SAMFIRE: multi-locus variant calling for time-resolved sequence data.

UNLABELLED: An increasingly common method for studying evolution is the collection of time-resolved short-read sequence data. Such datasets allow for the direct observation of rapid evolutionary processes, as might occur in natural microbial populations and in evolutionary experiments. In many circumstances, evolutionary pressure acting upon single variants can cause genomic changes at multiple nearby loci. SAMFIRE is an open-access software package for processing and analyzing sequence reads from time-resolved data, calling important single- and multi-locus variants over time, identifying alleles potentially affected by selection, calculating linkage disequilibrium statistics, performing haplotype reconstruction and exploiting time-resolved information to estimate the extent of uncertainty in reported genomic data. AVAILABILITY AND IMPLEMENTATION: C ++ code may be found at https://github.com/cjri/samfire/ CONTACT: chris.illingworth@gen.cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

Changes in mental state associated with prison environments: a systematic review.

OBJECTIVE: To develop an understanding of the stability of mental health during imprisonment through review of existing research evidence relating physical prison environment to mental state changes in prisoners. METHOD: A systematic literature search was conducted looking at changes in mental state and how this related to various aspects of imprisonment and the prison environment. RESULTS: Fifteen longitudinal studies were found, and from these, three broad themes were delineated: being imprisoned and aspects of the prison regime; stage of imprisonment and duration of sentence; and social density. Reception into prison results in higher levels of psychiatric symptoms that seem to improve over time; otherwise, duration of imprisonment appears to have no significant impact on mental health. Regardless of social density, larger prisons are associated with poorer mental state, as are extremes of social density. CONCLUSION: There are large gaps in the literature relating prison environments to changes in mental state; in particular, high-quality longitudinal studies are needed. Existing research suggests that although entry to prison may be associated with deterioration in mental state, it tends to improve with time. Furthermore, overcrowding, ever more likely as prison populations rise, is likely to place a particular burden on mental health services.

Computational studies of Family A and Family B GPCRs.

A full picture of the similarities between Family A and Family B GPCRs (G-protein coupled receptors) has been frustrated by the lack of clear homology between the respective sequences. Here, we review previous computational studies on GPCR dimerization in which the putative dimerization interfaces have been analysed using entropy, the ET (evolutionary trace) method and related methods. The results derived from multiple sequence alignments of Family A subfamilies have been mapped on to the rhodopsin crystal structure using standard alignments. Similarly, the results for the Family B alignments have been mapped on to the rhodopsin crystal structure using the 'cold-spot' alignment. For both Family A and Family B GPCRs, the sequence analysis indicates that there are functional sites on essentially all transmembrane helices, consistent with the parallel daisy chain model of GPCR oligomerization in which each GPCR makes interactions with a number of neighbouring GPCRs. The results are not too sensitive to the quality of the alignment. Molecular Dynamics simulations of the activation process within a single transmembrane bundle of the rhodopsin and the beta(2)-adrenergic receptor have been reviewed; the key observation, which is consistent with other computational studies, is that there is a translation and bending of helix 6, which contributes to a significant opening out of the intracellular face of the receptor, as shown in the accompanying movies. The simulations required the application of specific experiment-derived harmonic and half-harmonic distance restraints and so the application of such simulations to Family B GPCRs requires considerable care because of the alignment problem. Thus, in order to address the alignment problem, we have exploited the observation that GCR1, a plant GPCR, has homology with Family A, Family B and Family E GPCRs. The resulting alignment for transmembrane helix 3 is presented.

Translational regulation of the plant S-adenosylmethionine decarboxylase.

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.

Gene conversion within regulatory sequences generates maize r alleles with altered gene expression.

The maize r locus encodes a transcription factor that regulates the developmental expression of the plant pigment anthocyanin. In an unusual example of gene regulatory diversity, the R-sc (Sc, strong seed color) and the R-p (P, plant color) alleles of r have nonoverlapping tissue specificity and nonhomologous 5' flanking sequences. Heterozygotes between wild-type P and Sc mutants with Ds6 transposable element inserts (r-sc:m::Ds6 or sc:m) produce colored seed derivatives (Sc+) during meiotic recombination. The sc:m alleles with Ds6 insertion in 3' regions of r produce crossover Sc+ derivatives. sc:m alleles with Ds6 elements inserted in 5' regions produce rare Sc+ derivatives borne on nonrecombinant chromosomes. Among 52 such noncrossover Sc+ derivatives, 18 are indistinguishable from the Sc progenitor in phenotype and DNA sequence [Scp(+) alleles]. The remaining 34 derivatives have strong Sc+ expression, including darkly pigmented aleurone, scutellum, coleoptile, and scutellar node [Scp(e) alleles]. The coleoptile and scutellar node phenotypes are unique from either progenitor but are similar to those of some naturally occurring r alleles. Both classes of Sc+ derivatives are explained by gene conversion between the promoter region of Sc:124 and a homologous region located proximal to P. The recombinational intermediate formed between sc:m alleles and P results in deletion of the Ds6 element alone or both Ds6 and a nearby unrelated transposable element-like sequence.

Corneal biopsy with tissue micro-homogenisation for isolation of organisms in bacterial keratitis.

PURPOSE: To evaluate a novel two-stage technique to increase yield of bacteria isolated from infected corneal ulcers. METHODS: A new blade was designed to remove friable material from infected corneal ulcers. The new blade was used in combination with standard tissue micro-homogenisation equipment in a two-stage technique intended to distribute biopsy samples evenly between relevant agar plates. Patients with presumed-bacterial corneal ulcers underwent sequential corneal sampling using the new two-stage technique and a scalpel blade, used without micro-homogenisation (the order of sampling was varied between two groups). Bacterial isolation rates were compared using the chi-squared test. RESULTS: Twenty-four patients with presumed-bacterial corneal ulcers were studied. The overall positive bacterial isolation rate was 88%, with identical bacterial isolation rates for the new two-stage technique and the scalpel blade (71%). The new technique isolated bacteria from three ulcers that had initially been 'sterile' when sampled with a scalpel blade. Polymicrobial infections were identified in two ulcers with the new blade where only a single organism had been identified using the scalpel blade (not significantly different). CONCLUSIONS: The new two-stage technique shows promise for improving bacterial isolation rates from presumed-bacterial corneal ulcers.

Acanthamoeba keratitis.

Acanthamoeba species are an important cause of microbial keratitis that may cause severe ocular inflammation and visual loss. The first cases were recognized in 1973, but the disease remained very rare until the 1980s, when an increase in incidence mainly associated with contact lens wear was reported. There is an increased risk when contact lens rinsing and soaking solutions are prepared with nonsterile water and salt tablets. The clinical picture is often characterized by severe pain with an early superficial keratitis that is often treated as herpes simplex infection. Subsequently a characteristic radial perineural infiltration may be seen, and ring infiltration is common. Limbitis and scleritis are frequent. Laboratory diagnosis is primarily by culture of epithelial samples inoculated onto agar plates spread with bacteria. Direct microscopy of samples using stains for the cyst wall or immunostaining may also be employed. A variety of topically applied therapeutic agents are thought to be effective, including propamidine isethionate, clotrimazole, polyhexamethylene biguanide, and chlorhexidine. Various combinations of these and other agents have been employed, often resulting in medical cure, especially if treatment is commenced early in the course of the disease. Penetrating keratoplasty is preferably avoided in inflamed eyes, but may be necessary in severe cases to preserve the globe or, when the infection has resolved, to restore corneal clarity for optical reasons.

Acanthamoeba keratitis: risk factors and outcome.

AIMS/BACKGROUND: This study was initiated to investigate risk factors for and outcome of Acanthamoeba keratitis. METHODS: Results of treatment were studied in 22 patients (23 eyes) presenting to Bristol Eye Hospital between 1985 and February 1995. Details related to the use and disinfection of contact lenses were also obtained. An additional two patients who were seen at Bristol but mainly treated elsewhere were surveyed for contact lens related information only. RESULTS: The incidence of Acanthamoeba keratitis rose substantially in the 1990s: three patients presented before 1990, while the remaining 21 presented between January 1990 and February 1995. Eleven patients have presented since january 1994. All of the patients in this series were contact lens wearers, 16 (67%) using daily wear disposable contact lenses. Contact lens disinfection data were available in 22 patients of whom 11 (50%) were using chlorine disinfectant. Other types of disinfection were much less common. Four patients (18%) had not used any disinfectant. During the course of the series the average diagnostic delay has fallen markedly, although in 77% of patients a diagnosis of a viral keratitis, most commonly herpes simplex, was made on first presentation. All but three of the series were treated with a combination of polyhexamethylene biguanide and propamidine isethionate. Penetrating keratoplasty was performed in 9/23 eyes (39%); in all of these eyes diagnosis was delayed for at least 6 weeks. All but one of the eyes in the series achieved a visual acuity of 6/9 or better after treatment, and 18 eyes (78%) saw 6/6 or better. CONCLUSIONS: Most patients with Acanthamoeba keratitis can now expect a good visual result and cure by medical therapy alone is favoured by early diagnosis.

Analysis of maize brittle-1 alleles and a defective Suppressor-mutator-induced mutable allele.

A mutant allele of the maize brittle-1 (bt1) locus, brittle-1-mutable (bt1-m), was shown genetically and molecularly to result from the insertion of a defective Suppressor-mutator (dSpm) transposable element. An Spm-hybridizing restriction enzyme fragment, which cosegregates with the bt1-m allele and is absent from wild-type revertants of bt1-m, was identified and cloned. Non-Spm portions of it were used as probes to identify wild-type (Bt1) cDNAs in an endosperm library. The 4.3-kb bt1-m genomic clone contains a 3.3-kb dSpm, which is inserted in an exon and is composed of Spm termini flanking non-Spm sequences. RNA gel blot analyses, using a cloned Bt1 cDNA probe, indicated that Bt1 mRNA is present in the endosperm of developing kernels and is absent from embryo or leaf tissues. Several transcripts are produced by bt1-m. The deduced translation product from a 1.7-kb Bt1 cDNA clone has an apparent plastid transit peptide at its amino terminus and sequence similarity to several mitochondrial inner-envelope translocator proteins, suggesting a possible role in amyloplast membrane transport.

Gripe water: what is it? Why is it given?

Large numbers of babies are given gripe water for no valid reason or for only trivial symptoms, write Cynthia Illingworth and John Timmins. When a health visitor survey of Sheffield mothers and babies showed how widespread was the practice of giving gripe water, they decided to seek information about the nature of gripe water, the conditions which it is supposed to treat and its use by a representative number of women.

The emotional state of mothers in the first three months after the birth of their baby.

A questionnaire was sent to the 98 health visitors who had taken part in the postnatal questionnaire study, asking them for comments on its difficulties and benefits. In their answers they referred to their staffing difficulties, the mothers' language problems, doubt about the truthfulness and accuracy of the mothers' replies to their questionnaire, and to difficulties experienced in liaison with family doctors, hospital psychiatric units, social workers and other social services. Of the 69 health visitors who replied to the questionnaire, 48 (70 per cent) thought that the study had been helpful, 14 had mixed feelings and six replied in the negative. There was little doubt that the postnatal questionnaire study had greatly increased the health visitors' understanding of maternal emotional problems and depression, and the difficulties of helping the mothers. It helped the mothers to understand more of their own feelings, and to discuss them with the health visitors in order to seek advice.

BMX compared with ordinary bicycle accidents.

Three hundred new cases of bicycle accidents were seen in the accident and emergency department in 60 consecutive days. Fifty six per cent were related to ordinary cycles and 44% to BMX cycles. Significantly more children on BMX cycles were boys (94% v 76% on ordinary cycles). Those on BMX cycles were somewhat older and more had had previous accidents. By means of a proforma we investigated the nature and causes of the accidents, recorded the type of injury, and compared accidents on the two groups of bicycles. Forty children had fractures and the incidence on BMX machines was almost twice that on conventional bicycles as were serious injuries and admissions to hospital. Twenty one children had concussion, 18 broke teeth, 53 fell head first over handlebars, and 131 had injuries above the neck. Significantly more children on ordinary cycles (53%) had injuries above the neck than those on BMX cycles (31%). Difficulties and methods of preventing the increasing number of cycle accidents are discussed.

Injuries to children riding BMX bikes.

One hundred children presented over 40 days with BMX bike injuries, 40 of which had been sustained while trying to perform stunts. Injuries in this series were compared with previously reported injuries from accidents on ordinary bicycles. BMX bike injuries differed little from ordinary bike injuries except in the greater proportion of injuries due to stunts and the smaller incidence of head injuries.