In vitro cell lines were established from seven biologically distinct in vivo Dunning R3327 rat prostatic tumor sublines. Some of these in vitro cell lines (i.e., G, AT-1, AT-2) retain a low metastatic ability when inoculated back into syngeneic Copenhagen male rats, while others (i.e., AT-3, MAT-LyLu, MAT-Lu) retain a very high metastatic ability. A series of genetic (i.e., DNA content per cell, modal chromosomal number), as well as phenotypic parameters (i.e., morphology, 5 alpha-reductase, androgen receptor, estrogen receptor) were used to validate that the in vitro cell lines retained the major characteristics of the parental in vivo tumor sublines used for their respective establishment. A series of additional characteristics (i.e., morphology, growth rate, saturation density in surface culture, anchorage-dependent and -independent clonogenic potential) were compared between the high vs. the low metastatic in vitro cell lines to determine if a discriminatory parameter could be identified which reproducibly predicted the metastatic abilities of the particular prostatic cancer cell line. While the combination of the in vitro cell lines and their parental in vivo tumor subline will be a valuable tool for developing methods for predicting metastatic ability of prostate cancers, no single parameter yet measured is entirely successful in making this important distinction.

Inheritance of the active and inactive states of gene expression by individual cells is crucial for development. In fission yeast, mating-type region consists of three loci called matmatmatmatmatmation for transcriptionally active matmating-type switching were covariegated and were regulated by an epigenetic mechanism. Genetic analyses demonstrated that epigenetic states were remarkably stable not only in mitosis but also in meiosis and were linked to the mating-type region. This study indicates that different epigenetic states are heritable forms of chromatin organization at the mat region.

The influences of surfactants and medical drugs on the diameter size and uniformity of electrospun poly(L-lactic acid) (PLLA) fibers were examined by adding various surfactants (cationic, anionic, and nonionic) and typical drugs into the PLLA solution. Significant diameter reduction and uniformity improvement were observed. It was shown that the drugs were capsulated inside of the fibers and the drug release in the presence of proteinase K followed nearly zero-order kinetics due to the degradation of the PLLA fibers. Such ultrafine fiber mats containing drugs may find clinical applications in the future.

Mud volcanism is an important natural source of the greenhouse gas methane to the hydrosphere and atmosphere. Recent investigations show that the number of active submarine mud volcanoes might be much higher than anticipated (for example, see refs 3-5), and that gas emitted from deep-sea seeps might reach the upper mixed ocean. Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release are unknown. It is also unclear how efficiently methane-oxidizing microorganisms remove methane. Here we investigate the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72 degrees N, 14 degrees 44' E; 1,250 m water depth) to provide quantitative estimates of the in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities: aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a previously undescribed clade of archaea (ANME-3) associated with bacterial mats. We found that the upward flow of sulphate- and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to <40% of the total flux.

The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.

Energy taxis is widespread in motile bacteria and in some species is the only known behavioral response. The bacteria monitor their cellular energy levels and respond to a decrease in energy by swimming to a microenvironment that reenergizes the cells. This is in contrast to classical Escherichia coli chemotaxis in which sensing of stimuli is independent of cellular metabolism. Energy taxis encompasses aerotaxis (taxis to oxygen), phototaxis, redox taxis, taxis to alternative electron acceptors, and chemotaxis to a carbon source. All of these responses share a common signal transduction pathway. An environmental stimulus, such as oxygen concentration or light intensity, modulates the flow of reducing equivalents through the electron transport system. A transducer senses the change in electron transport, or possibly a related parameter such as proton motive force, and initiates a signal that alters the direction of swimming. The Aer and Tsr proteins in E. coli are newly recognized transducers for energy taxis. Aer is homologous to E. coli chemoreceptors but unique in having a PAS domain and a flavin-adenine dinucleotide cofactor that is postulated to interact with a component of the electron transport system. PAS domains are energy-sensing modules that are found in proteins from archaea to humans. Tsr, the serine chemoreceptor, is an independent transducer for energy taxis, but its sensory mechanism is unknown. Energy taxis has a significant ecological role in vertical stratification of microorganisms in microbial mats and water columns. It plays a central role in the behavior of magnetotactic bacteria and also appears to be important in plant-microbe interactions.

Electrospinning is a versatile and viable technique for generating ultrathin fibers. Remarkable progress has been made with regard to the development of electrospinning methods and engineering of electrospun nanofibers to suit or enable various applications. We aim to provide a comprehensive overview of electrospinning, including the principle, methods, materials, and applications. We begin with a brief introduction to the early history of electrospinning, followed by discussion of its principle and typical apparatus. We then discuss its renaissance over the past two decades as a powerful technology for the production of nanofibers with diversified compositions, structures, and properties. Afterward, we discuss the applications of electrospun nanofibers, including their use as "smart" mats, filtration membranes, catalytic supports, energy harvesting/conversion/storage components, and photonic and electronic devices, as well as biomedical scaffolds. We highlight the most relevant and recent advances related to the applications of electrospun nanofibers by focusing on the most representative examples. We also offer perspectives on the challenges, opportunities, and new directions for future development. At the end, we discuss approaches to the scale-up production of electrospun nanofibers and briefly discuss various types of commercial products based on electrospun nanofibers that have found widespread use in our everyday life.

The genetics of the mating-type (MAT) locus have been studied extensively in Saccharomyces cerevisiae, but relatively little is known about how this complex system evolved. We compared the organization of MAT and mating-type-like (MTL) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent HMR/HML cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active MAT locus and silent cassettes exists only in the Saccharomyces sensu stricto group and their closest relatives: Candida glabrata, Kluyveromyces delphensis, and Saccharomyces castellii. We identified C. glabrata MTL1 as the ortholog of the MAT locus of K. delphensis and show that switching between C. glabrata MTL1a and MTL1alpha genotypes occurs in vivo. The more distantly related species Kluyveromyces lactis has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as Candida albicans and Yarrowia lipolytica do not have silent cassettes. In Pichia angusta, a homothallic species, we found MATalpha2, MATalpha1, and MATa1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the MAT locus can be traced throughout hemiascomycete evolution and even to Neurospora, the gene content of the locus has changed with the loss of an HMG domain gene (MATa2) from the MATa idiomorph shortly after HO was recruited.

Current hypotheses suggest the Mre11 nuclease activity could be directly involved in double-strand break (DSB) resection in the presence of a large number of DSBs or limited to processing abnormal DNA ends. To distinguish between these possibilities, we used two methods to create large numbers of DSBs in Saccharomyces cerevisiae chromosomes, without introducing other substrates for the Mre11 nuclease. Multiple DSBs were created either by expressing the HO endonuclease in strains containing several HO cut sites embedded within randomly dispersed Ty1 elements or by phleomycin treatment. Analysis of resection by single-strand DNA formation in these systems showed no difference between strains containing MRE11 or the mre11-D56N nuclease defective allele, suggesting that the Mre11 nuclease is not involved in the extensive 5' to 3' resection of DSBs. We postulate that the ionizing radiation (IR) sensitivity of mre11 nuclease-defective mutants results from the accumulation of IR-induced DNA damage that is normally processed by the Mre11 nuclease. We also report that the processivity of 5' to 3' DSB resection and the yield of repaired products are affected by the number of DSBs in a dose-dependent manner. Finally, we show that the exonuclease Exo1 is involved in the processivity of 5' to 3' resection of an HO-induced DSB at the MAT locus.

Marrow adipose tissue (MAT) accumulates in diverse clinical conditions but remains poorly understood. Here we show region-specific variation in MAT adipocyte development, regulation, size, lipid composition, gene expression and genetic determinants. Early MAT formation in mice is conserved, whereas later development is strain dependent. Proximal, but not distal tibial, MAT is lost with 21-day cold exposure. Rat MAT adipocytes from distal sites have an increased proportion of monounsaturated fatty acids and expression of Scd1/Scd2, Cebpa and Cebpb. Humans also have increased distal marrow fat unsaturation. We define proximal 'regulated' MAT (rMAT) as single adipocytes interspersed with active haematopoiesis, whereas distal 'constitutive' MAT (cMAT) has low haematopoiesis, contains larger adipocytes, develops earlier and remains preserved upon systemic challenges. Loss of rMAT occurs in mice with congenital generalized lipodystrophy type 4, whereas both rMAT and cMAT are preserved in mice with congenital generalized lipodystrophy type 3. Consideration of these MAT subpopulations may be important for future studies linking MAT to bone biology, haematopoiesis and whole-body metabolism.

OBJECTIVE

To determine the prevalence and severity of asymmetry among independently ambulating stroke survivors and to establish the association between velocity and asymmetry.

METHODS

Descriptive analysis.

METHODS

Research gait laboratory in a Canadian hospital.

METHODS

Community-dwelling, independently ambulating participants (N=54) with chronic stroke.

METHODS

Not applicable.

METHODS

Overground gait velocity, symmetry ratios for temporal and spatial step parameters, and motor impairment of the foot and leg. Spatiotemporal parameters were collected with a pressure-sensitive mat. Motor impairment was measured clinically with the Chedoke-McMaster Stroke Assessment.

RESULTS

Thirty (55.5%) participants showed statistically significant temporal asymmetry and 18 (33.3%) exhibited statistically significant spatial asymmetry. Preferred velocity was negatively associated with temporal asymmetry (r=-.583, df=52, P<.001) but not spatial asymmetry (r=-.146, df=52, P=.29). Temporal asymmetry was also associated with motor recovery of the leg (r=-.644, df=35, P<.001) and foot (r=-.628, df=35, P<.001).

CONCLUSIONS

The results of the current study illustrate that temporal asymmetry can be found in many independently ambulating stroke patients. The work highlights the need for a standard assessment of poststroke gait symmetry in light of the complex relationship with motor impairment and velocity.

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

Primers were designed to amplify a 592-bp region within a conserved structural gene (g20) found in some cyanophages. The goal was to use this gene as a proxy to infer genetic richness in natural cyanophage communities and to determine if sequences were more similar in similar environments. Gene products were amplified from samples from the Gulf of Mexico, the Arctic, Southern, and Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of ca. 3,246 m in the Chuckchi Sea. Amplicons were separated by denaturing gradient gel electrophoresis, and selected bands were sequenced. Phylogenetic analysis revealed four previously unknown groups of g20 clusters, two of which were entirely found in freshwater. Also, sequences with >99% identities were recovered from environments that differed greatly in temperature and salinity. For example, nearly identical sequences were recovered from the Gulf of Mexico, the Southern Pacific Ocean, an Arctic freshwater cyanobacterial mat, and Lake Constance, Germany. These results imply that closely related hosts and the viruses infecting them are distributed widely across environments or that horizontal gene exchange occurs among phage communities from very different environments. Moreover, the amplification of g20 products from deep in the cyanobacterium-sparse Chuckchi Sea suggests that this primer set targets bacteriophages other than those infecting cyanobacteria.

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.

BACKGROUND

Understanding of local knowledge and practices relating to the newborn period, as locally defined, is needed in the development of interventions to reduce neonatal mortality. We describe the organisation of the neonatal period in Sylhet District, Bangladesh, the perceived threats to the well-being of neonates, and the ways in which families seek to protect them.

METHODS

We did 39 in-depth, unstructured, qualitative interviews with mothers, fathers, and grandmothers of neonates, and traditional birth attendants. Data on neonatal knowledge and practices were also obtained from a household survey of 6050 women who had recently given birth.

RESULTS

Interviewees defined the neonatal period as the first 40 days of life (chollish din). Confinement of the mother and baby is most strongly observed before the noai ceremony on day 7 or 9, and involves restriction of movement outside the home, sleeping where the birth took place rather than in the mother's bedroom, and sleeping on a mat on the floor. Newborns are seen as vulnerable to cold air, cold food or drinks (either directly or indirectly through the mother), and to malevolent spirits or evil eye. Bathing, skin care, confinement, and dietary practices all aim to reduce exposure to cold, but some of these practices might increase the risk of hypothermia.

CONCLUSIONS

Although fatalism and cultural acceptance of high mortality have been cited as reasons for high levels of neonatal mortality, Sylheti families seek to protect newborns in several ways. These actions reflect a set of assumptions about the newborn period that differ from those of neonatal health specialists, and have implications for the design of interventions for neonatal care.

Heterochromatin is a structurally compacted region of chromosomes in which transcription and recombination are inactivated. DNA replication is temporally regulated in heterochromatin, but the molecular mechanism for regulation has not been elucidated. Among heterochromatin loci in Schizosaccharomyces pombe, the pericentromeric region and the silent mating-type (mat) locus replicate in early S phase, whereas the sub-telomeric region does not, suggesting complex mechanisms for regulation of replication in heterochromatic regions. Here, we show that Swi6, an S. pombe counterpart of heterochromatin protein 1 (HP1), is required for early replication of the pericentromeric region and the mat locus. Origin-loading of Sld3, which depends on Dfp1/Dbf4-dependent kinase Cdc7 (DDK), is stimulated by Swi6. An HP1-binding motif within Dfp1 is required for interaction with Swi6 in vitro and for early replication of the pericentromeric region and mat locus. Tethering of Dfp1 to the pericentromeric region and mat locus in swi6-deficient cells restores early replication of these loci. Our results show that a heterochromatic protein positively regulates initiation of replication in silenced chromatin by interacting with an essential kinase.

The mating type of haploid yeast (a or alpha) is determined by information present at the MAT locus. Identical copies of a and alpha information are present at distal loci (HMR and HML), but transcription of these copies is repressed by the action, in trans, of four unlinked genes called SIR (silent information regulator). Repression by SIR also requires, in cis, DNA sequences called E which are found to the left of HML and HMR (but not MAT) and are greater than 1 kb from the mating-type gene promoters. SIR control can act on other promoters when they are brought near the E sequence, and thus the SIR gene products act in some general manner to repress transcription. We have determined the DNA sequence of two fragments which complement mutations in the SIR2 and SIR3 genes and show that these contain the structural genes by mapping the cloned sequences onto the yeast chromosome. The SIR2 and SIR3 coding sequences were identified by constructing gene disruptions and using these mutations to replace the normal chromosomal copies. Such null mutants of both SIR2 and SIR3 are defective in the position-effect control of the silent loci but have no other detectable phenotype. We have mapped the 5' and 3' ends of the SIR2 and SIR3 mRNAs and show that their level is unaffected by mutations in any of the four known SIR complementation groups.

Essential oils extracted from 10 medicinal plants were evaluated for larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species; Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. The essential oils of Juniperus macropoda and Pimpinella anisum were highly effective as both larvicidal and ovicidal. The essential oil of P. anisum showed toxicity against 4th instar larvae of A. stephensi and A. aegypti with equivalent LD95 values of 115.7 microg/ml, whereas it was 149.7 microg/ml against C. quinquefasciatus larvae. Essential oils of Zingiber officinale and Rosmarinus officinalis were found to be ovicidal and repellent, respectively towards the three mosquito species. The essential oil of Cinnamomum zeylanicum resulted into highest repellent (RD95) values of 49.6, 53.9 and 44.2 mg/mat against A. stephensi, A. aegypti and C. quinquefasciatus, respectively apart from oviposition-deterrent potential.

BACKGROUND

Candida species are microbial pathogens originally thought to be asexual, but several are now recognized as sexual or parasexual. Candida albicans, the most common fungus infecting humans, is an obligate diploid with a parasexual cycle involving mating, recombination, and genome reduction but no recognized meiosis. Others (C. lusitaniae, C. guilliermondii) are haploid, and their mating produces spores, suggestive of complete meiotic sexual cycles. However, comparative genomic analysis reveals that these species lack key meiotic components, including the recombinase Dmc1 and cofactors (Mei5/Sae3), synaptonemal-complex proteins (Zip1-Zip4/Hop1), and the crossover interference pathway (Msh4/5).

RESULTS

Here we elucidate the structure and functions of the mating-type (MAT) locus and establish that C. lusitaniae undergoes meiosis during its sexual cycle. The MAT-encoded a2 (high-mobility group) and alpha1 (alpha domain) factors specify a and alpha cell identity, whereas the a1 homeodomain protein drives meiosis and sporulation and functions without its canonical heterodimeric partner, alpha2. Despite the apparent loss of meiotic genes, C. lusitaniae undergoes meiosis during sexual reproduction involving diploid intermediates, frequent SPO11-dependent recombination, and whole-genome reduction generating haploid progeny. The majority of meiotic progeny are euploid, but approximately one-third are diploid/aneuploid.

CONCLUSIONS

The cell identity and meiotic pathways have been substantially rewired, and meiotic generation of both recombinant and aneuploid progeny may expand genetic diversity. These findings inform our understanding of sexual reproduction in pathogenic microbes and the evolutionary plasticity of the meiotic machinery, with implications for the sexual nature of C. albicans and the generation and consequences of aneuploidy in biology and medicine.

The mating-type locus of the haploid filamentous fungus Neurospora crassa is a regulatory region that controls entry into the sexual cycle and prevents formation of mixed mating-type heterokaryons in the vegetative phase. The locus consists of alternative sequences called A and a. The A mating-type DNA sequence of Neurospora crassa is composed of a region of 5301 base pairs that has little similarity to the sequence present at the mating-type locus in an a mating-type strain. However, the sequences flanking the mating-type locus in the A haploid and a haploid genome are essentially identical. The region of the A mating-type sequence required for expression of the heterokaryon incompatibility and sexual functions has been localized to a single open reading frame (ORF) encoding a polypeptide of 288 amino acids. Sequence analysis of sterile, heterokaryon-compatible mutants reveals frameshift mutations in this same ORF. The putative 288-amino acid product has a region of similarity to the MAT alpha 1 polypeptide of Saccharomyces cerevisiae.

Position-effect control at the silent matmatmatin-like structures. Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly. Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the matmatmat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases. Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities. We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state. This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest). In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase. Cell growth was not inhibited in arrested cells. Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase. However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose. These results suggest that the C terminus of the wild-type protein confers functional instability. cln-arrested cells are mating competent. However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains). These results are consistent with a specific CLN requirement for START transit.

Massive microbial mats covering up to 4-meter-high carbonate buildups prosper at methane seeps in anoxic waters of the northwestern Black Sea shelf. Strong 13C depletions indicate an incorporation of methane carbon into carbonates, bulk biomass, and specific lipids. The mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). If incubated in vitro, these mats perform anaerobic oxidation of methane coupled to sulfate reduction. Obviously, anaerobic microbial consortia can generate both carbonate precipitation and substantial biomass accumulation, which has implications for our understanding of carbon cycling during earlier periods of Earth's history.

Sonoporation, in the presence of ultrasound contrast agents (UCA), is a technique that permits the transfer of drugs, including genes, into cells. In this study, the size of the pores created by ultrasound application, and the duration of pore opening, have been characterized via indirect molecular probing and microscopic observation. Internalization of molecules with diameters up to 37 nm was efficient and generally well-tolerated; on the other hand, confocal microscopy revealed that 75 nm particles entered only a few cells when sonoporation was applied. In general, the larger the species to internalize, the poorer the transfer. Direct visualization of pores following insonification, using scanning electron microscopy, was hampered by the presence of numerous villi on the surface of the cells employed (MAT B III), and by the short duration of pore opening. Clearer observations of porated regions were possible using red blood cells. This research (i) confirms that sonoporation is a means with which to achieve macromolecule delivery into cells, and (ii) characterizes in some detail the phenomenon of ultrasound induction of transient pores in the cell membrane.