient survival times using Bonferroni-adjusted log-rank tests. RESULTS: Sporadic MSI-H colorectal cancers with promoter methylation of MLH1 (called MM) had a clinicopathological profile that was distinct from that of colorectal cancers of patients with germline mutations (Lynch syndrome, LS-associated) or somatic, Lynch-like mutations in mismatch repair genes. MM tumors had more insertions and deletions and more recurrent mutations in BRAF and RNF43 than LS-associated or Lynch-like MSI-H tumors. Eleven fusion kinases were exclusively detected in MM MSI-H colorectal cancers lacking oncogenic KRAS/BRAF missense mutations and were associated with worse post-relapse prognosis. We developed a simple method to identify MM tumors and applied it to a validation cohort of 28 MSI-H colorectal cancers, identifying 16 MM tumors and 2 fusion kinases. CONCLUSIONS: We discovered that fusion kinases are frequently observed among sporadic MM MSI-H colorectal cancers. The new method to identify MM tumors enables us to straightforwardly group MSI-H patients into candidates of LS or fusion kinase carriers.

The effectiveness of a Japanese style health program: comparison between Minowa, Japan and Rayong, Thailand.

AIM: This study aimed to appropriately establish a Japanese style healthcare program under the ISO9001:2008 (ISO: International Organization for Standardization) in Minowa, Japan and Rayong, Thailand to improve problem areas and inspect its effectiveness. Furthermore, we want to make this health promotion through ISO widely available in Asian countries and to make an international contribution. METHOD: We implemented a 6-month health program in the c Rayong city, Thailand, and a 12 month health program in the Minowa town, Japan. This study assessed findings from pedometry, anthropometry and blood pressure measurements, physical fitness, blood chemistry, and brain function tests. RESULTS: The comparisons were made using interaction effects between the participants in Rayong and Minowa. They showed significant differences in the 10-m obstacle walk, the 6-minute walk, HDL, the reverse and reverse differentiation time, the total number of forgets, the total number of mistakes, and the total number of brain function errors. CONCLUSIONS: To improve the brain function in participants from Rayong through this health education program, measures such as increasing the number of steps, adding muscular strength/stretching exercises, and home training every day is suggested.

Effects of triple-treatment trunk stretching on physical fitness and curvature of the spine.

BACKGROUND: Research on the effectiveness of treatment for low back disorders has been made, however yet no established method has been found. Therefore, we devised a triple-treatment trunk stretching program comprising the following three trunk stretching exercises: stretching using an unstable flex chair; stretching using a stretching bench; and stretching using a stretch pad. Our three-treatment trunk stretching program is based on the principles of static muscular stabilization of the spine and uses well-known physiological strength-training principles. In this study, we investigated the effects of triple-treatment trunk stretching on physical fitness levels and curvature of the spine measured by X-ray photographs. METHODS: Thirteen healthy male subjects (mean age, 26.3 ± 4.0 years; height, 173.5 ± 4.9 cm; weight, 64.9 ± 5.7 kg; BMI 21.6 ± 1.7) were enrolled in this study. In consideration of safety and simplicity, we applied the physical fitness test introduced by the Ministry of Education, Culture, Sports, Science and Technology of Japan for 65- to 79-year-olds. RESULTS: Triple-treatment trunk stretching led to significant improvements in sit-and-reach flexibility, 10-m obstacle course walking time, standing forward flexion, thoracolumbar extension and horizontal flexion. Significant improvements were also observed in the neutral angle of the curvature of the lower thoracic spine and the neutral angle of the curvature of the lower lumbar spine. CONCLUSIONS: Significant improvements were evident in the neutral angles of both the curvature of the lower thoracic spine and the curvature of the lower lumbar spine after triple-treatment trunk stretching. This suggests that triple-treatment trunk stretching can help improve the curvature of the spine and physical fitness.

MIWI2 targets RNAs transcribed from piRNA-dependent regions to drive DNA methylation in mouse prospermatogonia.

Argonaute/Piwi proteins can regulate gene expression via RNA degradation and translational regulation using small RNAs as guides. They also promote the establishment of suppressive epigenetic marks on repeat sequences in diverse organisms. In mice, the nuclear Piwi protein MIWI2 and Piwi-interacting RNAs (piRNAs) are required for DNA methylation of retrotransposon sequences and some other sequences. However, its underlying molecular mechanisms remain unclear. Here, we show that piRNA-dependent regions are transcribed at the stage when piRNA-mediated DNA methylation takes place. MIWI2 specifically interacts with RNAs from these regions. In addition, we generated mice with deletion of a retrotransposon sequence either in a representative piRNA-dependent region or in a piRNA cluster. Both deleted regions were required for the establishment of DNA methylation of the piRNA-dependent region, indicating that piRNAs determine the target specificity of MIWI2-mediated DNA methylation. Our results indicate that MIWI2 affects the chromatin state through base-pairing between piRNAs and nascent RNAs, as observed in other organisms possessing small RNA-mediated epigenetic regulation.