Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

新疆石河子棉蚜越冬卵的发育规律

棉蚜雌性蚜交配个体平均产卵量为 6 3± 1 1粒 ;未交配个体也产卵 ,卵亦变色 ,但卵量极少。越冬卵经 5 %NaOH浸泡 2 4h后卵壳透明 ,镜下观察可见受精卵胚胎发育到体躯分节期开始越冬。冬后胚胎发育仅处在颗粒团状的饱满卵应为无效卵 ,不会孵化 ;颜色发亮的卵处理后胚胎外貌清晰可见 ,此种卵当天即可破壳。棉蚜卵为滞育卵 ,春季降水对孵化有促进作用 ;室外棉蚜卵的饱满率和饱满卵的孵化率较低。该地区秋冬气候特点可能是石河子地区棉蚜卵低温条件下安全越冬的重要因素     

Nanoparticle analysis of cancer cells by light transmission spectroscopy

We have measured the optical properties of cancer and normal whole cells and lysates using light transmission spectroscopy (LTS). LTS provides both the optical extinction coefficient in the wavelength range from 220 to 1100 nm and (by spectral inversion using a Mie model) the particle distribution density in the size range from 1 to 3000 nm. Our current work involves whole cells and lysates of cultured human oral cells in liquid suspension. We found systematic differences in the optical extinction between cancer and normal whole cells and lysates, which translate to different particle size distributions (PSDs) for these materials. Specifically, we found that cancer cells have distinctly lower concentrations of nanoparticles with diameters less than 100 nm and have higher concentrations of particles with diameters from 100 to 1000 nm—results that hold for both whole cells and lysates. We also found a power-law dependence of particle density with diameter over several orders of magnitude.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.     

A low volume 3D-printed temperature-controllable cuvette for UV visible spectroscopy

We report the fabrication of a 3D-printed water-heated cuvette that fits into a standard UV visible spectrophotometer. Full 3D-printable designs are provided and 3D-printing conditions have been optimised to provide options to print the cuvette in either acrylonitrile butadiene styrene or polylactic acid polymers, extending the range of solvents that are compatible with the design. We demonstrate the efficacy of the cuvette by determining the critical micelle concentration of sodium dodecyl sulphate at 40 °C, the molar extinction coefficients of cobalt nitrate and dsDNA and by reproducing the thermochromic UV visible spectrum of a mixture of cobalt chloride, water and propan-2-ol.     

Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Abstract Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium , on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.     

Detection and survival of Campylobacter in chicken eggs

AIMS: Campylobacter jejuni, a food-borne human pathogen, is widespread in poultry; however, the sources of infection and modes of transmission of this organism on chicken farms are not well understood. The objective of this study was to determine if vertical transmission of C. jejuni occurs via eggs. METHODS AND RESULTS: Using a temperature differential method, it was shown that Campylobacter had limited ability to penetrate the eggshell. When C. jejuni was directly inoculated into the egg yolk and the eggs were stored at 18 degrees C, the organism was able to survive for up to 14 days. However, viability of C. jejuni was dramatically shortened when injected into the albumen or the air sac. When freshly laid eggs from Campylobacter-inoculated specific pathogen-free (SPF) layers were tested, C. jejuni-contamination was detected in three of 65 pooled whole eggs (5-10 eggs in each pool) via culture and PCR. However, the organism was not detected from any of the 800 eggs (80 pools), collected from the same SPF flock, but kept at 18 degrees C for 7 days before testing. Likewise, Campylobacter was not recovered from any of 500 fresh eggs obtained from commercial broiler-breeder flocks that were actively shedding Campylobacter in faeces. Also, none of the 1000 eggs from broiler breeders obtained from a commercial hatchery were positive for Campylobacter. CONCLUSIONS: These results suggest that vertical transmission of C. jejuni through the egg is probably a rare event and does not play a major role in the introduction of Campylobacter to chicken flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of Campylobacter transmission to chicken flocks should focus on sources of infection that are not related to eggs.     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro

The present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus-free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 μsec. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 μsec, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 μsec, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation. It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.     

Implementation of subcellular water mapping by electron energy loss spectroscopy in a medium-voltage scanning transmission electron microscope

The water concentration in biological cells plays a predominant role in cellular life. Using electron energy loss spectroscopy, the feasibility to measure the water content in cells has already been demonstrated. In this paper, we present an upgrade of water measurement in hydrated cryosections by spectrum imaging mode in a medium-voltage scanning transmission electron microscope. The electron energy loss spectra are recorded in spectrum imaging mode in a 2ⁿ×2ⁿ pixels array. Each spectrum is processed in order to determine the water mass content in the corresponding pixel. Then a parametric image is obtained in which grey levels are related to water concentration. In this image, it is possible to recognize the different subcellular compartments. By averaging the water concentration over the relevant pixels, we can determine the water mass content in the concerned subcellular compartment. As an example, we present water mass content measurement at subcellular level in rat hepatocytes.     

Effects of temperature on the post-diapause embryonic development and the hatching time in three grasshopper species (Orth., Acrididae)

A study was conducted to determine the effects of six constant temperatures (15, 20, 25, 30, 35 and 40°C) on the post‐diapause embryonic development and the hatching time in three grasshopper species –Omocestus haemorrhoidalis (Charp.), Calliptamus abbreviatus Ikonn. and Chorthippus fallax (Zub.) – from the Inner Mongolian steppe. The results indicate that the species differ in the developmental rates, survival curves and cumulative hatching probabilities. The eggs of O. haemorrhoidalis had the fastest developmental rate with a low developmental threshold temperature of 9.9°C and the sum of effective temperature (SET) 211.2 degree‐days (DD). The corresponding values were 10.9°C and 210.6 DD for C. abbreviatus, 10.5°C and 240.2 DD for Ch. fallax respectively. The SET at which 50% of post‐diapause eggs hatched were 252.0 DD for O. haemorrhoidalis, 262.8 DD for C. abbreviatus, and 273.3 DD for Ch. fallax. The predicted maximal hatch ability of O. haemorrhoidalis (91.17%), C. abbreviatus (75.67%) and Ch. fallax (94.07%) occurred at 23.7, 29.0 and 31.3°C, respectively. The thermal death points of each species were reached at 43.3, 45.0 and 48.6°C. The optimal temperature ranges were 12.2–35.2°C for O. haemorrhoidalis, 21.7–36.3°C for C. abbreviatus and 20.9–41.7°C for Ch. fallax respectively. These results suggest that O. haemorrhoidalis adapt to hatch at a lower temperature range, C. abbreviatus adapt to mid‐temperature range, while Ch. fallax adapt to hatch at a higher temperature range. Although the SET of Ch. fallax is more than that of the other two species, it is not sufficient to explain the hatching sequence of the species in springtime. The results also indicate that Ch. fallax and O. haemorrhoidalis have wider adaptive temperature range than C. abbreviatus.     

Nitrosomethylurea disturbs differentiation of mouse embryonic lungs in organ cultures

We have studied the effect of nitrosomethylurea (NMU) on the differentiation of early rudiments of mouse embryonic lungs (12th day of embryogenesis) explanted into an organ culture. We have demonstrated that nontoxic doses of NMU are capable of accelerating normal lung differentiation both at the early (increase in the number of epithelial buds) and at the late (increase in the number of explants with regions of well-developed alveoli) stages of cultivation. However, NMU induces disturbances of differentiation, which appear as polycystic structures and hyperplastic nodules generally absent in the control.     

Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10⁰ TCID₅₀ MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10⁻⁵ TCID₅₀ MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.     

Changes in embryonic 8-cell nuclei transferred by means of cell fusion to mouse eggs.

Metaphase II and activated mouse oocytes were fused with 8-cell blastomeres, and morphological changes in the transferred nuclei were followed using light and electron microscopy. In metaphase II oocytes, blastomere nuclei underwent premature chromosome condensation (PCC) typical for S-phase nuclei: chromatin pulverization. Then an abortive spindle was formed without evident microtubule organizing centers. Blastomere chromosomes condensed to a lesser degree than meiotic chromosomes and lacked mature functional, trilaminar kinetochores. After parthenogenetic activation of these oocytes, blastomere chromosomes followed, in synchrony with oocyte chromatin, a similar route of changes (anaphase, telophase) and then reformed interphase nuclei of the pronuclear type. Remodeling of 8-cell nucleus thus occurred, but the integrity of the chromatin set was frequently disturbed by formation of micronuclei. If blastomere fusion with oocytes was done close to activation (either before or after parthenogenetic stimulation), the chances of remodeling of the nuclei decreased, because PCC was not regularly induced in all oocytes. In hybrids produced 60 min or later after oocyte activation, blastomere nuclei were maintained in interphase without any structural modifications. Multiple experiments in the mouse have shown that the nuclei from 8-cell stage transferred to enucleated oocytes and egg cells are not capable of substituting for pronuclear functions. Possible reasons for impaired functional reprogramming of 8-cell nucleus in the mouse are discussed in light of our present findings on the morphology of nuclei transferred before and after oocyte activation.     

Ultrastructure and resistance to water loss in eggs of Acanthoscelides obtectus Say (Coleoptera: Bruchidae)

The mature oöcyte of Acanthoscelides obtectus is surrounded by three envelopes: an external layer, a chorion and a vitelline membrane. The external layer is secreted by the walls of the lateral oviducts. The chorion and vitelline membrane are secreted by the follicular cells. The vitelline membrane becomes very compact during the hour following fertilization and laying. The chorion is composed of three layers, one of which has a paracrystalline ultrastructure.Mature, unfertilized, chorion-containing oöcytes, whose vitelline membranes are loose, dehydrate rapidly in a dry atmosphere after laying or after removal from the lateral oviducts. Fertilized eggs are quite resistant to desiccation: after 12 days at 25°C and 5% relative humidity, viable larvae are obtained.The compact vitelline membrane is the most effective protection against dehydration. The chorion and the external layer are much less effective in preventing water loss from the egg.The retention of eggs in the lateral oviducts does not seem to lead to any modification of the structure of their envelopes.     

Spectroscopic diagnosis of chronic fatigue syndrome by visible and near-infrared spectroscopy in serum samples

To investigate visible and near-infrared (Vis-NIR) spectroscopy enabling chronic fatigue syndrome (CFS) diagnosis, we subjected sera from CFS patients as well as healthy donors to Vis-NIR spectroscopy. Vis-NIR spectra in the 600-1100 nm region for sera from 77 CFS patients and 71 healthy donors were subjected to principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) to develop multivariate models to discriminate between CFS patients and healthy donors. The model was further assessed by the prediction of 99 masked other determinations (54 in the healthy group and 45 in the CFS patient group). The PCA model predicted successful discrimination of the masked samples. The SIMCA model predicted 54 of 54 (100%) healthy donors and 42 of 45 (93.3%) CFS patients of Vis-NIR spectra from masked serum samples correctly. These results suggest that Vis-NIR spectroscopy for sera combined with chemometrics analysis could provide a promising tool to objectively diagnose CFS.     

Differences between Fragaria clones by fourth derivative room temperature spectroscopy of intact leaves

Clones of Fragaria were characterized by the attenuance (absorption) spectra of their photosynthetic apparatus. This method uses fourth derivative analysis of spectra of intact lamina of detached leaves at room temperature (20–25 C). Twenty-one different species and hybrids of Fragaria were examined. F. chiloensis accessions frequently have characteristic fourth derivative spectra distinct from that of other Fragaria species. These spectra are characterized by a strong band that appears to be photosystem I component Ca 693; and is correlated with a higher chlorophyll b content per unit area of leaf, and a lower chlorophyll a/b ratio.Abbreviations Chl chlorophyll - chiloensis Fragaria chiloensis - 4th deriv fourth derivative - PSI photosystem I - PSH photosystem II