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Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMale gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.

RBM (RNA-binding motif) protein is a marker of male germ cells. This protein is encoded by the Azoospermia factor region-b (AZF-b) of the human Y chromosome and is expressed exclusively in the male germ cell line, that is, spermatogonia, spermatocytes, and round spermatids. The authors analyzed the expression of the RBM gene in germ cell tumors and in the seminiferous tubules in the vicinity of these tumors to identify the presence of IGCN. Sections from testicular germ cell tumors of 21 patients were stained with anti-RBM antibody by using an immunohistochemical method. Distal tubules showing spermatogenesis were immunopositive for RBM protein. All of the germ cell tumors studied were completely immunonegative for RBM. Defined areas of IGCN also showed an absence of RBM expression. Tubules with spermatocyte-like cells, which were expected to express RBM, did not express this protein. This result enabled the identification of tubules as being IGCN. RBM is a novel marker consistently expressed in normal male germ cells but not in malignant germ cell tumors or IGCN. Thus, the absence of RBM expression in germ cells provides a new diagnostic tool of preinvasive malignancy of the testis.

This paper describes the potential for wet oxidation (WO) processes to treat activated sludge under stoichiometric oxygen conditions. Tests were carried out at temperatures ranging from 200 to 300°C at retention times of 10, 30 and 60 min. Temperature proved to be the most significant parameter, followed by retention time and oxygen overpressure. A considerable reduction in sludge volume and COD was generally obtained, producing an effluent liquor with a high oxygen demand and relatively stable residual solids. Simplified empirical equations were developed from the experimental data to describe the transformation pattern of the organic and inorganic components of the activated sludge in a WO environment at different temperatures.

Objective: To review the current literature on genes known to affect fertility in the human and mouse. Design: A literature review was performed and key articles were chosen for focus in the areas of genes with effects only on spermatogenesis and oogenesis, with an emphasis on Y-chromosome–encoded gene families and spermatogenesis. In addition, studies describing genes deleted in transgenic mice were incorporated. Result(s): Several gene families on the Y chromosome are implicated in spermatogenic failure, but the link between the genetic lesion and the resulting defect is unclear. Many mouse genes involved in repair and DNA damage monitoring have specific effects on gametogenesis in and around meiosis. Conclusion(s): Many genes are involved only in gametogenesis, and some of these are beginning to be understood in terms of their functions. An even larger number of genes is required for gametogenesis, and other functions and mouse models give insights important for human disease.

The survival rates of enteric bacteria (i.e. Escherichia coli and enterococci) exposed to a sunlight simulator were determined at two different depths of sea water in varying conditions of light intensity, turbidity, sewage content, degree of mixing and temperature under controlled laboratory conditions. The combined effect of these factors on bacterial die-off was evaluated by statistical analyses carried out on the experimental data obtained according to the central composite experimental design. The magnitude and the general character of the influence of the factors were determined. The variability of bacterial mortality due to the effect of light was shown to depend on the variability of the intensity of light and the other microenvironmental factors (i.e. turbidity, sewage content, and mixing) influencing the depth profile of the light intensity and bacterial concentration. All factors except temperature were found to exert a significant influence on bacterial die-off under light conditions. Higher rates of die-off were brought about by high levels of light intensity and mixing with low levels of turbidity and sewage content of the water. No appreciable difference was found between the survival capabilities of E. coli and enterococci on exposure to light.

The formation of ethanol from CO and H2 over a CuOZnO methanol catalyst was studied in a fixed-bed microreactor by measuring the isotopic distribution of the carbon in the product ethanol when 13C methanol was added to the feed. The purpose of this work was to determine whether experimental support could be obtained for any of the various mechanisms proposed in the literature for the synthesis of ethanol over methanol catalysts. The methanol was added at six partial pressures so that the ratio of methanol to CO ranged from 1:34 to 1:250. For each feed ratio the isotopic composition of the reaction products was determined at various contact times from 0.5 to 10 s, and the composition at zero contact time was determined by extrapolation. All four isotopic species possible for ethanol, 12CH312CH2OH, 12CH313CH2OH, 13CH312CH2OH, and 13CH313CH2OH, were observed; there was no scrambling of the carbon atoms of ethanol. Contrary to what is expected for the formation of ethanol either by condensation or by homologation of methanol, the fraction of carbon-13 in the ethanol at zero contact time was a function of the partial pressure of 13C methanol in the feed. Also, at low contact times a large fraction of the product ethanol was doubly labeled which is incompatible with the formation of ethanol by carbonylation of formaldehyde, methoxide, or other C1 species. The isotopic distribution of the ethanolic carbon was consistent with a mechanism that involves a C1 species that is an intermediate in the formation of both ethanol and methanol.

The reaction of linear primary alcohols over CuO/ZnO/Al 2 O 3 was studied under nitrogen and under carbon monoxide at 285 °C and 1 to 65 atm. The principal products for alcohols with n carbon atoms were, in addition to the corresponding aldehydes, esters with 2 n carbon atoms, ketones with 2 n carbon atoms, and ketones with 2 n −1 carbon atoms. Under nitrogen the esters predominated, but under 65 atm of carbon monoxide the principal condensation products were ketones with 2 n carbon atoms. Isotopic labeling studies showed that carbon from CO was not incorporated into the esters and ketones, that oxygen, on the other hand, was exchanged between CO and the reaction products, and that the ketones were formed by an aldol-type mechanism. The changes in selectivity observed under carbon monoxide are attributed to changes in the catalyst surface resulting from the removal of lattice oxygen by the CO and the formation of oxygen vacancies.

The 15,650 base-pair mitochondrial genome of the sea urchin Strongylocentrotus purpuratus has been cloned and sequenced. It exhibits a novel organization that suggests the primacy of post-transcriptional gene regulation. The same 13 polypeptides, two rRNAs and 22 tRNAs are encoded as in other animal mitochondrial DNAs, but are organized with extreme economy; non-coding information between genes is almost completely absent, some stop codons are generated post-transcriptionally and tRNA sequences are interspersed between only a minority of other structural genes. The genome uses a variant genetic code, in which AAA specifies asparagine, ATA isoleucine, TGA tryptophan and AGN serine, and has an unusual pattern of codon bias. The order of genes shows several differences from that of vertebrates. The genes for the large (16 S) ribosomal RNA and for NADH dehydrogenase subunit 4L (ND4L) are in different positions, located respectively between those encoding ND2 and cytochrome oxidase subunit I (COI) and between COI and COII. This organization is conserved amongst at least four regular echinoids diverging by some 225 million years. Most tRNA genes are also in different positions. The only long unassigned sequence in the genome (121 base-pairs) is located within a cluster of 15 tRNA genes. It contains elements resembling some of those found in the displacement (D) loop of vertebrate mtDNAs, notably polypurine/polypyrimidine tracts that may play a role in regulating transcription and the initiation of replication. The separation of the ribosomal RNA genes from each other and from the putative control region imposes special demands on the transcription of the genome.

Clusters of Ru, Ni, and a series of RuNi and RuCu bimetallics in a Y-type zeolite were investigated as catalysts for the reaction of CO with H2 to form CH4. Although the particle size of the Ru clusters indicates that they remained in the large cavities of the zeolite, the turnover numbers for several samples were in agreement with those observed for RuAl2O3. By contrast, the nickel clusters were located on the external surface of the zeolite, and the turnover number was more than an order of magnitude less than that observed for NiAl2O3. The presence of ruthenium in bimetallic clusters caused the average particle size to decrease significantly, relative to the values which were observed for pure nickel or pure copper. The turnover numbers for the RuNi zeolites decreased linearly with respect to the addition of Ni, but the decrease in initial activity was much more severe upon addition of Cu to the RuY zeolite. The supported ruthenium catalysts exhibited a marked decline in activity during the reaction, but the addition of excess nickel (1:7 atomic ratio) brought about a stabilization in activity which even exceeded the stability of the supported Ni catalysts. The decrease in activity is attributed to the build-up of excess carbon which results from the dissociation of CO. The presence of Ni is believed to moderate the activity of ruthenium for this reaction, thus providing more sites for dissociative chemisorption of H2.

A primary thrust of optical lithography is the use of shorter wavelengths of light in order to produce smaller geometries for VLSI device production. Excimer lasers have been identified as likely candidates to extend optical lithography into the 0.4-0.3 micron regime. This paper describes an excimer laser optical approach for the projection imaging of sub-micron geometries. An excimer laser-based imaging and ablation system is described in detail along with initial results obtained with resist materials. The optical system employs inter-changeable lenses, with varying numerical apertures, reduction ratios, and field sizes. The data presented shows how excimer laser energy at the 248nm and 193nm wavelengths can produce 0.4-0.5 um resolution using direct photoablative decomposition of the resist layer. Attenuation of the beam allows the system to be used for conventional latent image exposure and wet development with deep UV resists. Small-field chrome-on-quartz reticles at 15x and 36x are used to project device structures, resolution patterns and alignment marks. SEM photos show the resolution capabilities across the effective field of the system. Patterns as small as 0.3um are resolvable using this system.

Recently it was reported that the addition of small incremental amounts of cobalt (0-2%) to a conventional Cu/ZnO methanol catalyst systematically destroyed the methanol synthesis activity of the catalyst. The authors have now studied the influence on the same catalyst of several other group VIII metals and of metals of the lower groups. This note summarizes the effects produced by these metals on the catalytic properties of Cu/ZnO under methanol synthesis conditions. 4 references.