rTMS concurrent with cognitive training rewires AD brain by enhancing GM-WM functional connectivity: a preliminary study.

Repetitive transcranial magnetic stimulation (rTMS) and cognitive training for patients with Alzheimer's disease (AD) can change functional connectivity (FC) within gray matter (GM). However, the role of white matter (WM) and changes of GM-WM FC under these therapies are still unclear. To clarify this problem, we applied 40 Hz rTMS over angular gyrus (AG) concurrent with cognitive training to 15 mild-moderate AD patients and analyzed the resting-state functional magnetic resonance imaging before and after treatment. Through AG-based FC analysis, corona radiata and superior longitudinal fasciculus (SLF) were identified as activated WM tracts. Compared with the GM results with AG as seed, more GM regions were found with activated WM tracts as seeds. The averaged FC, fractional amplitude of low-frequency fluctuation (fALFF), and regional homogeneity (ReHo) of the above GM regions had stronger clinical correlations (r/P = 0.363/0.048 vs 0.299/0.108, 0.351/0.057 vs 0.267/0.153, 0.420/0.021 vs 0.408/0.025, for FC/fALFF/ReHo, respectively) and better classification performance to distinguish pre-/post-treatment groups (AUC = 0.91 vs 0.88, 0.65 vs 0.63, 0.87 vs 0.82, for FC/fALFF/ReHo, respectively). Our results indicated that rTMS concurrent with cognitive training could rewire brain network by enhancing GM-WM FC in AD, and corona radiata and SLF played an important role in this process.

Development and validation of a nomogram for predicting cognitive frailty in patients on maintenance haemodialysis.

AIMS: This study aimed to construct a nomogram for predicting the risk of cognitive frailty in patients on maintenance haemodialysis. DESIGN: An explorative cross-sectional design was adopted. METHODS: From April 2022 to July 2022, 496 participants were recruited from five haemodialysis centres in Qingdao, Shandong Province, China. Participants with cognitive frailty were screened by Frailty Phenotype scale and Mini-Mental State Examination. Least Absolute Shrinkage and Selection Operator (LASSO) regression and multivariate logistic regression were utilized to determine predictors. The predictive performance of the nomogram was validated by calibration and discrimination. Decision curve analysis was used to assess clinical utility. Internal validation was implemented using 1000 bootstrap samples to mitigate overfitting. RESULTS: The prevalence of cognitive frailty was 17.5% (n = 87). Six risk predictors, namely health empowerment, alexithymia, age, educational level, marital status and dialysis vintage, were screened and used to develop a nomogram model. The nomogram had satisfactory discrimination and calibration, and decision curve analysis revealed considerable clinical utility. CONCLUSIONS: A nomogram incorporated with the six risk predictors was developed, and it exhibited excellent prediction performance. The nomogram may strengthen the effective screening of patients at high risk of cognitive frailty. IMPACT: This study established a tool for healthcare staff to predict cognitive frailty probability and identify risk factors in patients on maintenance haemodialysis. The nomogram can meet the needs of personalized care and precision medicine simultaneously. PATIENT OR PUBLIC CONTRIBUTION: Data were collected from patients on maintenance haemodialysis by using questionnaire survey. REPORTING METHOD: STROBE checklist was used.

Phase retrieval via nonlocal complex-domain sparsity.

Phase retrieval is indispensable for a number of coherent imaging systems. Owing to limited exposure, it is a challenge for traditional phase retrieval algorithms to reconstruct fine details in the presence of noise. In this Letter, we report an iterative framework for noise-robust phase retrieval with high fidelity. In the framework, we investigate nonlocal structural sparsity in the complex domain by low-rank regularization, which effectively suppresses artifacts caused by measurement noise. The joint optimization of sparsity regularization and data fidelity with forward models enables satisfying detail recovery. To further improve computational efficiency, we develop an adaptive iteration strategy that automatically adjusts matching frequency. The effectiveness of the reported technique has been validated for coherent diffraction imaging and Fourier ptychography, with ≈7 dB higher peak SNR (PSNR) on average, compared with conventional alternating projection reconstruction.

Image-free single-pixel object detection.

Recently developed image-free sensing techniques have achieved remarkable performance in various vision tasks. However, existing image-free methods still cannot simultaneously obtain the category, location, and size information of all objects. In this Letter, we report a novel image-free single-pixel object detection (SPOD) technique. SPOD enables efficient and robust multi-object detection directly from a small number of measurements, eliminating the requirement for complicated image reconstruction. Different from the conventional full-size pattern sampling method, the reported small-size optimized pattern sampling method achieves higher image-free sensing accuracy with fewer pattern parameters (∼1 order of magnitude). Moreover, instead of simply stacking CNN layers, we design the SPOD network based on the transformer architecture. It can better model global features and reinforce the network's attention to the targets in the scene, thus improving the object detection performance. We demonstrate the effectiveness of SPOD on the Voc dataset, which achieves a detection accuracy of 82.41% mAP at a sampling rate of 5% with a refresh rate of 63 f.p.s.

Structural Insight into Polymerase Mechanism via a Chiral Center Generated with a Single Selenium Atom.

DNA synthesis catalyzed by DNA polymerase is essential for all life forms, and phosphodiester bond formation with phosphorus center inversion is a key step in this process. Herein, by using a single-selenium-atom-modified dNTP probe, we report a novel strategy to visualize the reaction stereochemistry and catalysis. We capture the before- and after-reaction states and provide explicit evidence of the center inversion and in-line attacking SN2 mechanism of DNA polymerization, while solving the diastereomer absolute configurations. Further, our kinetic and thermodynamic studies demonstrate that in the presence of Mg2+ ions (or Mn2+), the binding affinity (Km) and reaction selectivity (kcat/Km) of dGTPαSe-Rp were 51.1-fold (or 19.5-fold) stronger and 21.8-fold (or 11.3-fold) higher than those of dGTPαSe-Sp, respectively, indicating that the diastereomeric Se-Sp atom was quite disruptive of the binding and catalysis. Our findings reveal that the third metal ion is much more critical than the other two metal ions in both substrate recognition and bond formation, providing insights into how to better design the polymerase inhibitors and discover the therapeutics.

Synthesis and anti-tumor activity evaluation of salinomycin C20-O-alkyl/benzyl oxime derivatives.

Seventeen C20-O-alkyl/benzyl oxime derivatives were synthesized by a concise and effective method. Most of these derivatives showed tens to several hundred nanomolar IC50 values against HT-29 colorectal, HGC-27 gastric and MDA-MB-231 breast cancer cells, whose antiproliferative activity is 15-240 fold better than that of salinomycin. The C20-oxime etherified derivatives can coordinate potassium ions, and further adjust the cytosolic Ca2+ concentrations in HT-29 cells. The significant improvement of the potency should be attributed to the better ion binding and transport ability of the modified derivatives. In addition, the C20-O-alkyl/benzyl oxime derivatives showed much better selectivity indexes (SI) than salinomycin, indicating that they present lower neurotoxic risk.

The probiotic potential of Lactobacillus plantarum strain RW1 isolated from canine faeces.

AIM: To evaluation the probiotic potential of Lactobacillus plantarum strain RW1 isolated from healthy dogs for its further utilization as a dietary supplement for dogs. METHODS AND RESULTS: This study aimed to evaluate the probiotic potential of L. plantarum strain RW1 isolated from canine faeces. After confirming by conventional and then by 16S rRNA sequencing, the identified strain RW1 was in vitro screened for its survivability in simulated gastrointestinal conditions, low pH, bile salts and adhesion to gut epithelial tissues, growth inhibitory effects on common pathogens and anti-inflammatory potential by measuring the mRNA expression level of IL-6, IL-8, IL-1ß in Salmonella-infected MODE-K cells. Furthermore, the effects on epithelial barrier function and host defensin peptide (beta-defensin 3) was studied by measuring the mRNA expression level of tight junction protein (occludin) and beta-defensin 3 in MODE-K cells. The strain RW1 showed a considerable potential to survive in simulated gastrointestinal environmental conditions, low pH and high bile salt concentrations along with good adhesion to MODE-K cell line. Pathogenic bacterial growth and their adhesion to MODE-K cell line were significantly inhibited by the strain RW1. Real-time PCR analyses demonstrated that the strain RW1 inhibited Salmonella-induced pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) production and reinforced the expression of tight junction protein (occludin). The strain RW1 did not induce mRNA expression of beta-defensin 3. CONCLUSION: Based on in vitro results, the strain RW1 has the potential to be used as a probiotic supplement in dogs. However, further study involving in vivo health effects is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotics have many side effects and nowadays the probiotics are considered as a potential alternative to antibiotics. This study evaluates the probiotic potential of dog isolated L. plantarum strain RW1 to use it as a dietary supplement in dogs feeding to control infectious diseases.

Effects of radiofrequency field from 5G communications on the spatial memory and emotionality in mice.

The rapid development of 5G network technology has gained much popularity as well as concerns about its adverse effects. In this study, we investigated the effects of 4.9 GHz (one of working frequencies of 5G communication) radiofrequency (RF) field on emotional behaviours and spatial memory in adult male mice. Open field test (OFT), tail suspension test (TST) and Y maze were used to evaluate anxiety, depression-like behaviour and spatial memory ability, respectively. It was found that the anxiety-like behaviour and spatial memory ability of mice did not change, but the depression-like behaviour was induced in mice after 4.9 GHz RF exposure. In addition, the number of neurons significantly reduced and the level of pyroptosis obviously increased in amygdala rather than hippocampus. These results suggested that 4.9 GHz RF exposure could induce depression-like behaviour, which might be associated with the neuronal pyroptosis in amygdala.

Cognitive Dysfunction in Migraineurs.

Background and Objectives: Migraines are one of the most common types of primary headaches in neurology. Many studies to date have investigated cognitive impairment in migraineurs, but the results are inconsistent. This study aimed to investigate the cognitive function of migraineurs and explore the influencing factors. Material and Methods: A total of 117 patients with primary headaches (87 with migraine and 30 with tension-type headache (TTH)) and 30 healthy controls were enrolled. General information and data on headache clinical characteristics, and assessments of headache-related disability, psychological symptoms, and cognitive function were collected for statistical analysis. Results: The Montreal Cognitive Assessment (MoCA) total score and the scores of visuospatial and executive functions, language, and delayed recall in the migraine and TTH groups were significantly lower than those in the healthy control group (all p < 0.05). The MoCA total score did not correlate with Headache impact test-6, Migraine Disability Assessment Questionnaire, Patient Health Questionnaire-9, or Generalized Anxiety Disorder Questionnaire-7 scores in migraineurs (all p > 0.0125). The multiple linear regression analysis showed that age and duration of attack had a major influence on the overall and various fields of cognition in migraineurs. Conclusion: The study confirmed the impairment of cognitive function in patients with migraine and TTH, and found that the duration of attack had an effect on cognitive function in migraineurs.

[Pregnancy Outcomes and Prognosis of Fetal Ventriculomegaly: A Retrospective Cohort Study].

Objective: To evaluate the pregnancy outcomes and neurodevelopment prognosis of subjects prenatally diagnosed with fetal ventriculomegaly (VM). Methods: All the subjects with VM diagnosed by ultrasound and were admitted and treated at West China Second Hospital, Sichuan University between March 2011 and September 2020 were retrospectively enrolled for a chohort study, while non-VM subjects of the same period were selected with a random number table to form the control group. Pregnancy outcomes of the two groups were compared, and the fetuses of both groups were followed up after birth for further assessment and comparison of their neurodevelopmental prognosis. Results: The live birth rate of the VM group was lower than that of the control group (77.63% [229/295] vs. 94.31% [265/281], P<0.001). Furthermore, the proportion of subjects that were transferred to NICU for monitoring and observation after birth was higher in the VM group than that of the control group (20.96% [48/229] vs. 4.53% [12/265], P<0.001). During the follow-up, it was found that the rate of neurodevelopmental abnormalities of the VM group was significantly higher than that of the control group (11.79% [27/229] vs. 1.90% [5/265], P<0.001). Moreover, neurodevelopmental abnormalities of VM fetuses were correlated to the following factors, the degree of VM ( P=0.010), intrauterine progression of VM ( P=0.024), and whether the postnatal cranial ultrasound result was suggestive of VM ( P=0.001). In addition, postnatal cranial ultrasound suggestive of VM was found to be an independent risk factor for neurodevelopmental abnormalities ( OR=9.434, 95% CI: 1.791-49.688, P=0.008). Conclusion: VM reduces the fetal live birth rate and may increase the risks of neurodevelopmental abnormalities after birth. All VM fetuses should be closely followed up for neurodevelopment status after birth, especially those with severe VM, intrauterine progression, and postnatal cranial ultrasound indicative of VM.

Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91-135 aa, S1 gene; 377-434 aa, S2 gene; 647-671 aa, S3 gene; 951-971 aa, S4 gene; 207-227 aa) and two T-cell epitopes in tandem: T-E1-E2-E3-E4-T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

Relationship between illness perception, fear of progression and quality of life in interstitial lung disease patients: A cross-sectional study.

AIMS AND OBJECTIVES: This study aimed to investigate whether fear of progression mediates the association between illness perception and quality of life among interstitial lung disease patients. BACKGROUND: So far, the physiological treatment of interstitial lung disease is limited. In addition to immunosuppressants such as glucocorticoids, two anti-fibrosis drugs (pirfenidone and nintedanib) have shown moderately beneficial effects on slowing the progression of interstitial lung disease fibrosis. However, none of these drugs has shown reliable or strong beneficial effects on improving quality of life. Psychological care and mental health support strategies focusing on improving patients' quality of life are particularly important. DESIGN: A cross-sectional study. METHODS: A convenience sample of patients suffering from interstitial lung disease were enrolled from August to December 2019. Data including sociodemographic and clinical characteristics, illness perception, fear of progression and quality of life were collected. The descriptive analysis and Pearson correlations were analysed by SPSS 26.0 (IBM Corp.). PROCESS v3.4 (by Andrew F. Hayes) macro was applied to analyse the mediating effects. We used the STROBE checklist to report the results. RESULTS: Both illness perception and fear of progression were correlated with quality of life. Fear of progression mediated the association between illness perception and quality of life. The indirect effect was 0.121, and the proportion of intermediary effect in the main effect was 26.36%. CONCLUSION: Interstitial lung disease patients experience relatively poor quality of life and fear of progression exerts a mediating role between illness perception and quality of life. RELEVANCE TO CLINICAL PRACTICE: This study alerts medical staff to pay attention to negative illness perception and excessive fear, which is helpful to formulate effective interventions to manage interstitial lung disease patients' quality of life.

Discovery and structure-activity relationship study of phthalimide-phenylpyridine conjugate as inhibitor of Wnt pathway.

Aberrant Wnt signaling has been implicated in a variety of disease. Inhibition of the Wnt pathway is an attractive approach for developing new therapeutics for the treatment of various types of fibrosis and cancers. We have discovered the phthalimide-phenylpyridine conjugate as a novel hit compound for the Wnt pathway inhibitors from cellular screening. The structure-activity relationship of these compounds suggested both of the substituent group on the phthalimide fragment and the structure of the linker were critical to the inhibitory activity. The most potent compound was about 10-folds more potent than the hit compound, with IC50 value of 0.28 ±â€¯0.01 µM.

Moonlighting Phosphatase Activity of Klenow DNA Polymerase in the Presence of RNA.

RNA is a key player in the cellular central dogma, including RNA transcription and protein synthesis. However, it is unknown whether RNA can directly interfere with DNA synthesis. Recently, we have found in vitro that while binding to DNA polymerase nonspecifically, RNA can transform DNA polymerase to display a moonlighting activity, dNTP phosphatase, in turn interfering with DNA synthesis. This phosphatase activity removes the γ-phosphate from dNTPs (generating dNDPs) and subsequently removes the ß-phosphate from the formed dNDPs (generating dNMPs), confirmed by the noncleavable α,ß-CH2-dGTP and ß,γ-CH2-dGTP analogues. We also found that dGTP is the best substrate for the phosphatase, and the dNTP phosphatase activity is sensitive to the reaction medium. In addition, we have revealed that RNA can tune the activity of closely related proteins and give rise to new catalytic functions with subtle differences. Moreover, we have demonstrated in vitro that at the lower dNTP level, this phosphatase can directly inhibit DNA synthesis by dNTP depletion, though the phosphatase activity is 690-fold slower than the polymerase activity. Our observation in vitro suggests a plausible strategy for RNA to directly interfere with DNA polymerase and DNA synthesis in vivo.

Clinical and Biological Implications of Mutational Spectrum in Acute Myeloid Leukemia of FAB Subtypes M0 and M1.

BACKGROUND/AIMS: Acute myeloid leukemia (AML) of French-American-British (FAB) subtypes M0 and M1 are both poorly differentiated AML, but their mutational spectrum and molecular characteristics remain unknown. This study aimed to explore the mutational spectrum and prognostic factors of AML-M0 and M1. METHODS: Sixty-five AML patients derived from The Cancer Genome Atlas (TCGA) database were enrolled in this study. Whole-genome sequencing was performed to depict the mutational spectrum of each patient. Clinical characteristics at diagnosis, including peripheral blood (PB) white blood cell counts (WBC), blast percentages in PB and bone marrow (BM), FAB subtypes and the frequencies of known recurrent genetic mutations were described. Survival was estimated using the Kaplan-Meier methods and log-rank test. Univariate and multivariate Cox proportional hazard models were constructed for event-free survival (EFS) and overall survival (OS), using a limited backward elimination procedure. RESULTS: Forty-six patients had more than five recurrent genetic mutations. FLT3 had the highest mutation frequency (n=20, 31%), followed by NPM1 (n=18, 28%), DNMT3A (n=16, 25%), IDH1 (n=14, 22%), IDH2 (n=12, 18%), RUNX1 (n=11, 17%) and TET2 (n=7, 11%). Univariate analysis showed that age ≥60 years and TP53 mutations had adverse effect on EFS (P=0.015, P=0.036, respectively) and OS (P=0.003, P=0.004, respectively), WBC count ≥50×109/L and FLT3-ITD negatively affected EFS (P=0.003, P=0.034, respectively), whereas NPM1 mutations had favorable effect on OS (P=0.035) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) on EFS and OS (all P< 0.001). Multivariate analysis suggested that allo-HSCT and NPM1 mutations were independent favorable prognostic factors for EFS and OS (all P< 0.05), WBC count ≥50×109/L was an independent risk factor for EFS (P=0.002) and TP53 mutations for OS (P=0.043). CONCLUSIONS: Our study provided new insights into the mutational spectrum and molecular signatures of AML-M0 and M1. We proposed that FLT3-ITD, NPM1 and TP53 be identified as markers for risk stratification of AML-M0 and M1. Patients with AML-M0 and M1 would likely benefit from allo-HSCT.

Internal Duplications of DH, JH, and C Region Genes Create an Unusual IgH Gene Locus in Cattle.

It has been suspected for many years that cattle possess two functional IgH gene loci, located on Bos taurus autosome (BTA) 21 and BTA11, respectively. In this study, based on fluorescence in situ hybridization and additional experiments, we showed that all functional bovine IgH genes were located on BTA21, and only a truncated µCH2 exon was present on BTA11. By sequencing of seven bacterial artificial chromosome clones screened from a Hostein cow bacterial artificial chromosome library, we generated a 678-kb continuous genomic sequence covering the bovine IGHV, IGHD, IGHJ, and IGHC genes, which are organized as IGHVn-IGHDn-IGHJn-IGHM1-(IGHDP-IGHV3-IGHDn)3-IGHJn-IGHM2-IGHD-IGHG3-IGHG1-IGHG2-IGHE-IGHA. Although both of two functional IGHM genes, IGHM1 and IGHM2, can be expressed via independent VDJ recombinations, the IGHM2 can also be expressed through class switch recombination. Likely because more IGHD segments can be involved in the expression of IGHM2, the IGHM2 gene was shown to be dominantly expressed in most tissues throughout different developmental stages. Based on the length and identity of the coding sequence, the 23 IGHD segments identified in the locus could be divided into nine subgroups (termed IGHD1 to IGHD9). Except two members of IGHD9 (14 nt in size), all other functional IGHD segments are longer than 30 nt, with the IGHD8 gene (149 bp) to be the longest. These remarkably long germline IGHD segments play a pivotal role in generating the exceptionally great H chain CDR 3 length variability in cattle.

The Influence of Oxygen Atoms on Conformation and π-π Stacking of Ladder-Type Donor-Based Polymers and Their Photovoltaic Properties.

A novel ladder-type donor pyran-bridged indacenodithiophene (IDTP) is developed by introducing two oxygen atoms into indacenodithiophene unit. IDTP possesses a twisted backbone and leads to facially asymmetric arrangement of side chains, resulting in enhanced local π-π stacking of according polymer poly[(5,5,11,11-tetrakis(4-octylphenyl)-5,11-dihydrothieno[2',3':5,6]pyrano[3,4-g]thieno[3,2-c]isochromene)-alt-4,7-(5-fluoro-2,1,3-benzothiadiazole)] (PIDTP)-FBT, which shows extended absorption range. Moreover, oxygen atoms render deeper highest occupied molecular orbital (HOMO) levels of poly[indacenodithiophene-alt-4,7-(5-fluoro-2,1,3-benzothiadiazole)] (PIDTP)-FBT compared with PIDT-FBT, therefore bringing a higher open-circuit voltage (Voc ).

Adipose-derived stem cells induce autophagic activation and inhibit catabolic response to pro-inflammatory cytokines in rat chondrocytes.

OBJECTIVE: Adipose-derived stem cells (ADSCs) have been demonstrated to have an anti-apoptosis effect on chondrocytes; However, their effect on autophagic activation remains unclear. We sought to explore whether ADSCs can activate autophagy and inhibit IL-1ß- and lipopolysaccharide (LPS)-induced catabolism in chondrocytes. METHODS: ADSCs and chondrocytes were collected from SD rats. The biologic characteristics of ADSCs were analyzed by flow cytometric analysis, Oil red O and Alizarin Red staining. Autophagic level and autophagic flux were revealed by Western blotting for LC3-II and SQSTM1/P62, MDC (monodansylcadaverine) staining and mRFP-GFP-LC3 analysis. The mTOR pathway was investigated by Western blotting for p-mTOR. The mRNA level of matrix metalloproteinases (MMPs) and thrombospondin motifs (ADAMTSs) was detected by real-time PCR. RESULTS: The typical surface markers and differentiation potentials of ADSCs were proved. ADSCs enhanced the expression of LC3-II/LC3-I and reduced SQSTM1 levels in IL-1ß-induced chondrocytes after 24 and 48 h co-culturing and in LPS-induced chondrocytes after 48 h co-culturing respectively. mRFP-GFP-LC3 analysis suggested that autophagosomes and autolysosomes were formed earlier in IL-1ß-treated chondrocytes than in LPS-treated chondrocytes. Bafilomycin A1 treatment further increased the LC3-II/LC3-I level in chondrocytes in co-culture with ADSCs. The mTOR pathway was inhibited in the chondrocytes in co-culture with ADSCs. Finally, ADSCs inhibited the increase of MMPs and ADAMTSs in chondrocytes induced by IL-1ß and LPS. CONCLUSIONS: ADSCs seem able to activate autophagy and inhibit catabolism in chondrocytes in an inflammation environment, and the mTOR pathway might be involved in the autophagy activation.

Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes.

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.

Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice.

Intraperitoneal busulfan injections are used to prepare recipients for spermatogonial stem cell (SSC) transplantation but they are associated with haematopoietic toxicity. Testicular injections of busulfan have been proposed to overcome this limitation. To date, testicular injections have not been studied in the mouse model. Therefore, in the present study we used ICR mice as recipients for SSC transplantation and prepared these mice by testicular injection of busulfan on both sides (2, 3, 4 or 6mgkg-1 per side). Following this, donor germ cells expressing red fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted into recipients via the efferent duct on Days 16-17 after busulfan treatment. Positive control mice were prepared by intraperitoneal injection of 40mgkg-1 busulfan and negative control mice were treated with bilateral testicular injection of 50% dimethyl sulfoxide. On Day 49 after transplantation, recipient mice that were RFP-positive by in vivo imaging were mated with ICR female mice. Donor-derived germ cell colonies with red fluorescence were observed on Day 60 after transplantation, and donor-derived offspring were obtained. The results demonstrated that endogenous germ cells were successfully eliminated in the seminiferous tubules via testicular busulfan administration, and that exogenous SSCs successfully undergo spermatogenesis in the testes of recipient mice prepared by testicular injections of busulfan. In addition to its effects on recipient preparation, this method was safe in rodents and could possibly be adapted for use in other species.