Methane- and dissolved organic carbon-fueled microbial loop supports a tropical subterranean estuary ecosystem.

Subterranean estuaries extend inland into density-stratified coastal carbonate aquifers containing a surprising diversity of endemic animals (mostly crustaceans) within a highly oligotrophic habitat. How complex ecosystems (termed anchialine) thrive in this globally distributed, cryptic environment is poorly understood. Here, we demonstrate that a microbial loop shuttles methane and dissolved organic carbon (DOC) to higher trophic levels of the anchialine food web in the Yucatan Peninsula (Mexico). Methane and DOC production and consumption within the coastal groundwater correspond with a microbial community capable of methanotrophy, heterotrophy, and chemoautotrophy, based on characterization by 16S rRNA gene amplicon sequencing and respiratory quinone composition. Fatty acid and bulk stable carbon isotope values of cave-adapted shrimp suggest that carbon from methanotrophic bacteria comprises 21% of their diet, on average. These findings reveal a heretofore unrecognized subterranean methane sink and contribute to our understanding of the carbon cycle and ecosystem function of karst subterranean estuaries.

Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane.

Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.

Dissolved oxygen saturation controls PAH biodegradation in freshwater estuary sediments.

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in terrestrial and aquatic environments and can represent a significant constituent of the carbon pool in coastal sediments. We report here the results of an 18-month seasonal study of PAH biodegradation and heterotrophic bacterial production and their controlling biogeochemical factors from 186 sediment samples taken in a tidally influenced freshwater estuary. For each sampling event, measurements were averaged from 25-45 stations covering approximately 250 km(2). There was a clear relationship between bacterial production and ambient temperature, but none between production and bottom water dissolved oxygen (DO) % saturation or PAH concentrations. In contrast with other studies, we found no effect of temperature on the biodegradation of naphthalene, phenanthrene, or fluoranthene. PAH mineralization correlated with bottom water DO saturation above 70% (r(2) > 0.99). These results suggest that the proportional utilization of PAH carbon to natural organic carbon is as much as three orders of magnitude higher during cooler months, when water temperatures are lower and DO % saturation is higher. Infusion of cooler, well-oxygenated water to the water column overlying contaminated sediments during the summer months may stimulate PAH metabolism preferentially over non-PAH organic matter.

Three days of incubation may be sufficient for routine blood cultures with BacT/Alert FAN blood culture bottles.

BacT/Alert FAN blood culture bottles have been shown to enhance the recovery of bacteria and yeast from blood compared with standard BacT/Alert bottles. It is well established that standard BacT/Alert blood culture bottles require no more than 5 days of incubation for the detection of routine bacteria and yeast. It is less clear, however, whether FAN bottles also routinely require 5 days of incubation. To address this question, we recently reviewed the results of 17,887 blood culture sets collected in FAN blood culture bottles at Geisinger Medical Center. Of these cultures, 1,780 were positive for bacteria or yeast, yielding a total of 1,242 clinically significant isolates. The numbers of isolates recovered on days 1, 2, 3, 4, and 5 were as follows: (values in parentheses are percentages of total significant isolates): 877 (71%), 269 (22%), 65 (5%), 18 (1%) and, 13 (1%), respectively. In total, 97.5% of all clinically significant isolates were detected in the first 3 days of incubation. Of the 31 significant isolates detected on day 4 or 5 of incubation, 17 were detected in concurrent blood cultures within the first 3 days of incubation. Chart reviews were conducted for the 13 patients with the remaining 14 isolates detected on day 4 or 5 to determine whether therapy was changed due to this blood culture result. Therapy was changed for only 1 patient. These results suggest that it may not be necessary to routinely incubate FAN blood culture bottles for more than 3 days.

Timed killing kinetic studies of the interaction between ciprofloxacin and beta-lactams against gram-negative bacilli.

Timed killing kinetic studies were performed with ciprofloxacin in combination with aztreonam, ceftazidime, piperacillin/tazobactam, and ticarcillin/clavulanic acid against three isolates each of Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa. Each antimicrobial agent in the combination was tested at its MIC and at one-half and one-quarter of its MIC. Colony counts were determined at 0, 3, 5, and 7 hours. Synergy occurred most frequently at 7 hours and, when present, was most likely to occur when ciprofloxacin and the beta-lactam were tested at concentrations equal to their respective MICs. Synergy after 3 hours of incubation was not predictive of a synergestic interaction at 5 or 7 hours. Antagonism was noted in several instances, particularly when ciprofloxacin and the beta-lactam were combined at one-quarter of their respective MICs.

Controlled clinical evaluation of BACTEC Plus Aerobic/F and BacT/Alert Aerobic FAN bottles for detection of bloodstream infections.

A total of 7,190 blood culture sets were obtained from adult patients with a suspected bloodstream infection. A 20-ml sample of blood was distributed equally between the aerobic FAN bottle which was monitored in the BacT/Alert system and a Plus Aerobic/F bottle which was monitored in the BACTEC 9240 system. A total of 988 positive cultures were obtained from 483 patients; however, only 453 positive cultures from 173 patients met the criteria for volume ( > or = ml per bottle) and clinical significance on the basis of concurrent case review required for data analysis. There were 25 and 68 false positives from the FAN and Plus Aerobic/F bottles, respectively. There were no statistically significant differences between systems in the number of positive cultures or septic episodes by species; however, the total number of Enterobacteriaceae and Pseudomonas aeruginosa isolates combined was significantly greater in the FAN bottle (P = 0.04). Detection times did not differ significantly between systems for positive cultures; however, episodes of Staphylococcus aureus bacteremia were detected significantly more rapidly from the FAN bottle (P = 0.005). There was no significant difference between systems in the detection of bloodstream infections in patients receiving antibiotics at the time of blood culture.

Controlled clinical comparison of Isolator and BACTEC 9240 Aerobic/F resin bottle for detection of bloodstream infections.

A controlled clinical comparison was carried out with the BACTEC 9240 Aerobic/F resin bottle and the Isolator system with adult patients suspected of having bloodstream infections. A total of 10,500 paired specimens were collected, of which 1,122 from 520 patients were positive. There were 68 false-positive BACTEC bottles; 259 positive cultures that were excluded from analysis because the bottle, the Isolator, or both failed to meet the minimum volume criterion of 8 ml of blood; and 207 positive cultures that were excluded because the isolates were found to be clinically insignificant or of indeterminate clinical significance on the basis of patient assessment. A total of 656 positive cultures from 258 patients formed the basis of the analysis. Significantly more Staphylococcus aureus isolates (P = 0.03), Staphylococcus epidermidis isolates (P = 0.03), members of the family Enterobacteriaceae (P = 0.03), and Pseudomonas aeruginosa isolates (P = 0.04) were recovered from the resin bottle, and there was no category of organism that was recovered significantly more frequently from the Isolator system. With patients receiving antibiotics at the time of blood culture, S. aureus, S. epidermidis, and gram-negative bacilli were recovered significantly more frequently from the resin bottle. No significant differences between systems were found with cultures from patients not receiving antibiotics at the time of blood culture. Only 12 clinically significant organisms were recovered from the bottle on terminal subcultures, and only 1 of these had not been previously isolated from another blood culture set (10 of the 12) or from the companion Isolator (1 of 12). The Aerobic/F resin bottle continuously monitored in the BACTEC 9240 instrument proved to be superior to the Isolator in overall yield of organisms causing bloodstream infection in adults and required less technician time for specimen processing and examination than the Isolator system.

Stimulation of MHC class I transcription by interferon-gamma involves a non-A, non-C kinase in addition to protein kinase C.

The signal pathways by which interferon-gamma (IFN-gamma) is able to up-regulate major histocompatibility complex (MHC) class I transcription were studied in two human hematopoietic tumor cell lines, K562 and Ramos. These studies suggest that the IFN-gamma signal is transduced via an H7- and staurosporine-sensitive kinase that is distinct from protein kinase C (PKC) and protein kinase A (PKA) in both cell types. Ramos cells appear to utilize an additional pathway involving double-stranded RNA-dependent protein kinase. PKC and possibly PKA appear to be involved in one or more intersecting pathways by which agonists of these kinases are able to act synergistically with IFN-gamma, but activation of these latter pathways is neither necessary nor sufficient for induction of MHC class I transcription. Modulation of G-protein- and Ca2+-calmodulin-associated pathways and arachidonic acid metabolism had no effect on constitutive or IFN-gamma-stimulated class I transcription. The class I stimulatory factor produced in response to IFN-gamma treatment appears to have a short t1/2. The identity of this factor is unknown, but is likely to be distinct from known mediators of IFN-stimulated transcription. Gene and cell-type specificity in the signal transduction pathways utilized by IFN-gamma implies that such pathways may be useful targets for experimental and therapeutic manipulation.

The effects of multisensory structured language instruction on native language and foreign language aptitude skills of at-risk high school foreign language learners.

Research findings suggest that most students who have foreign language learning problems have language-based difficulties and, in particular, phonological processing problems. Authors of the present study examined pre- and posttest scores on native language and foreign language aptitude tests of three groups of at-risk high school students enrolled in special, self-contained sections of first-year Spanish. Two groups were instructed using a multisensory structured language (MSL) approach. One of the groups was taught in both English and Spanish (MSL/ES), the other only in Spanish (MSL/S). The third group (NO-MSL) was instructed using more traditional second language teaching methodologies. Significant gains were made by the MSL-ES group on measures of native language phonology, vocabulary, and verbal memory and on a test of foreign language aptitude; the MSL/S group made significant gains on the test of foreign language aptitude. No significant gains on the native language or foreign language aptitude measures were made by the NO-MSL group. Implications for foreign language classroom instruction of at-risk students are discussed.

Identifying native language difficulties among foreign language learners in college: a "foreign" language learning disability?

The present study compared successful and unsuccessful college foreign language learners on measures of intelligence, foreign language aptitude, native oral and written language, and math. Unsuccessful students had received petitions to waive the foreign language requirement. No significant differences between groups were found on intelligence and reading comprehension. Significant differences were found on the Modern Language Aptitude Test, on tests of written and oral language in the syntactic and phonological domains, and on math calculation. Authors suggest that students with foreign language learning difficulties may have underlying native language problems manifested especially in the areas of syntax and phonology. Suggestions for diagnosing a foreign language disability are made.

Linguistic coding deficits in foreign language learners.

As increasing numbers of colleges and universities require a foreign language for graduation in at least one of their degree programs, reports of students with difficulties in learning a second language are multiplying. Until recently, little research has been conducted to identify the nature of this problem. Recent attempts by the authors have focused upon subtle but ongoing language difficulties in these individuals as the source of their struggle to learn a foreign language. The present paper attempts to expand upon this concept by outlining a theoretical framework based upon a linguistic coding model that hypothesizes deficits in the processing of phonological, syntactic, and/or semantic information. Traditional psychoeducational assessment batteries of standardized intelligence and achievement tests generally are not sensitive to these linguistic coding deficits unless closely analyzed or, more often, used in conjunction with a more comprehensive language assessment battery. Students who have been waived from a foreign language requirement and their proposed type(s) of linguistic coding deficits are profiled. Tentative conclusions about the nature of these foreign language learning deficits are presented along with specific suggestions for tests to be used in psychoeducational evaluations.

In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.

An optional 46-base-pair G + C-rich element (GC cluster) in the coding region of the yeast mitochondrial var1 gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var1 GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the var1 gene in mitochondrial DNA isolated from unmated haploid rho+ and rho- strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient var1 DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var1 GC cluster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.

Kinetic and segregational analysis of mitochondrial DNA recombination in yeast.

A pair of yeast strains of opposite mating type was constructed to contain polymorphisms at three loci on the mitochondrial genome--the 21 S rRNA gene, var1, and cob--such that parental and recombinant forms of these genes could be easily detected by Southern blot analysis. These polymorphisms were used to measure in a single cross gene conversions at the 21 S rRNA and var1 loci and a reciprocal recombination at cob. For all three loci, recombination initiates at about the same time, 4 to 6 h after mixing cells, and increases with similar kinetics over a 24-h period. The segregation of parental and recombinant forms of these genes was then followed by pedigree analysis. The results, which show a high variance in the distribution of parental and recombinant forms of all three genes in cells derived from both the first bud and the mother zygote, are consistent with the segregation of a small number of mitochondrial DNA molecules from the zygote to diploid buds. Based on these results and previous experiments of this type, a limited "zone of mixing" of parental mitochondrial DNA molecules probably exists in the zygote. The extent of sampling from this zone, together with the intrinsic properties of the recombination events themselves, is likely to determine the observed pattern of recombination of mitochondrial DNA sequences at the population level.

Transcriptional activation of an embryonic globin gene in adult erythroid cells in vivo.

We have studied the mechanism and level of activation of the normally silent rho globin gene in adult erythroid cells of anemic chickens treated with 5-azacytidine and sodium butyrate. We have shown that while demethylation of the rho globin gene does not result in high levels of specific mRNA, demethylation does appear to be a prerequisite for sodium butyrate to cause such an effect. Runoff nuclear transcription assays were used to demonstrate that 5-azacytidine plus sodium butyrate treatment act to cause transcriptional activation of the rho gene in adult animals. However, the data also show that there may be post-transcriptional down-regulation of mRNA levels in adult red cells, since the level of rho gene nuclear runoff relative to beta exceeds the corresponding stable mRNA ratios for the two gene products. These data are consistent with a model in which developmental switching of the chicken globin genes may involve both transcriptional regulation and one or more levels of post-transcriptional regulation. Our studies of histone acetylation in erythroid cells from butyrate treated animals show that no bulk changes in histone acetylation can be detected. While transient localized changes in the fast kinetic form of histone acetylation cannot be excluded, these results are consistent with the hypothesis that the transcriptional activation of the rho gene by sodium butyrate in this animal model may be mediated by some trans-acting transcription regulating factor. Thus, the requirement for both 5-azacytidine and sodium butyrate for gene activation could reflect a mechanism which involves both an alteration in a specific regulatory DNA recognition sequence (by demethylation) and a change in either the specificity or amount of a stimulatory (or inhibitory) trans acting factor. These proposed mechanisms are directly testable with currently available techniques.

Activation of a chicken embryonic globin gene in adult erythroid cells by 5-azacytidine and sodium butyrate.

Adult White Leghorn chickens were rendered anemic by injection with 1-acetyl-2-phenylhydrazine and then treated with parenteral 5-azacytidine, and levels of embryonic globin RNA in circulating reticulocytes were measured. A very small but detectable amount of correctly initiated embryonic p-type globin RNA was detected in reticulocytes from birds treated with 5-azacytidine, while none was detected in reticulocytes from those receiving only phenylhydrazine or phenylhydrazine plus 1-beta-D-arabinofuranosylcytosine (cytosine arabinonucleoside). An attempt to increase embryonic globin RNA induction by treatment with parenteral sodium butyrate after 7 days of 5-azacytidine administration resulted in a 5- to 10-fold increase in the level of embryonic globin RNA. However, sodium butyrate did not induce embryonic gene expression when given alone or after treatment with cytosine arabinonucleoside. Sodium butyrate treatment also caused a DNase I-hypersensitive site to be exposed at the 5' end of the rho-globin gene only after 5-azacytidine induced demethylation of several CpG sites in and around the gene. The implications of this model of gene activation in vivo are discussed in the context of multistep gene regulation.