A long non-coding RNA GATA6-AS1 adjacent to GATA6 is required for cardiomyocyte differentiation from human pluripotent stem cells.

Long noncoding RNAs (lncRNAs) are crucial in many cellular processes, yet relatively few have been shown to regulate human cardiomyocyte differentiation. Here, we demonstrate an essential role of GATA6 antisense RNA 1 (GATA6-AS1) in cardiomyocyte differentiation from human pluripotent stem cells (hPSCs). GATA6-AS1 is adjacent to cardiac transcription factor GATA6. We found that GATA6-AS1 was nuclear-localized and transiently upregulated along with GATA6 during the early stage of cardiomyocyte differentiation. The knockdown of GATA6-AS1 did not affect undifferentiated cell pluripotency but inhibited cardiomyocyte differentiation, as indicated by no or few beating cardiomyocytes and reduced expression of cardiomyocyte-specific proteins. Upon cardiac induction, the knockdown of GATA6-AS1 decreased GATA6 expression, altered Wnt-signaling gene expression, and reduced mesoderm development. Further characterization of the intergenic region between genomic regions of GATA6-AS1 and GATA6 indicated that the expression of GATA6-AS1 and GATA6 were regulated by a bidirectional promoter within the intergenic region. Consistently, GATA6-AS1 and GATA6 were co-expressed in several human tissues including the heart, similar to the mirror expression pattern of GATA6-AS1 and GATA6 during cardiomyocyte differentiation. Overall, these findings reveal a previously unrecognized and functional role of lncRNA GATA6-AS1 in controlling human cardiomyocyte differentiation.

DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in Staphylococcus aureus.

Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the -10 element of the P farR promoter, (ii) in palindrome PAL1 immediately in the 3' direction of P farR , or (iii) within PAL2 upstream of the predicted P farE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in P farR Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded.IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.

Effects of Cleft Width and Veau Type on Incidence of Palatal Fistula and Velopharyngeal Insufficiency After Cleft Palate Repair.

BACKGROUND: Postoperative fistulae and velopharyngeal insufficiency (VPI) are 2 important complications after cleft palate repair. The effects of preoperative cleft width on outcomes after cleft palate repair have been rarely studied. METHODS: A retrospective review of all patients undergoing primary cleft palatoplasty by a single surgeon between 2004 and 2011 was performed. Primary outcomes were palatal fistula and VPI, defined as the need for corrective surgery after failing conservative speech-language therapy. Logistic regression analysis was performed to identify factors associated with the primary outcomes. RESULTS: One hundred seventy-seven patients (84 men and 93 women) were identified. Median age at repair was 10 months with median follow-up of 3.80 years. Preoperative cleft width was 10 mm or less for 72 (41%) patients, 11 to 14 mm for 54 (30%) patients, and 15 mm or greater for 51 (29%) patients. Palatal fistula was observed in 8 (4.5%) patients, but required surgical repair in only 2 (1.1%). Fistula was overall associated with Veau IV classification (odds ratio, 8.13; P < 0.01) but not with cleft width. Velopharyngeal insufficiency needing surgical intervention occurred in 9 patients (7.38% of patients older than 4 years) and was associated with increasing cleft width (odds ratio, 1.29; P = 0.011). Outcomes were similar for patients undergoing surgery in the earlier and later halves of the study. CONCLUSIONS: This retrospective review is one of the first from the United States to explore the associations between measured cleft width and outcomes after palatoplasty. Overall rates of palatal fistula and VPI were low, corroborating previous studies showing good outcomes with the 2-flap palatoplasty. After adjusting for multiple variables including Veau type, cleft width was associated with higher VPI rates but not with fistula formation. Cleft width is a unique preoperative factor that should be considered and studied as a potential predictor of outcomes.

A rapid beverage intake questionnaire can detect changes in beverage intake.

UNLABELLED: Attention on beverage intake, specifically sugar-sweetened beverages (SSB), has increased in recent years. A brief valid, reliable and sensitive assessment tool for quantifying beverage consumption and determining its influence on weight status could help to advance research on this topic. The valid and reliable 15-item beverage questionnaire (BEVQ-15) estimates mean daily intake of water, SSB and total beverages (g, kcal) across multiple beverage categories. OBJECTIVE: to determine the ability of the BEVQ-15 to detect changes in beverage intake over time. Participants (n=70; age=37±2 yr; BMI=24.5±0.4 kg/m(2)) underwent two randomly assigned 30-day periods (intervention, increased water and fruit juice consumption; control, increased solid fruit consumption), with a 30-day washout phase between feeding periods. The BEVQ-15 was administered at the beginning and end of each period. Reliability was assessed by Pearson's correlations, paired sample t tests and Cronbach's alpha. Paired sample t tests and repeated measures ANOVA were used to evaluate sensitivity to change. Sixty-nine participants completed all study sessions. Reliability was acceptable for most beverages (range: R(2)=0.52-0.95, P<0.001), but not for energy drinks. Increases in water (g), juice (kcal, g) and total beverage (g) were detected during the intervention period (P<0.001); no changes in these variables were detected in the control period. The BEVQ-15 demonstrates the ability to detect changes in beverage intake over time. This brief (~2 min), self-administered, valid, reliable and sensitive beverage intake assessment tool may be used by researchers and practitioners who evaluate and intervene upon beverage intake patterns in adults.

Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania.

Background. T-SPOT.TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allow delayed processing, but has not been extensively field tested. Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1-3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25 µL of T Cell Xtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TB and culture was 95.4% (95%CI; 87.1-99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using T Cell Xtend reagent was 96.8% (95%CI; 88.8-99.6%). Conclusions. When T Cell Xtend reagent is added to specimens held overnight at recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis.

Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium.

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium: DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or "twin" formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.