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Homologous recombination-mediated gene targeting has greatly contributed to genetic analysis in a wide range of species, but is highly inefficient in human cells because of overwhelmingly frequent random integration events, whose molecular mechanism remains elusive. Here we show that DNA polymerase θ, despite its minor role in chromosomal DNA repair, substantially contributes to random integration, and that cells lacking both DNA polymerase θ and DNA ligase IV, which is essential for non-homologous end joining (NHEJ), exhibit 100% efficiency of spontaneous gene targeting by virtue of undetectable levels of random integration. Thus, DNA polymerase θ-mediated end joining is the sole homology-independent repair route in the absence of NHEJ and, intriguingly, their combined absence reveals rare Alu - Alu recombination events utilizing a stretch of homology. Our findings provide new insights into the mechanics of foreign DNA integration and the role of DNA polymerase θ in human genome maintenance. Homologous recombination mediated gene targeting is highly inefficient in human cells due to random integration events, Here the authors show that dual repression of polymerase θ and DNA ligase IV eliminate random integration events.

We investigated the association of adipose tissue insulin resistance with blood pressure and hypertension incidence, comparing it with hepatic and skeletal muscle insulin resistance. The cross-sectional analysis included 6892 general health checkup examinees (mean age: 69.3 years; 51.3% women and 48.7% men) who had no cardiovascular disease. Of those, 3948 normotensive participants (mean age: 68.4 years; 54.8% women and 45.2% men) were enrolled in the retrospective cohort analysis. The adipose insulin resistance index (Adipo-IR) was calculated as the product of fasting serum insulin and free fatty acid levels. A high adipo-IR, high homeostasis model assessment of insulin resistance (HOMA-IR), and low Matsuda index were indicated based on the optimal cutoff values in a receiver operating characteristic curve analysis. Adipo-IR (β = 0.096, P < 0.001), HOMA-IR (β = 0.052, P < 0.001), and Matsuda index (β = −0.055, P < 0.001) were associated with systolic blood pressure in the cross-sectional analysis. Over a mean 5.3-year follow-up period, 1310 participants developed hypertension. A high adipo-IR (adjusted OR, 1.29; 95% CI, 1.11–1.51), but not HOMA-IR or Matsuda index, was significantly associated with the incidence of hypertension. Moreover, the combination of high adipo-IR with high HOMA-IR or low Matsuda index showed no higher odds of hypertension than a high adipo-IR alone. These results suggest that insulin resistance is associated with blood pressure control regardless of the tissue in which it occurs; however, the risk of hypertension is determined by insulin resistance in adipose tissue rather than in liver or muscle tissue.

Background: Tobacco exposure during pregnancy is associated with several adverse outcomes in infants. We investigated the association between tobacco exposure during pregnancy (both active and second-hand) and various infections in infants up to 1 year.Methods: This prospective cohort study used a fixed dataset (jecs-an-20180131) from the Japan Environment and Children’s Study of registered births in Japan during 2011–2014 that included 104,065 fetal records from enrolled pregnant women. Based on the participants’ responses to the questionnaire on smoking status, mothers were first divided into “never smoked,” “quit smoking,” and “current smoker” groups and then into “no second-hand smoking (SHS)” and “SHS” groups. Infectious diseases included central nervous system infection, otitis media (OM), upper respiratory tract infection (URTI), lower respiratory tract infection (LRTI), gastroenteritis (GI), and urinary tract infection. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated using logistic regression analysis and adjusted for maternal, socioeconomic, and postnatal confounding factors.Results: Among the 73,205 newborns enrolled, multivariable analysis revealed that the aOR of LRTI and GI was 1.20 (95% CI, 1.07–1.33) and 1.18 (95% CI, 1.04–1.35), respectively, for the “current smoker with/without SHS” group compared with the “never smoked without SHS” group. “Quit smoking without SHS” was not associated with the risk of LRTI. SHS was associated with an increased risk of OM, URTI, LRTI, and GI, especially with LRTI and GI.Conclusion: Exposure to tobacco smoke during pregnancy was associated with an increased risk of OM, URTI, LRTI, and GI in infants during their first year of life.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development. The authors add to our knowledge of the transcriptional regulation of the meiotic program in mice spermatocytes, showing ZFP541 regulates meiotic prophase and transition to the division phase by being the target for upstream factors MEIOSIN/STRA8.

SRY is the master regulator of male sex determination in eutherian mammals. In mice, Sry expression is transcriptionally and epigenetically controlled in a developmental stage-specific manner. The Sry promoter undergoes demethylation in embryonic gonadal somatic cells at the sex-determining period. However, its molecular mechanism and in vivo significance remain unclear. Here, we report that the Sry promoter is actively demethylated during gonadal development, and TET2 plays a fundamental role in Sry demethylation. Tet 2-deficient mice showed absence of 5-hydroxymethylcytosine in the Sry promoter. Furthermore, Tet2 deficiency diminished Sry expression, indicating that TET2-mediated DNA demethylation regulates Sry expression positively. We previously showed that the deficiency of the H3K9 demethylase Jmjd1a compromises Sry expression and induces male-to-female sex reversal. Tet2 deficiency enhanced the sex reversal phenotype of Jmjd1a -deficient mice. Thus, TET2-mediated active DNA demethylation and JMJD1A-mediated H3K9 demethylation contribute synergistically to sex determination.

Monoclonal gammopathy (M-proteinemia) is a premalignant plasma cell disorder. The prevalence of M-proteinemia increases with age and is affected by genetic or environmental factors. Atomic bomb (A-bomb) survivors in Hiroshima are in an age range when they are susceptible to M-proteinemia. The prevalence and incidence of M-proteinemia in Hiroshima A-bomb survivors were investigated for 30 years (1989–2018) to examine the influence of radiation exposure. The overall prevalence of M-proteinemia among 38,602 A-bomb survivors was 2.4%. M-proteinemia prevalence at age 70 years and monoclonal gammopathy of undetermined significance (MGUS) incidence were not associated with radiation exposure category. Males had a 2.30-fold higher prevalence and a 2.08-fold higher incidence than females. The risk of incidence for MGUS was 4.32-fold higher in persons aged < 10 years at the time of the A-bombing and 2.56-fold higher in those aged 10–19 years compared with those aged over 30 years. IgG type M-proteinemia was common and the IgM type developed 5–8 years later than other immunoglobulin types. Exposure to radiation was not clearly associated with the prevalence of M-proteinemia or incidence of MGUS in Hiroshima A-bomb survivors. However, males and those aged < 20 years at A-bombing had higher susceptibility to MGUS.

: Peripheral nerve ultrasound is used to evaluate nerve enlargement in immune-mediated neuropathies; however, assessment can be challenging because the distribution and severity of nerve enlargement vary among patients and are often accompanied by indistinct nerve boundaries and heterogeneous echogenicity. Although deep learning-based segmentation has been reported, most studies have focused on limited regions or single anatomical sites, primarily in compressive neuropathies. This study aimed to evaluate the performance of temporal deep learning-based segmentation for assessing diffuse or focal nerve enlargement in immune-mediated neuropathies using continuous ultrasound scans. : Twenty-five healthy participants and five patients with immune-mediated neuropathy and nerve enlargement were included. Continuous ultrasound scanning from the wrist to below the elbow was performed. A static DeepLabV3+ model and temporal models incorporating convolutional long short-term memory (ConvLSTM) or Temporal Mamba were constructed and compared. : In healthy participants, segmentation performance was comparable across models. In contrast, in patients with nerve enlargement, temporal models demonstrated higher Dice coefficients and reduced frame-to-frame variability. The ConvLSTM-based model showed the highest performance, with mean Dice coefficients ranging from 0.87 to 0.92. : Temporal deep learning showed potential for nerve segmentation in selected cases with nerve enlargement associated with immune-mediated neuropathies. Temporal models achieved improved segmentation performance and reduced frame-to-frame variability in these preliminary cases. This approach may facilitate more consistent quantitative ultrasound evaluation and warrants further validation in larger cohorts.

Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.

IntroductionLittle is known about the risk of hypertension in patients with the early stage of type 2 diabetes. We investigated the risk of hypertension in participants with newly diagnosed type 2 diabetes and prediabetes.Research design and methodsThis is a retrospective cohort study consisting of 2136 middle-aged participants (1022 with normal fasting glucose/normal glucose tolerance (NFG/NGT), 418 with impaired fasting glucose (IFG), 466 with impaired glucose tolerance (IGT) and 230 with diabetes) and 3426 elderly participants (1762 with NFG/NGT, 599 with IFG, 781 with IGT, and 284 with diabetes). All participants underwent 75 g oral glucose tolerance tests at baseline.ResultsOver a median 59-month follow-up period, 459 middle-aged and 1170 elderly participants developed hypertension. In middle-aged participants, the odds of incident hypertension were significantly higher in those with IFG (OR 1.40; p=0.019), IGT (OR 1.49; p=0.004), and diabetes (OR 1.55; p=0.013) than those with NFG/NGT, which was no longer significant after adjustment for body mass index. Subgroup analysis showed that the risk of hypertension was significantly higher in diabetes than NFG/NGT only in participants without obesity. Conversely, obesity was a risk factor of hypertension only in those with IFG and NFG/NGT. In elderly participants, there was no difference in the risk of hypertension among the NFG/NGT, IFG, IGT and diabetes groups.ConclusionsThe risk of hypertension is modest in participants with newly diagnosed type 2 diabetes and prediabetes. Our findings suggest that the early stages of type 2 diabetes and prediabetes may be a key period for reducing hypertension, given the pronounced risk of hypertension in patients with diabetes reported in previous studies. In terms of reducing the risk for hypertension, obesity treatment might be advantageous in the early stages rather than the advanced stages of impaired glucose metabolism.

BackgroundAnimal models of nephrotic syndrome (NS) revealed that tight junction (TJ)-like structures are generated together with a concomitant decrease in slit diaphragms (SDs). Claudins (CLDNs) are capable of forming TJ strands and thereby the backbone of TJs. We showed the ectopic expression of CLDN2 in podocytes in pediatric NS, and detected its localization.MethodsRenal frozen specimens were obtained by biopsy from 49 pediatric patients: 21 subjects with MCD, 18 with FSGS, and 10 with IgA nephritis (IgA-N). CLDN2 expression was observed by immunohistochemistry and the CLDN2-positive area was calculated. Moreover, its localization was detected using immunoelectron microscopy.ResultsCLDN2 is ectopically detected in cases with MCD and FSGS before remission. The CLDN2-stained region in MCD and FSGS glomeruli before remission was significantly greater than that after remission as well as in IgA-N patients. Immunoelectron microscopy revealed that CLDN2 was concentrated along newly formed TJs in podocytes.ConclusionThe same pathological findings in terms of ectopic CLDN2 expression in podocytes were shown in cases with MCD and FSGS before remission. Immunofluorescence and immunoelectron studies of CLDN2 appear to afford a powerful tool for the diagnosis of primary NS. In addition, CLDN2 expression level may be related to disease status.