Convenient method to prepare neutral sulfide solution for cultivation of phototrophic sulfur bacteria

A device is described for the preparation and storage of sterile neutral sulfide solution which is required for the repeated addition of this substrate to growing cultures of phototrophic sulfur bacteria.     

Younger genes are less likely to be essential than older genes, and duplicates are less likely to be essential than singletons of the same age

Recently duplicated genes are believed to often overlap in function and expression. A priori, they are thus less likely to be essential. Although this was indeed observed in yeast, mouse singletons and duplicates were reported to be equally often essential. This contradiction can only partly be explained by experimental biases. We herein show that older genes (i.e., genes with earlier phyletic origin) are more likely to be essential, regardless of their duplication status. At a given phyletic gene age, duplicates are always less likely to be essential compared with singletons. The "paradoxical" high essentiality among mouse gene duplicates is then caused by different age profiles of singletons and duplicates, with the latter tending to be derived from older genes.     

Determinants of exclusive breastfeeding practice to infants aged less than six months in Offa district,Southern Ethiopia: a cross-sectional study

Background

World Health Organization (WHO) recommends timely initiation of breastfeeding after birth and only feeding breast milk to infants during the first 6 months of life. It was estimated that exclusive breastfeeding can reduce infant deaths by 13%. The practice of exclusive breastfeeding is suboptimal in many parts of Ethiopia to a varied extent. Factors associated with exclusive breastfeeding practice and the proportion of its practice was not well documented in Offa district. Therefore, this study was aimed to assess the determinants of exclusive breastfeeding in the first 6 months of life in Offa district, Southern Ethiopia.

Methods

A community-based cross-sectional study was carried out in 396 mothers of infants younger than 6 months using random sampling. Data were collected from mothers of the infants by trained interviewers. Exclusive breastfeeding was measured by the history of infant feeding in the prior 24 h. Pretested and structured questionnaires adopted from standard questionnaires and Ethiopia linkages modules were used. Multivariate logistic regression was used to identify factors significantly influencing exclusive breastfeeding practice.

Results

Based on findings of this study of 396 participants, the proportion of exclusive breastfeeding was 78.0% and awareness of exclusive breastfeeding and bottle feeding were 85.6 and 6.1% respectively. About 6% of infants were given prelacteal feeds. The number of infants fed cow milk was 12.9%, formula 7.8%, water 8.5%, fruits and semisolids 1.5%, over 24 h prior to the survey. The initiation of breastfeeding within one h (AOR 2.2; 95% CI 1.1, 4.27), attending formal education (AOR 4; 95% CI 2.20, 7.25), having an awareness on the benefits of exclusive breastfeeding (AOR 6; 95% CI 3.10, 11.70) and knowledge of colostrum feeding (AOR 2.1; 95% CI 1.11, 4.27) had a statistically significant association with exclusive breastfeeding in the study area.

Conclusions

The practice of exclusive breastfeeding as well as awareness was worthy in Offa district. Additionally the proportion of bottle feeding use was small. However, feeding other than breast milk was associated with the perception that breast milk alone was insufficient for their child. Strategies on promoting exclusive breastfeeding practice must focus on strengthening women’s education and awareness creation activities further.

     

Fungal community reveals less dispersal limitation and potentially more connected network than that of bacteria in bamboo forest soils

A central aim of this microbial ecology research was to investigate the mechanisms shaping the assembly of soil microbial communities. Despite the importance of bacterial and fungal mediation of carbon cycling in forest ecosystems, knowledge concerning their distribution patterns and underlying mechanisms remains insufficient. Here, soils were sampled from six bamboo forests across the main planting area of Moso bamboo in southern China. The bacterial and fungal diversities were assessed by sequencing 16S rRNA and ITS gene amplicons, respectively, with an Illumina MiSeq. Based on structural equation modelling, dispersal limitation had strongest impact on bacterial beta diversity, while the mean annual precipitation had a smaller impact by directly or indirectly mediating the soil organic carbon density. However, only the mean annual temperature and precipitation played direct roles in fungal beta diversity. Moreover, the co‐occurrence network analyses revealed a possibly much higher network connectivity in the fungal network than in the bacteria. With less dispersal limitation, stronger environmental selection and a potentially more connected network, the fungal community had more important roles in the soil carbon metabolisms in bamboo forests. Fungal beta diversity and the clustering coefficient explained approximately 14.4% and 6.1% of the variation in the carbon metabolic profiles among sites, respectively, but that of bacteria only explained approximately 1.7% and 1.8%, respectively. This study explored soil microbial spatial patterns along with the underlying mechanisms of dispersal limitation, selection and connectivity of ecological networks, thus providing novel insights into the study of the distinct functional traits of different microbial taxa.     

Simple, sensitive, and quick protocol to detect less than 1 ng of bacterial lipopolysaccharide

Detection and quantitation of lipopolysaccharide (LPS) are important for quality assurance of pharmaceutical, biopharmaceutical products and for several research and industrial aspects. The widely available assays for LPS detection are the normal, or chromogenic, and the synthetic LAL (Limulus amebocyte lysate). Unfortunately, both assays are expensive and could not distinguish between different types of bacterial LPS, while LPS silver nitrate staining requires more than 20 hr and can only detect 5 ng LPS. The current modified protocol was able to detect less than 0.5 ng LPS in a polyacrylamide-urea gel. The procedure is rapid, inexpensive, and reproducible. It depends on introducing two modifications to improve the staining sensitivity in sample buffer and staining procedure. The results revealed that excluding the reducing agent sucrose to use instead glycerol, and replacing SDS with DOC, as well as incorporation of 4 M urea in stacking and separating gels, increased the sensitivity up to 150 pg. In summary, the gels were fixed, carbohydrates moieties were oxidized to create active aldehydes, and the gels were silver stained using non-ammoniacal silver nitrate.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.     

Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA−) is less than that of its isogenic strain L33 (RecA+) but comparable to that of two S. meliloti wild-type isolates

Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA⁻) and L33 (RecA⁺) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA⁺ strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA⁻ strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release. Received: 4 November 1996 / Received revision: 9 January 1997 / Accepted: 17 January 1997     

Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides

The innate immunity factor lactoferrin harbours two antimicrobial moieties, lactoferricin and lactoferrampin, situated in close proximity in the N1 domain of the molecule. Most likely they cooperate in many of the beneficial activities of lactoferrin. To investigate whether chimerization of both peptides forms a functional unit we designed a chimerical structure containing lactoferricin amino acids 17-30 and lactoferrampin amino acids 265-284. The bactericidal activity of this LFchimera was found to be drastically stronger than that of the constituent peptides, as was demonstrated by the need for lower dose, shorter incubation time and less ionic strength dependency. Likewise, strongly enhanced interaction with negatively charged model membranes was found for the LFchimera relative to the constituent peptides. Thus, chimerization of the two antimicrobial peptides resembling their structural orientation in the native molecule strikingly improves their biological activity.     

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND-FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)–FD complex and the flower meristem identity gene, LEAFY ( LFY ). Two redundant functioning homeobox genes, PENNYWISE ( PNY ) and POUND-FOOLISH ( PNF ), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT . Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy , a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.     

Physical anomalies and developmental delays in nonhuman primate infants exposed to weekly doses of ethanol during gestation

Ethanol was orally administered once per week to 54 gravid pigtailed macaques (Macaca nemestrina) in doses of 0.0, 0.3, 0.6, 1.2, 1.8, 2.5 or 4.1 gm/kg from the 1st week in gestation or in doses of 2.5, 3.3, or 4.1 gm/kg from the 5th week. Mean maternal mean peak plasma ethanol concentrations (MPPEC) ranged from 24 +/- 6 mg/dl at the 0.3 gm/kg dose to 549 +/- 71 mg/dl at the 4.1 gm/kg dose. Thirty-three viable infants were followed from birth to 6 months of age and assessed for growth, health, congenital anomalies and developmental rate. Facial anomalies, growth deficiency, or central nervous system dysfunction were found in 57% of the alcohol-exposed animals. No animal showed all the features of the human fetal alcohol syndrome. Ten of the twelve animals (83%) with mean MPPEC above 140 mg/dl had evidence of a teratogenic impact. The animals with full gestational exposure to ethanol and mean MPPEC between 140 and 249 mg/dl had much more severe and consistent cognitive abnormalities than the animals with delayed gestational exposures, even though the latter were exposed to mean MPPEC between 260 and 540 mg/dl. Conclusions from this study included: 1) ethanol-related behavioral teratogenesis occurred without accompanying physical anomalies, 2) measurable teratogenic effects from weekly exposures occurred only at intoxicating doses of ethanol, and 3) early gestational exposure to ethanol appeared to be more damaging to cognitive function than later and considerably greater alcohol exposure.     

Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.     

Comparison of DNA damage responses following equimutagenic doses of UVA and UVB: a less effective cell cycle arrest with UVA may render UVA-induced pyrimidine dimers more mutagenic than UVB-induced ones

Mechanisms of UVA-mutagenesis remain a matter of debate. Earlier described higher rates of mutation formation per pyrimidine dimer with UVA than with UVB and other evidence suggested that a non-pyrimidine dimer-type of DNA damage contributes more to UVA- than to UVB-mutagenesis. However, more recently published data on the spectra of UVA-induced mutations in primary human skin cells and in mice suggest that pyrimidine dimers are the most common type of DNA damage-inducing mutations not only with UVB, but also with UVA. As this rebuts a prominent role of non-dimer type of DNA damage in UVA-mutagenesis, we hypothesized that the higher mutation rate at UVA-induced pyrimidine dimers, as compared to UVB-induced ones, is caused by differences in the way UVA- and UVB-exposed cells process DNA damage. Therefore, we here compared cell cycle regulation, DNA repair, and apoptosis in primary human fibroblasts following UVB- and UVA-irradiation, using the same physiologic and roughly equimutagenic doses (100-300 J m(-2) UVB, 100-300 kJ m(-2) UVA) we have used previously for mutagenesis experiments with the same type of cells. ELISAs for the detection of pyrimidine dimers confirmed that much fewer dimers were formed with these doses of UVA, as compared to UVB. We found that cell cycle arrests (intra-S, G1/S, G2/M), mediated at least in part by activation of p53 and p95, are much more prominent and long-lasting with UVB than with UVA. In contrast, no prominent differences were found between UVA and UVB for other anti-mutagenic cellular responses (DNA repair, apoptosis). Our data suggest that less effective anti-mutagenic cellular responses, in particular different and shorter-lived cell cycle arrests, render pyrimidine dimers induced by UVA more mutagenic than pyrimidine dimers induced by UVB.     

Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein

The Coomassie brilliant blue G assay for proteins described by Bradford (1976) (Anal. Biochem.72, 248) was reexamined. It was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 μg/ml of solution. This unchanging extinction coefficient, $\text{A}{}_{595}\text{=}\text{0.60 ± 0.01}\text{10}\text{μ}\text{g}$ of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 μg/ml permits the application of Beer's Law for accurate determinations of ≤0.5 to 50 μg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the A₅₉₅ of the dye-reagent.