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This article from PC Pro features readers' comments on various topics. One reader discusses the recent CrowdStrike/Windows outage and how it affected their work as a nurse. They highlight the challenges they faced in accessing patient records and prescribing medication without the necessary software. Another reader raises concerns about the installation of external software on business-critical systems and the need for better testing procedures. The article also includes comments on aging technology and the importance of simplicity in tech for older individuals. Finally, the article presents a readers' poll on optimism and pessimism about the future of technology, with mixed views expressed. [Extracted from the article]

Hybrid embryonic stem (ES)–like clones were generated by fusion of murine ES cells with somatic cells that carried a neo resistance gene under the transcriptional control of the Oct-4 promoter. The Oct-4 promoter was reactivated in hybrid ES cells formed by fusion with fetal fibroblasts, and all hybrid colonies were of ES rather than fibroblast phenotype, suggesting efficient reprogramming of fibroblast chromosomes. Like normal diploid murine ES cells, hybrid lines expressed alkaline phosphatase activity and formed differentiated cells derived from the three embryonic germ layers both in vitro and in vivo. Treatments thought to affect nuclear transfer efficiency (ES cell confluence and serum starvation of primary embryonic fibroblasts) were investigated to determine whether they had an effect on reprogramming in cell hybrids. Serum starvation of primary embryonic fibroblasts increased hybrid colony number 50-fold. ES cells were most effective at reprogramming when they contained a high proportion of cells in the S and G2/M phases of the cell cycle. These data suggest that nuclear reprogramming requires an initial round of somatic DNA replication of quiescent chromatin in the presence of ES-derived factors produced during S and G2/M phases.

AbstractAir samples of particulate matter (PM) with an aerodynamic diameter less than 10 µm (PM10) were collected from six sites in Bangkok, Thailand, using high-volume air samplers. Daily samples were taken at intervals of 12 days from November 1999 to November 2000. Size-selected sampling using a multislit Andersen size-fractionated cascade impactor was undertaken at one site in central Bangkok to identify particulate size distribution. The annual average PM10concentration at all six sites exceeded the Thailand National Ambient Air Quality Standard (NAAQS) of 50 µg/m3. The daily PM10concentrations at heavy traffic roadside areas ranged between 30 and 160 µg/m3. The highest PM10level occurred during the winter period (November–February), which is the dry season. From our results, which are based on a 1-yr survey, it can be observed that the particulate concentrations are associated with traffic volumes and seasonal factors (temperature and rainfall). The relative importance of size fractions in contributing to PM load is presented and discussed. Twenty polycyclic aromatic hydro-carbons (PAHs) associated with PM have been identified and quantified. The summed PAHs based on the 20 species had an average concentration of 60 ng/m3. Benzo(e)pyrene, indeno(123cd)pyrene, and benzo(ghi)perylene were the major compounds with average concentrations of 8, 10, and 13 ng/m3, respectively. Results indicate that more than 97% of PAHs were found in the small particulate size range of <0.95 µm.

A novel, rapid, and simple capillary electrophoresis (CE) method has been developed for the determination of aspirin by on-line treatment with alkali. Such on-line reaction with 0.20 mol/L NaOH solution for 2.5 min at 45°C automatically and reproducibly converts aspirin into its hydrolysis product salicylic acid. Analysis is carried out in less than 12 min after conversion of aspirin in a flow injection (FI) system coupled to a CE instrument via a split-flow interface cell, and a sampling frequency of 9 h^–1 is achievable. The on-line conversion method has been used to determine aspirin in four commercial pharmaceutical preparations, and the results are satisfactory.

A novel, rapid, and simple capillary electrophoresis (CE) method has been developed for the determination of aspirin by on‐line treatment with alkali. Such on‐line reaction with 0.20 mol/L NaOH solution for 2.5 min at 45°C automatically and reproducibly converts aspirin into its hydrolysis product salicylic acid. Analysis is carried out in less than 12 min after conversion of aspirin in a flow injection (FI) system coupled to a CE instrument via a split‐flow interface cell, and a sampling frequency of 9 h–1is achievable. The on‐line conversion method has been used to determine aspirin in four commercial pharmaceutical preparations, and the results are satisfactory.

Mutations in the WT1 gene are found in a subset of Wilms' tumours and in certain other disorders such as Denys-Drash syndrome. The WT1 gene product is a zinc finger transcription factor for which many target genes have been suggested. Here we utilise gene targeting to generate cells containing only truncated forms of WT1, in which the DNA-binding region is disrupted. Examination of gene expression in these cells using cDNA macroarrays suggests two novel WT1 transcriptional targets, cyclin G1 (Ccng1), and insulin-like growth factor binding protein 4 (Igfbp4).

An on-line microwave oven was employed in this study to accelerate the coordination reactions between Rh(III), Pd(II), and 2-(3,5-dicloro-2-pyridyl-azo)-5-dimethylaminoaniline (3,5-diCl-PADMA) in HOAc-NaOAc media. Two sensitive flow-injection spectrophotometric methods have been established for the determination of Rh and sequential determination of Rh and Pd, respectively. Two important parameters in on-line Microwave-FIA, T1 and T2, were investigated. The difference in the effect of the microwave irradiation on the reactions was explained. The proposed methods are applied to the determination of Rh in the waste and sequential determination of Pd and Rh in artificial samples with satisfactory results. © 2000 John Wiley & Sons, Inc. Lab Robotics and Automation 12:91–96, 2000

Abstract: Airborne viable and total fungal spores were sampled inside and outside 80 houses in the Latrobe Valley, Victoria, Australia as part of a larger indoor environmental study. Each residence was visited six times over a period of 1 year for sample collection, and fungal spore samples were collected from at least three indoor sites and from an outdoor site. Viable spores were sampled using an Andersen sampler, while total spores were assessed using a Burkard spore trap. Identification of fungal colonies to genera level was performed in two seasons; winter and late spring. The most common fungal genera/groups wereCladosporium, Penicillium, and yeasts, both indoors and outdoors in winter and late spring. Outdoor levels were higher than those indoors throughout the year, and a significant seasonal variation in spore levels was seen both indoors and outdoors with overall maxima in summer. Contrary to this trend, the levels ofAspergillus, yeasts,Cephalosporium andGliocladium were higher in winter. Most fungal genera were found in greater concentrations outdoors compared to indoors, butPenicillium was more common indoors. Outdoor spore levels were a significant influence on indoor levels, but seasonal differences suggest that other influences are important.

Abstract: We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.

Gene targeting using embryonal stem cells has been used to generate strains of mice with inactivating mutations at the Rb-1 and p53 tumour suppressor loci. Mice heterozygous for a null allele of Rb-1 do not show retinoblastomas but instead develop pituitary tumours. Homozygotes die at between 10 and 14 days’ gestation and show increased levels of both cell division and cell death by apoptosis in the haematopoietic and nervous systems. This is consistent with the view that the Rb-1 gene product plays a general role in the maturation of precursor cells. In contrast, mice het- erozygous for a null allele of p53 are predisposed to a spectrum of tumours, while the corresponding homozygotes are viable but show a very high tumour incidence. Thymo- cytes from p53 homozygotes, unlike wild-type thymocytes, do not show increased levels of apoptosis following treatment with DNA-damaging agents, while response to its induction by other agents is unaltered. Similarly, epithelial cells from the crypts of both small and large intestine of p53- deficient mice are resistant to the induction of apoptosis by γ -irradiation. In contrast, two other early responses of wild- type crypts to γ -irradiation, namely the G2 block and the reduction in bromodeoxyuridine incorporation, are both largely intact in p53-deficient mice. These observations are consistent with the view that p53 is responsible for monitoring DNA damage so that damaged cells can be either repaired or eliminated prior to division.

The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.

Immunosurgical isolation of inner cell masses (ICMs) from sheep embryos was most efficient at the expanded, zona-intact blastocyst stage (day 7 to 8 post oestrus) before migration of endoderm cells beyond the boundary of the ICM across the blastocoelic surface of the trophectoderm. When cultured under conditions which allow the isolation of embryonal stem (ES) cell lines from mouse ICMs, sheep ICMs attached, spread and developed areas of both ES cell-like and endoderm-like cells. After prolonged culture only endoderm-like cells were evident. The implications for the isolation of ES cell lines from sheep embryos and possible species-specific requirements are discussed.

We examined the gap-junctional communication properties of a communication-defective cell line R5/3 and its communication-competent revenant H2T12. For these studies, we carried out microelectrode impalements to monitor ionic coupling and dye coupling. Our dye-injection experiments revealed that the H2T12 cells are much more efficient in dye coupling than the R5/3 cells. This latter observation is in agreement with the previous finding that the H2T12 cells are much better metabolically coupled than the R5/3 cells. With ionic coupling measurements, however, both cell lines exhibited similar levels of cell–cell coupling. The R5/3 cells demonstrated an ionic coupling coefficient of 0·19 ±0·011 (S.E.M.) and H2T12 a coupling coefficient of 0·25 ± 0·009 (S.E.M.). These results in conjunction with observations from other studies indicate that the different experimental approaches for monitoring gap-junctional communication may have different levels of sensitivity for detecting as opposed to measuring the level of cell—cell coupling.

Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts1–3, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts4,5. Recently, ES cells with multiple integrations of retro viral sequences have been used to introduce these sequences into the germ-line of chimaeric mice5, demonstrating an alternative to the microinjection of fertilized eggs6for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch–Nyhan syndrome7, which is characterized by mental retardation and self-mutilation.

The mouse Pim-1 gene encodes two cytoplasmic serine-threonine—specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (Eμ-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-ni-trosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow—derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.

Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alpbafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used.Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative.The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.

In tumors and embryoid bodies of mouse teratoma a correlation has been established between specific activity of alkaline phosphatase (EC 3.1.3.1) and content of embryonal carcinoma, the stem cell of the tumor. A histochemical study of embryoid bodies has shown that high levels of the enzyme are confined to embryonal carcinoma. Fifteen tissue culture lines could be classified into three groups: (a) lines identifiable as pluripotential embryonal carcinoma by their morphology, tumorigenicity, and capacity to differentiate in vivo; (b) nullipotential embryonal carcinoma, resembling pluripotential embryonal carcinoma in morphology and malignancy but giving rise to undifferentiated tumors; and (c) lines of apparently nonmalignant somatic cells. Both types of embryonal carcinoma possess levels of alkaline phosphatase 5- to a 100-fold higher than the somatic cell lines. The embryonal carcinoma enzyme resembles the enzymes from kidney and placenta in kinetics of thermal inactivation and sensitivity to the inhibitor L-phenylalanine, but is distinguishable from the alkaline phosphatases of liver and intestine. These findings are discussed in relation to the use of teratoma for the study of cell differentiation.

Two recent developments suggest a route to predetermined alterations in mammalian germlines. These are, first, the characterization of mouse embryonic stem (ES) cells1that can still enter the germline after genetic manipulation in culture2,3and second, the demonstration that homologous recombination between a native target chromosomal gene and exogenous DNA can be used in culture to modify specifically the target locus4. We here use gene targetting functionally to correct the mutant hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in the ES cell line which has previously been isolated and used to produce an HPRT-deficient mouse5. This modification of a chosen gene in pluripotent ES cells demonstrates the feasibility of this route to manipulating mammalian genomes in predetermined ways.

A letter to the editor is presented that reflects on what chemists do when they retire.

A simple, rapid, and accurate method for the separation and determination of ephedrine and pseudoephedrine using direct UV absorbance detection has been developed by the combination of flow injection with capillary electrophoresis for the first time. The buffer solution used is a 40mM borate solution with the pH adjusted to 9.5 using a 2M NaOH solution. The linear calibration range is 50 to1000 µg/mL (r = 0.9996) for both analytes, and the recoveries are 91.2–108.2% for ephedrine and 92.6–107.3% for pseudoephedrine, respectively. The relative standard deviation of the peak area is 1.6% for ephedrine and 1.3% for pseudoephedrine (n = 6) at a concentration of 500 µg/mL, respectively. A series of samples is injected repeatedly without current interruption and subsequent rinsing, and the contents of these two alkaloids in three marketed drugs and the medical plant, Ephedra sinica, are determined with satisfactory results by this method.

The Pim-1 proto-oncogene encodes a highly conserved serine/threonine phosphokinase (1–5) which is predominantly expressed in hematopoietic organs and gonads in mammals (6-9). Overexpression of Pim-1 predisposes to lymphomagenesis in mice (8, 10, 11). To develop a further understanding of Pim-1 in molecular terms, as well as in terms of its potential role in hematopoietic development, we have generated mice deficient in Pim-1 function. P/m-7-deficient mice are ostensibly normal, healthy and fertile. Detailed comparative analyses of the hematopoietic systems of the mutant mice and their wild-type littermates showed that they are indistinguishable for most of the parameters studied. Our analyses revealed one unexpected phenotype that correlated with the level of Pim-1 expression: Pim-1 deficiency correlated with a erythrocyte microcytosis, whereas overexpression of Pim-1 in E/i-P/7n-7-transgenic mice resulted in erythrocyte macrocytosis. In order to confirm that the observed decrease in erythrocyte Mean Cell Volume (MCV) was attributable to the Pim-1 deficiency, we developed mice transgenic for a Pim-1 gene construct with its own promoter and showed that this transgene could restore the low erythrocyte Mean Cell Volume observed in the Pirn-/-deficient mice to near wild-type levels. These results might be relevant to the observed involvement of the Pim-1 gene in mouse erythroleukemogenesis (12). The surprising lack of a readily observed phenotype in the lymphoid compartment of the P/m-7-deficient mice, suggests a heretofore unrecognized degree of in vivo functional redundancy of this highly conserved proto-oncogene.