Parallel reverse genetic screening in mutant human cells using transcriptomics

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.     

Cotton roots and vitamin-requiring and amino acid-requiring bacteria

Summary The rhizosphere effects of diploid and amphidiploid strains of cotton in black cotton soils have been investigated with reference to vitamin-requiring bacteria and amino acid-requiring bacteria. Influence of cotton roots on amino acid-requiring bacteria was found to be greater than on vitamin-requiring bacteria. The rhizosphere effects of diploid strains were greater than those of amphidiploid strains. The effects were also influenced by the initial proportions of these bacterial groups in the two soils. The rhizosphere effects of inoculated plants were lower than the respective healthy controls; but those of apparently healthy (disease escapes) diploid plants and those of inoculated amphidiploid plants in PLD soil (wilt-sick) were strikingly higher than the healthy controls. Differences in the rhizosphere effects between diploid and amphidiploid plants could be correlated with the amino acid and vitamin exudations by roots of these strains, but not with reference to soil types.Part of Doctoral Thesis, University of Madras.     

Root surface fungi of Crotalaria

Summary An agar-slide technique which provides scope for development up to sporulation stage of fungi occurring on root surfaces, aiding better evaluation of root surface mycoflora is described. As, by this method, it is possible to observe the pattern of attachment of spores to sporophores, identification is facilitated.Results of a study of the root surface fungi ofCrotolaria juncea L. seedlings healthy and DEMV infected from the day after sowing till the infected plants withered away, for different root lengths from the base of the stem, employing this technique are reported here and discussed.The following genera of fungi, seldom observed on soil dilution plates in a parallel study, were recorded in this study: Acremonium, Cephalotrichum, Cunninghamella, Drechslera, Fusarium, Memnoniella, Pullularia and some phycomycetes.Memoir No.40 from the Centre for Advanced Studies in Botany, University Botany Laboratory, Madras-5, India.     

Respiratory and calcium-accumulating properties of muscle mitochondria of some aquatic arthropods

Mitochondria were isolated from the muscles ofStreptocephalus dichotomus, Caridina nilotica, Paratelphusa hydrodromous, Sphaerodema rusticum, andEnithares rogersi, all of which belong to the phylum arthropoda, and their properties studied. All mitochondria showed qualitatively similar respiratory and Ca^2* uptake capacities but there was much quantitative variation. There emerges an apparent correlation between locomotor activity of the organism and the respiratory activity of the mitochondria. Similar correlation is found between the calcium need of the animal and the Ca^2* uptake capacity of the mitochondria. The results also suggest an inverse correlation between respiration and Ca^2* uptake.     

Decision-making tools for Frankliniella occidentalis management in strawberry: consideration of target markets

The western flower thrips (WFT), Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), a cosmopolitan pest of many crops, is considered a major pest of low tunnel and greenhouse strawberries. The extent of damage to strawberry is unclear because different studies have produced contradictory results. Also, economic thresholds published for WFT in strawberry vary greatly, and most fail to incorporate economic factors. This study was aimed at developing a decision‐making tool for WFT management in strawberries in Israel. Toward this end, economic injury levels (EIL) and economic thresholds were calculated, based on target markets (export vs. domestic). Results indicate that serious infestation of ripe berries may cause a dull, rough appearance, and the fruit may be soft and have a reduced shelf life, rendering it unsuitable for export. Most fruit damage occurred at green and turning‐red stages of development. Two decision‐making tools were developed, one for winter, when WFT populations increase slowly but crop value is high (export market); and the second for spring, when the pest increases rapidly but crop value is low (local markets). Economic thresholds of 10 and 24 WFT/flower were calculated for winter and spring strawberries, respectively, based on direct thrips damage to fruit. This calculation does not take into account the recorded WFT damage to flowers, or its role in facilitating Botrytis cinerea fruit infection. Western flower thrips has proved only an occasional economic pest in Israeli strawberries, and no routine control measures are warranted. Furthermore, augmentative releases of Orius laevigatus or Neoseilus cucumeris against WFT are not justified in this system, because Orius colonizes strawberry fields spontaneously in high numbers when no broad spectrum insecticides are used.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.