An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

In situ bioremediation of monoaromatic pollutants in groundwater: a review

Monoaromatic pollutants such as benzene, toluene, ethylbenzene and mixture of xylenes are now considered as widespread contaminants of groundwater. In situ bioremediation under natural attenuation or enhanced remediation has been successfully used for removal of organic pollutants, including monoaromatic compounds, from groundwater. Results published indicate that in some sites, intrinsic bioremediation can reduce the monoaromatic compounds content of contaminated water to reach standard levels of potable water. However, engineering bioremediation is faster and more efficient. Also, studies have shown that enhanced anaerobic bioremediation can be applied for many BTEX contaminated groundwaters, as it is simple, applicable and economical.

This paper reviews microbiology and metabolism of monoaromatic biodegradation and in situ bioremediation for BTEX removal from groundwater under aerobic and anaerobic conditions. It also discusses the factors affecting and limiting bioremediation processes and interactions between monoaromatic pollutants and other compounds during the remediation processes.     

Two-phase systems: potential for in situ extraction of microalgal products

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted.Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.     

An unprecedented (3,4)-connected self-penetrating network of zinc complex: In situ formation of a tetradentate N-heterocyclic ligand under POMs-mediated hydrothermal conditions

The reaction of lacunary polyanion [α-H₂P₂W₁₂O₄₈]¹²⁻, zinc acetate, bpp (1,3-bis(4-pyridyl)propane) and BTC (1,3,5-benzenetricarboxylate) in an acidic aqueous solution led to the isolation of a new complex [Zn₃(BTC)₂(chtpy)(H₂O)₂] · 2H₂O (1), which crystallizes in the space group C2/c with a = 28.988(6) Å, b = 11.254(2) Å, c = 17.347(4) Å, V = 4682.5(2) Å³, Z = 4. X-ray diffraction analysis reveals that there exists the generation of in situ ligand through simultaneous dehydrogenative coupling and hydroxylation of an N-heterocyclic ligand in complex 1. More interestingly, it exhibits an unprecedented (3,4)-connected (8².10)₂(8³)₂(8⁴.10.12)(8⁵.10) topology which has been reported for the first time and shows self-penetrating nature at the same time.     

The effect of feedback regulation and in situ product removal on the conversion of sugar to cycloheximide by Streptomyces griseus

An addition of cycloheximide to cycloheximide-producing Streptomyces griseus cultures resulted in reductions in the production rate and in the conversion of sugar into cycloheximide. In situ cycloheximide adsorption was observed to enhance: total cycloheximide titers; productivities; and the conversion of sugar to cycloheximide. During the secondary metabolite-producing phase, sugar consumption was observed to be linearly dependent on cycloheximide productivity. From this analysis a true product yield and maintenance coefficient were estimated to be 0.08 g cycloheximide/g glucose and 0.028 g glucose/g cell-h, respectively. The sixfold difference between this true product yield and a theoretical value obtained from knowledge of the biosynthetic pathway is discussed. Since the maintenance sugar requirement for cycloheximide production is large, stimulation of biosynthesis through in situ adsorption significantly increases the overall efficiency of sugar conversion to this secondary metabolite.     

Biomass measurement online: the performance of in situ measurements and software sensors

Biomass measurement is one of the most critical measurements in biotechnological processes. The technologies developed for the measurement of biomass in situ have developed over the years. Because it has been over 10 years since the last review concentrating on practical issues concerning biomass measurements, it is time to evaluate recent developments in the field. This review concentrates on the applications of dielectric spectroscopy, optical density, infrared spectroscopy, and fluorescence for in situ measurement of biomass. The advantages offered by these methods and an economic way of estimating biomass concentration, the software sensors, are considered.     

Degradation of xenobiotic compounds in situ: Capabilities and limits

Abstract: Exploiting microorganisms for remediation of waste sites is a promising alternative to groundwater pumping and above ground treatment. The objective of in situ bioremediation is to stimulate the growth of indigenous or introduced microorganisms in regions of subsurface contamination, and thus to provide direct contact between microorganisms and the dissolved and sorbed contaminants for biotransformation. Subsurface microorganisms detected at a former manufactured gas plant site contaminated with coal tars mineralized significant amounts of naphthalene (8–43%) and phenanthrene (3–31%) in sediment-water microcosms incubated for 4 weeks under aerobic conditions. Evidence was obtained for naphthalene mineralization (8–13%) in the absence of oxygen in field samples. These data suggest that biodegradation of these compounds is occurring at the site, and the prospects are good for enhancing this biodegradation. Additional batch studies demonstrated that sorption of naphthalene onto aquifer materials reduced the extent and rate of biodegradation, indicating that desorption rate was controlling the biodegradation performance.     

In situ bioremediation of hydrocarbon in soil

Laboratory and field experiments were carried out for bioremediation of soils contaminated by fuel oil and motor oil. Bioventing was combined with the application of selected bacteria and dissolved nutrients. In the field experiments, soil gas was evacuated by air pumps from the permeable boreholes. The process was followed by both soil and gas analysis. Biodegradation of oil contamination and the microbial activity was measured by the oil and cell concentration in the soil. In 2 months, the oil content decreased considerably, and the cell number increased by one order of magnitude or more. The evacuated gas was tested for CO₂, O₂ and volatilized hydrocarbon content. The CO₂ level proves the presence of biodegradation: a permanent high value about ten times higher than normal, could be measured for 2 months, followed by a slow decrease in the third month. Volatilized hydrocarbon content was the highest in the first 2 d. After a continuous decrease, it dropped under the threshold of measurability for the third month. Selective biodegradation of hydrocarbon mixtures (oily wastes) was investigated as well: gas Chromatographic oil analysis showed the changes in the oil composition. The appropriate microflora was working in an ideal commensalism, and as a result, all of the hydrocarbon components were degraded nearly to the same extent.     

In vitro dissolution method for evaluation of buprenorphine in situ gel formulation: A technical note

Conclusion  The in situ gel formulation of buprenorphine showed sustained drug release for a prolonged period of time. The drug release from RG 502 followed a linear pattern throughout the dissolution without any significant burst release. The amount of buprenorphine released during the first 30 minutes, irrespective of the type of Resomer or dissolution medium, was less than 3%. Drug release continued over 55 days in phosphate buffer and 35 days in Tween 80. The in vitro dissolution method developed during this study was capable of identifying formulation differences and thus will be useful for routine drug delivery research, particularly in situ gel formulation development research. In situ gel formulations are routinely compared using animal models,²¹, so development of such an in vitro method will expedite formulation evaluation. Published: August 3, 2007     

An in situ hybridization screen for the rapid isolation of differentially expressed genes

To rapidly isolate genes specifically expressed during medaka development we generated a cDNA library enriched for genes expressed in the head region of the developing embryo. Clones were spotted on filters automatically and preselected for abundantly expressed genes by hybridizing them with a probe derived from RNA of undifferentiated totipotent cells. Of the nonhybridizing clones 153 were chosen randomly and further analyzed by whole-mount in situ hybridization. There were 67 selected clones differentially expressed in the developing embryos, and 48 of these were expressed in the developing head. Differentially expressed genes were either of novel type or showed homology to known genes containing DNA binding motifs or to putative housekeeping genes. Received: 1 December 1998 / Accepted: 17 May 1999     

Diatoms as indicators: The influences of experimental nitrogen enrichment on diatom assemblages in sub-Arctic streams

Indices using diatoms are widely used to assess water quality, but are usually constructed from field correlations and not tested through rigorous experimentation. We tested experimentally the performance of the Sørensen and the Shannon indices, and the trophic diatom index (TDI). Nitrogen was naturally limiting in the eight remote sub-Arctic streams used and we measured the effects of experimental nitrogen enrichment on diatom assemblages. Diatom densities increased significantly in the enriched reaches but there was no significant difference in invertebrate density between control and treatment reaches. Grazing effects were thus controlled for. Diversity within streams (Shannon index) was significantly reduced by nutrient addition but the Sørensen index did not change. The trophic diatom index (TDI), which is presumed to reflect nutrient concentration, was not influenced by nutrient addition and generally the values were low in both control and treatment reaches. Densities of the diatom genera Achnanthes and Gomphonema increased significantly with enrichment while those of Nitzschia and Fragilaria decreased significantly. Less abundant diatom species, which collectively constituted around 40% in relative abundance in the control reaches, were around 15–18% in treatment reaches. Growth forms were altered by the nutrients. Diatoms attached by mucilage pads were more abundant in treated reaches compared with control reaches. Motile diatoms became scarcer. The size of diatom species was unaffected by nutrient enrichment. This study showed that it is important to test experimentally indices that are developed for particular habitats before using them elsewhere.     

Two methods of haploidization in pear,Pyrus communis L.: greenhouse seedling selection and in situ parthenogenesis induced by irradiated pollen

Seedlings of 12 crosses involving pear varieties or hybrids were observed for the presence of haploid plants. On the basis of phenotypic characteristics, 17 plants corresponded to the haploid condition and, of these, 12 were determined by chromosome counting to be haploid (2n=x=17). In addition, and in order to induce in situ parthenogenesis, several pear varieties were pollinated with a selected clone carrying a homozygous dominant marker gene for the colour of red. This pollen had previously been irradiated with -rays of cobalt 60 at 0, 200, 250 and 500 Grays. The immature embryos were cultured in vitro, whereby 1 haploid and two mixoploid plants were obtained. Numerous diploid plants with the maternal phenotype were also obtained, and their genetic origin was subsequently studied by means of isozyme analysis.     

玉米两个RFLP标记的原位单杂交与共杂交定位的比较

ＲＦＬＰ标记ｂｎ１８．２３和 ｕｍｃ１１１位于玉米遗传日第 ４连锁群近端,彼此密切连锁但次序尚未确定。用生物素标记对它们进行了原位单杂交和共杂交的比较定位。在植物中,这类原位共杂交的研究为首次报道。在单一探针的原位杂交中 ｕｍｃ１１１被定位在第 １、 ４和９染色体长上,与着丝粒的百分距离分别是７．３６±２．６５、６３．６７±１．０７、４７．８７±２．９０。ｂｎ１８．２３被定位在第４和８染色体长臂上,与着丝粒的百分距离分别是８７．４２±２．４５和２７．６０±１．７５,清楚地表明了这两个标记在第４染色体上的次序。ｂｎ１８．２３和ｕｍｃ１１１分别与编码过氧化氢酶的ｃａｔ３基因和编码丝氨酸／苏氨酸蛋白激酶的ｃｄｅ２Ａ基因紧密连锁。根据供试ＲＦＬＰ标记检出位点推断了基因ｃｄｃ２Ａ和ｃａｔ３的物理位置。原位共杂交在第 ４染色体长臂上同时显示出了ｕｍｃ１１１和ｂｎ１８．２３两个标记的杂交信号,它们的位置分别与单一探针原位杂交的位置基本吻合。这为低拷贝或单一拷贝等小片段ＤＮＡ物理定位的可靠性以及它们共杂交的可行性提供了令人信服的证据。     

In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.     

Relevance to prenatal diagnosis of the identification of a human Y/autosome translocation by Y-chromosome-specific in situ hybridisation

Routine cytogenetic analysis of an amniotic fluid sample revealed a large brightly fluorescent region in the short arm of chromosome 14 in an otherwise normal male karyotype (46,XY,14p+ + +). This site was also present in the paternal karyotype. In situ hybridisation to a Y-chromosome-specific DNA probe confirmed that the father had a Y/14 translocation. The incidence of two hybridisation bodies (large hybridisation sites), detecting both the translocated Y chromatin and the normal Y chromosome, was lower in interphase nuclei (44.3%) than in metaphase spreads (95.2%). The relevance of these observations to the potential use of in situ hybridisation to interphase nuclei for prenatal diagnosis is discussed.     

Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

Aneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one-half-domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one-domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one-half-domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley-Liss, Inc.