Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n = 2), follicular phase (F, n = 4), ovulation (O, n = 2), early luteal (EL, n = 7) and mid/late luteal phase (MLL, n = 2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II + III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P < 0.05; O vs. A: P < 0.001), in EL (61%; O vs. EL: P < 0.01), or in MLL (0%; F vs. MLL: P < 0.05; O vs. MLL: P < 0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P < 0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.     

Defective Autophagy in Parkinson’s Disease: Role of Oxidative Stress

Parkinson??s disease (PD) is a paradigmatic example of neurodegenerative disorder with a critical role of oxidative stress in its etiopathogenesis. Genetic susceptibility factors of PD, such as mutations in Parkin, PTEN-induced kinase 1, and DJ-1 as well as the exposure to pesticides and heavy metals, both contribute to altered redox balance and degeneration of dopaminergic neurons in the substantia nigra. Dysregulation of autophagy, a lysosomal-driven process of self degradation of cellular organelles and protein aggregates, is also implicated in PD and PD-related mutations, and environmental toxins deregulate autophagy. However, experimental evidence suggests a complex and ambiguous role of autophagy in PD since either impaired or abnormally upregulated autophagic flux has been shown to cause neuronal loss. Finally, it is generally believed that oxidative stress is a strong proautophagic stimulus. However, some evidence coming from neurobiology as well as from other fields indicate an inhibitory role of reactive oxygen species and reactive nitrogen species on the autophagic machinery. This review examines the scientific evidence supporting different concepts on how autophagy is dysregulated in PD and attempts to reconcile apparently contradictory views on the role of oxidative stress in autophagy regulation. The complex relationship between autophagy and oxidative stress is also considered in the context of the ongoing search for a novel PD therapy.     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids

Oligonucleotide microarrays or oDNA chips are effective decoding and analytical tools for genomic sequences and are useful for a broad range of applications. Therefore, it is desirable to have synthesis methods of DNA chips that are highly flexible in sequence design and provide high quality and general adoptability. We report herein, DNA microarray synthesis based on a flexible biochip method. Our method simply uses photogenerated acid (PGA) in solution to trigger deprotection of the 5′-OH group in conventional nucleotide phosphoramidite monomers (i.e. PGA-gated deprotection), with the rest of the reactions in the synthesis cycle the same as those used for routine synthesis of oligonucleotides. The complete DNA chip synthesis process is accomplished on a regular DNA synthesizer that is coupled with a UV-VIS projection display unit for performing digital photolithography. Using this method, oDNA chips containing probes of newly discovered genes can be quickly and easily synthesized at high yields in a conventional laboratory setting. Furthermore, the PGA-gated chemistry should be applicable to microarray syntheses of a variety of combinatorial molecules, such as peptides and organic molecules.     

Stereotactic radiotherapy for liver oligometastases

The liver is the first metastatic site in 15–25% of colorectal cancer patients and one of the first metastatic sites for lung and breast cancer patients.A computed tomography (CT ) scan with contrast medium is a standard procedure for assessing liver lesions but magnetic resonance imaging (MRI) characterizes small lesions better thanks to its high soft-tissue contrast. Positron emission tomography with computed tomography (PET-CT ) plays a complementary role in the diagnosis of liver metastases. Triphasic (arterial, venous and time-delayed) acquisition of contrast-medium CT images is the first step in treatment planning. Since the liver exhibits a relatively wide mobility due to respiratory movements and bowel filling, appropriate techniques are needed for target identification and motion management. Contouring requires precise recognition of target lesion edges. Information from contrast MRI and/or PET-CT is crucial as they best visualize metastatic disease in the parenchyma. Even though different fractionation schedules were reported, doses and fractionation schedules for liver stereotactic radiotherapy (SRT ) have not yet been established. The best local control rates were obtained with BED₁₀ values over 100 Gy. Local control rates from most retrospective studies, which were limited by short follow-ups and included different primary tumors with intrinsic heterogeneity, ranged from 60% to 90% at 1 and 2 years. The most common SRT-related toxicities are increases in liver enzymes, hyperbilirubinemia and hypoalbuminemia. Overall, late toxicity is mild even in long-term follow-ups.     

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.     

The Forgotten Role of Central Volume in Low Frequency Oscillations of Heart Rate Variability

The hypothesis that central volume plays a key role in the source of low frequency (LF) oscillations of heart rate variability (HRV) was tested in a population of end stage renal disease patients undergoing conventional hemodialysis (HD) treatment, and thus subject to large fluid shifts and sympathetic activation. Fluid overload (FO) in 58 chronic HD patients was assessed by whole body bioimpedance measurements before the midweek HD session. Heart Rate Variability (HRV) was measured using 24-hour Holter electrocardiogram recordings starting before the same HD treatment. Time domain and frequency domain analyses were performed on HRV signals. Patients were retrospectively classified in three groups according to tertiles of FO normalized to the extracellular water (FO/ECW%). These groups were also compared after stratification by diabetes mellitus. Patients with the low to medium hydration status before the treatment (i.e. 1st and 2nd FO/ECW% tertiles) showed a significant increase in LF power during last 30 min of HD compared to dialysis begin, while no significant change in LF power was seen in the third group (i.e. those with high pre-treatment hydration values). In conclusion, several mechanisms can generate LF oscillations in the cardiovascular system, including baroreflex feedback loops and central oscillators. However, the current results emphasize the role played by the central volume in determining the power of LF oscillations.     

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle‐stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre‐ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct‐specific glycoprotein 1, heat‐shock protein family A member 5, α‐l ‐fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.