Spontaneous Overactivation of Xenopus Frog Eggs Triggers Necrotic Cell Death.

The excessive activation of frog eggs, referred to as overactivation, can be initiated by strong oxidative stress, leading to expedited calcium-dependent non-apoptotic cell death. Overactivation also occurs spontaneously, albeit at a low frequency, in natural populations of spawned frog eggs. Currently, the cytological and biochemical events of the spontaneous process have not been characterized. In the present study, we demonstrate that the spontaneous overactivation of Xenopus frog eggs, similarly to oxidative stress- and mechanical stress-induced overactivation, is characterized by the fast and irreversible contraction of the egg's cortical layer, an increase in egg size, the depletion of intracellular ATP, a drastic increase in the intracellular ADP/ATP ratio, and the degradation of M phase-specific cyclin B2. These events manifest in eggs in the absence of caspase activation within one hour of triggering overactivation. Importantly, substantial amounts of ATP and ADP leak from the overactivated eggs, indicating that plasma membrane integrity is compromised in these cells. The rupture of the plasma membrane and acute depletion of intracellular ATP explicitly define necrotic cell death. Finally, we report that egg overactivation can occur in the frog's genital tract. Our data suggest that mechanical stress may be a key factor promoting egg overactivation during oviposition in frogs.

Oxidative Stress-Induced Overactivation of Frog Eggs Triggers Calcium-Dependent Non-Apoptotic Cell Death.

Excessive activation of frog eggs (overactivation) is a pathological process that renders eggs unfertilizable. Its physiological inducers are unknown. Previously, oxidative stress was shown to cause time- and dose-dependent overactivation of Xenopus laevis frog eggs. Here, we demonstrate that the oxidative stress-induced egg overactivation is a calcium-dependent phenomenon which can be attenuated in the presence of the selective calcium chelator BAPTA. Degradation of cyclin B2, which is known to be initiated by calcium transient in fertilized or parthenogenetically activated eggs, can also be observed in the overactivated eggs. Decline in mitochondrial membrane potential, ATP depletion and termination of protein synthesis manifest in the eggs within one hour of triggering overactivation. These intracellular events occur in the absence of caspase activation. Furthermore, plasma membrane integrity is compromised in the overactivated eggs, as evidenced by ATP leakage and egg swelling. In sum, our data demonstrate that oxidative stress-induced overactivation of frog eggs causes fast and dramatic disruption of cellular homeostasis, resulting in robust and expedited cell death by a calcium-dependent non-apoptotic mechanism.

Polarization-insensitive local-oscillator-carrier loopback modulation for a cost-effective and high-port-count wavelength-routing optical switch.

A wavelength-routing optical switch uses a wavelength-tunable laser at each input port, and this transmitter implements output port selection by tuning the wavelength that is associated with each output port. With coherent transmission, loopback modulation of a local oscillator (LO) carrier generated at the output port can eliminate the need for a wavelength-tunable laser. However, loopback modulation can be unstable since the power fluctuates because fiber traversal by the light creates polarization rotation. Here, we propose a simple polarization-alignment circuit and verify its effectiveness in creating a high-port-count optical switch system. The proposed circuit consists of passive components and aligns the polarization state of the supplied LO carrier to be linearly polarized along the x-direction of a TE-input dual-polarization (DP) IQ modulator. The circuit is shown to yield stable modulation with Q-variation of less than 0.8 dB, regardless of any birefringence along the transmission path. The proposal's effectiveness is verified in optical switch system experiments with DP-QPSK signals; 1,856 × 1,856 switch scale is achieved with loopback modulation.

Protein pI and Intracellular Localization.

The protein isoelectric point (pI) can be calculated from an amino acid sequence using computational analysis in a good agreement with experimental data. Availability of whole-genome sequences empowers comparative studies of proteome-wide pI distributions. It was found that the whole-proteome distributions of protein pI values are multimodal in different species. It was further hypothesized that the observed multimodality is associated with subcellular localization-specific differences in local pI distributions. Here, we overview the multimodality of proteome-wide pI distributions in different organisms focusing on the relationships between protein pI and subcellular localization. We also discuss the probable factors responsible for variation of the intracellular localization-specific pI profiles.

Design and verification of a LO bank enabled by fixed-wavelength lasers and fast tunable silicon ring filters for creating large scale optical switches.

The fast and widely tunable wavelength bank is a key enabler in creating wavelength-routing optical switches that do not use fast wavelength tunable lasers. A cost-effective design criterion needs to be developed before it can be applied to intra data center networks. In this paper, we develop a systematic method for designing a wavelength bank that yields high port-count and fast wavelength-routing optical switches for intra data center application. The wavelength bank is created with fixed-wavelength laser sources and wavelength-tunable filters with rapid wavelength selectivity. To optimize the optical switching system that uses the wavelength bank for supplying local oscillator (LO) lights for coherent detection, various parameters are analyzed, including effective bandwidth, laser output power, loss distribution, splitter port count, and optical amplifier gain. We carry out numerical simulations for optimizing the tradeoff between system performance and cost. To verify the designed wavelength bank, a silicon ring filter is newly fabricated with an average fiber-to-fiber insertion loss of 5.3 dB over a 22-nm bandwidth. Using 256-Gb/s DP-QPSK signals, experiments demonstrate a 1,024×1,024 optical switch that uses a fabricated silicon ring filter. The effectiveness of the scalable and fast-tunable LO bank is verified by achieving 262.1-Tb/s switch throughput with switching time under 18 µs.

Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants.

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

Loss of H3K27 trimethylation is frequent in IDH1-R132H but not in non-canonical IDH1/2 mutated and 1p/19q codeleted oligodendroglioma: a Japanese cohort study.

Oligodendrogliomas are defined by mutation in isocitrate dehydrogenase (NADP(+)) (IDH)1/2 genes and chromosome 1p/19q codeletion. World Health Organisation diagnosis endorses testing for 1p/19q codeletion to distinguish IDH mutant (Mut) oligodendrogliomas from astrocytomas because these gliomas require different treatments and they have different outcomes. Several methods have been used to identify 1p/19q status; however, these techniques are not routinely available and require substantial infrastructure investment. Two recent studies reported reduced immunostaining for trimethylation at lysine 27 on histone H3 (H3K27me3) in IDH Mut 1p/19q codeleted oligodendroglioma. However, the specificity of H3K27me3 immunostaining in this setting is controversial. Therefore, we developed an easy-to-implement immunohistochemical surrogate for IDH Mut glioma subclassification and evaluated a validated adult glioma cohort. We screened 145 adult glioma cases, consisting of 45 IDH Mut and 1p/19q codeleted oligodendrogliomas, 30 IDH Mut astrocytomas, 16 IDH wild-type (Wt) astrocytomas, and 54 IDH Wt glioblastomas (GBMs). We compared immunostaining with DNA sequencing and fluorescent in situ hybridization analysis and assessed differences in H3K27me3 staining between oligodendroglial and astrocytic lineages and between IDH1-R132H and non-canonical (non-R132H) IDH1/2 Mut oligodendroglioma. A loss of H3K27me3 was observed in 36/40 (90%) of IDH1-R132H Mut oligodendroglioma. In contrast, loss of H3K27me3 was never seen in IDH1-R132L or IDH2-mutated 1p/19q codeleted oligodendrogliomas. IDH Mut astrocytoma, IDH Wt astrocytoma and GBM showed preserved nuclear staining in 87%, 94%, and 91% of cases, respectively. A high recursive partitioning model predicted probability score (0.9835) indicated that the loss of H3K27me3 is frequent to IDH1-R132H Mut oligodendroglioma. Our results demonstrate H3K27me3 immunohistochemical evaluation to be a cost-effective and reliable method for defining 1p/19q codeletion along with IDH1-R132H and ATRX immunostaining, even in the absence of 1p/19q testing.

Dissection of the Ovulatory Process Using ex vivo Approaches.

Ovulation is a unique physiological phenomenon that is essential for sexual reproduction. It refers to the entire process of ovarian follicle responses to hormonal stimulation resulting in the release of mature fertilization-competent oocytes from the follicles and ovaries. Remarkably, ovulation in different species can be reproduced out-of-body with high fidelity. Moreover, most of the molecular mechanisms and signaling pathways engaged in this process have been delineated using in vitro ovulation models. Here, we provide an overview of the major molecular and cytological events of ovulation observed in frogs, primarily in the African clawed frog Xenopus laevis, using mainly ex vivo approaches, with the focus on meiotic oocyte maturation and follicle rupture. For the purpose of comparison and generalization, we also refer extensively to ovulation in other biological species, most notoriously, in mammals.

Prevalence and Possible Predictors of Drug-Resistant Pseudomonas aeruginosa in External Ocular Infections: A Single-Center, Retrospective, Cross-Sectional Study.

Objective Ocular infections due to multi-drug-resistant Pseudomonas aeruginosa (MDRP) have been reported in recent years. This study was undertaken to determine the culture-positivity rate of P. aeruginosa and drug resistance among patients with suspected external ocular infections and to predict systemic risk factors for drug resistance in P. aeruginosa. Methods This retrospective, single-center, cross-sectional study involved 781 consecutive patients who provided samples for aerobic culture to test for a suspected external ocular infection. DRP was defined as a strain resistant to one or two of three antibiotics, levofloxacin, gentamicin, and imipenem; MDRP was defined as that which was resistant to all three. Results Among 108 patients in whom gram-negative bacilli were observed, P. aeruginosa was isolated from nine patients, including three DRP-positive cases; no MDRP was isolated. P. aeruginosa was not isolated from those <69 years of age. Among patients in whom gram-negative bacilli were detected, the isolation rate of P. aeruginosa was 0 for patients aged ≤64 and 0.1 for those aged ≥65, indicating a significant difference. For patients with gram-negative bacilli, the DRP-positivity rate was significantly higher for hospitalized patients than outpatients. Thus, in addition to being geriatric, being hospitalized was a risk factor for DRP infection among patients with gram-negative bacilli. All the P. aeruginosa strains isolated were susceptible to colistin. Conclusions Our findings may suggest that once gram-negative bacilli are isolated in an elderly hospitalized patient, possible DRP infection and the topical use of colistin should be taken into consideration even before the results of culture and susceptibility testing are obtained.

Toward the understanding of biology of oocyte life cycle in Xenopus Laevis: No oocytes left behind.

BACKGROUND: For the past more than 25 years, we have been focusing on the developmental and reproductive biology of the female gametes, oocytes, and eggs, of the African clawed frog Xenopus laevis. METHODS: The events associated with the life cycle of these cells can be classified into the four main categories: first, oogenesis and cell growth in the ovary during the first meiotic arrest; second, maturation and ovulation that occur simultaneously and result in the acquisition of fertilization competence and the second meiotic arrest; third, fertilization, that is sperm-induced transition from egg to zygote; and fourth, egg death after spontaneous activation in the absence of fertilizing sperm. MAIN FINDINGS: Our studies have demonstrated that signal transduction system involving tyrosine kinase Src and other oocyte/egg membrane-associated molecules such as uroplakin III and some other cytoplasmic proteins such as mitogen-activated protein kinase (MAPK) play important roles for successful ovulation, maturation, fertilization, and initiation of embryonic development. CONCLUSION: We summarize recent advances in understanding cellular and molecular mechanisms underlying life cycle events of the oocytes and eggs. Our further intention is to discuss and predict potentially promising impact of the recent findings on the challenges facing reproductive biology and medicine, as well as societal contexts.

Long-Term Outcomes of a Surgical Technique in Management of Subconjunctival Orbital Fat Prolapse.

Objective This retrospective, single-center, interventional study presented the long-term results of a novel surgical technique for the management of subconjunctival orbital fat prolapse. Methods Nine eyes of seven consecutive patients were surgically repaired using the technique involving connective tissue repair and were intended to be followed-up for more than five years. Results All surgeries performed were uneventful and esthetic enhancement was achieved for each patient. No postoperative complications were noted. There was no recurrence throughout the follow-up period; six eyes of four patients were followed-up for more than five years. Conclusions This technique with connective tissue repair achieved good long-term results.

In Vitro Reconstruction of Xenopus Oocyte Ovulation.

Progesterone is widely used to induce maturation of isolated fully grown oocytes of the African clawed frog, Xenopus laevis. However, the hormone fails to release oocytes from the layer of surrounding follicle cells. Here, we report that maturation and follicle rupture can be recapitulated in vitro by treating isolated follicular oocytes with progesterone and low doses of the matrix metalloproteinase (MMP), collagenase, which are ineffective in the absence of the steroid. Using this in vitro ovulation model, we demonstrate that germinal vesicle breakdown (GVBD) and oocyte liberation from ovarian follicles occur synchronously during ovulation. Inhibition of the MAPK pathway in these experimental settings suppresses both GVBD and follicular rupture, whereas inhibition of MMP activity delays follicular rupture without affecting GVBD. These results highlight importance of MAPK and MMP activities in the ovulation process and provide the first evidence for their involvement in the release of oocytes from ovarian follicles in frogs. The in vitro ovulation model developed in our study can be employed for further dissection of ovulation.

Localization-specific distributions of protein pI in human proteome are governed by local pH and membrane charge.

BACKGROUND: Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail. RESULTS: In this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression (R2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles. CONCLUSIONS: Our study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome.

Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs.

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.

Biochemical Hallmarks of Oxidative Stress-Induced Overactivation of Xenopus Eggs.

Egg overactivation occurs with a low frequency in the populations of naturally ovulated frog eggs. At present, its natural inducers, molecular mechanisms, and intracellular events remain unknown. Using microscopic and biochemical analyses, we demonstrate here that high levels of hydrogen peroxide-induced oxidative stress can cause time- and dose-dependent overactivation of Xenopus eggs. Lipofuscin accumulation, decrease of soluble cytoplasmic protein content, and depletion of intracellular ATP were found to take place in the overactivated eggs. Progressive development of these processes suggests that egg overactivation unfolds in a sequential and ordered fashion.

Design and evaluation of quasi-Nyquist WDM networks utilizing widely deployed wavelength-selective switches.

Photonic networks based on wavelength-selective switches (WSSs) can transport wavelength-division-multiplexed (WDM) signals in a cost-effective manner. To accommodate the ever-increasing network traffic, the spectral efficiency should be maximized by minimizing the bandwidth of the guardbands inserted between WDM signals. Quasi-Nyquist WDM systems are seen as offering the highest spectral efficiency in a feasible way. However, highly dense WDM systems suffer from the signal-spectrum narrowing induced by the non-rectangular passbands of WSSs. Furthermore, widely deployed WSSs cannot process quasi-Nyquist WDM signals since the signal-alignment granularity does not match the passband resolution of the WSSs. This paper proposes a network architecture that enables quasi-Nyquist WDM networking with widely deployed WSSs. Through intensive network analyses based on computer simulations, we confirm that it has 30.8% higher spectral efficiency than conventional networks. Its feasibility is verified by transmission experiments on 72-channel 32-Gbaud/400-Gbps dual-carrier dual-polarization 16-ary quadrature-amplitude-modulation signals aligned with 66.6-GHz spacing in the full C-band. The net fiber capacity of 28.8 Tbps, the transmission distance of 900 km, and the hop count of 9 are attained by our proposed quasi-Nyquist WDM networking scheme.

Reconstitution of Intracellular Calcium Signaling in Xenopus Egg Extracts.

Generation of calcium signal in the cytoplasm of fertilized or parthenogenetically activated eggs has been extensively studied in the intact eggs of several biological species. Calcium transient was found to elicit a plethora of biochemical and cellular events in these cells. Remarkably, intracellular calcium signaling can also be reconstituted in cell-free environment. In this chapter, we describe the methods that allow reconstitution, detection, and quantification of the calcium signal in cell-free extracts of Xenopus oocytes and eggs.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

Activity and intracellular localization of senescence-associated ß-galactosidase in aging Xenopus oocytes and eggs.

Senescence-associated ß-galactosidase (SA-ß-gal) serves as a marker of senescence in aging somatic cells. However, little is known about SA-ß-gal dynamics in aging gamete cells. To address this issue, here we investigated activity and intracellular localization of SA-ß-gal in freshly obtained and aging oocytes and eggs of the African clawed frog Xenopus laevis. Data base mining revealed the presence of several homologous ß-galactosidase sequences in the annotated Xenopus genome. Some of them were predicted to contain an N-terminal signal peptide sequence, suggesting enzyme translocation to cellular organelles. Biochemical and microscopic analyses confirmed SA-ß-gal localization in the particulate and cytosolic fractions of oocytes and eggs. SA-ß-gal activity was found to reside predominantly within a fraction of dense cytoplasmic granules that were extensively stained with the lysosome-specific dye LysoTracker Green DND-26 and had an average size of 8.9 ±â€¯5.6 µm. These features identify the SA-ß-gal-containing granules as a subpopulation of yolk platelets, specialized late endosomes or lysosomes that accumulate and store processed protein in frog oocytes. Further analysis revealed an increase of SA-ß-gal activity in Xenopus eggs, but not in oocytes, aged in vitro over 48 h. Our data suggest that endosomal acidification during egg aging may be responsible for this increase.

Postovulatory cell death: why eggs die via apoptosis in biological species with external fertilization.

Spawned unfertilized eggs have been found to die by apoptosis in several species with external fertilization. However, there is no necessity for the externally laid eggs to degrade via this process, as apoptosis evolved as a mechanism to reduce the damaging effects of individual cell death on the whole organism. The recent observation of egg degradation in the genital tracts of some oviparous species provides a clue as to the physiological relevance of egg apoptosis in these animals. We hypothesize that egg apoptosis accompanies ovulation in species with external fertilization as a normal process to eliminate mature eggs retained in the genital tract after ovulation. Furthermore, apoptosis universally develops in ovulated eggs after spontaneous activation in the absence of fertilization. This paper provides an overview of egg apoptosis in several oviparous biological species, including frog, fish, sea urchin, and starfish.