A near-deterministic mutational hotspot in Pseudomonas fluorescens is constructed by multiple interacting genomic features.

Mutation - whilst stochastic - is frequently biased toward certain loci. When combined with selection this results in highly repeatable and predictable evolutionary outcomes. Immotile variants of the bacterium Pseudomonas fluorescens (SBW25) possess a 'mutational hotspot' that facilitates repeated occurrences of an identical de novo single nucleotide polymorphism when re-evolving motility, where ≥95% independent lines fix the mutation ntrB A289C. Identifying hotspots of similar potency in other genes and genomic backgrounds would prove valuable for predictive evolutionary models, but to do so we must understand the genomic features that enable such a hotspot to form. Here we reveal that genomic location, local nucleotide sequence, gene strandedness and presence of mismatch repair proteins operate in combination to facilitate the formation of this mutational hotspot. Our study therefore provides a framework for utilising genomic features to predict and identify hotspot positions capable of enforcing near-deterministic evolution.

The social evolution of dispersal with public goods cooperation.

Selection can favour the evolution of individually costly dispersal if this alleviates competition between relatives. However, conditions that favour altruistic dispersal also mediate selection for other social behaviours, such as public goods cooperation, which in turn is likely to mediate dispersal evolution. Here, we investigate - both experimentally (using bacteria) and theoretically - how social habitat heterogeneity (i.e. the distribution of public goods cooperators and cheats) affects the evolution of dispersal. In addition to recovering the well-known theoretical result that the optimal level of dispersal increases with genetic relatedness of patch mates, we find both mathematically and experimentally that dispersal is always favoured when average patch occupancy is low, but when average patch occupancy is high, the presence of public goods cheats greatly alters selection for dispersal. Specifically, when public goods cheats are localized to the home patch, higher dispersal rates are favoured, but when cheats are present throughout available patches, lower dispersal rates are favoured. These results highlight the importance of other social traits in driving dispersal evolution.

Bacterial motility confers fitness advantage in the presence of phages.

Dispersal provides the opportunity to escape harm and colonize new patches, enabling populations to expand and persist. However, the benefits of dispersal associated with escaping harm will be dependent on the structure of the environment and the likelihood of escape. Here, we empirically investigate how the spatial distribution of a parasite influences the evolution of host dispersal. Bacteriophages are a strong and common threat for bacteria in natural environments and offer a good system with which to explore parasite-mediated selection on host dispersal. We used two transposon mutants of the opportunistic bacteria, Pseudomonas aeruginosa, which varied in their motility (a disperser and a nondisperser), and the lytic bacteriophage ФKZ. The phage was distributed either in the central point of colony inoculation only, thus offering an escape route for the dispersing bacteria; or, present throughout the agar, where benefits of dispersal might be lost. Surprisingly, we found dispersal to be equally advantageous under both phage conditions relative to when phages were absent. A general explanation is that dispersal decreased the spatial structuring of host population, reducing opportunities for parasite transmission, but other more idiosyncratic mechanisms may also have contributed. This study highlights the crucial role the parasites can play on the evolution of dispersal and, more specifically, that bacteriophages, which are ubiquitous, are likely to select for bacterial motility.

High-quality RNA from cells isolated by laser capture microdissection.

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.

Effectiveness of a two-dose regimen of prostaglandin administration in inducing luteolysis without adverse side effects in mares.

Our objectives were to determine whether repeated administration of prostaglandin F2alpha (PGF2alpha) to simulate the endogenous mode of secretion would be more effective than a single injection in inducing luteolysis and enable use of smaller doses less likely to cause adverse side effects. The main study comprised 43 dioestrous mares, who were given im. either a single 10 mg dose of natural PGF2alpha (n = 22) or 2 doses of 0.5 mg PGF2, 24 h apart (n = 21). The intensity of side effects was assessed in 8 dioestrous mares given 5, 1.5, 0.5 or 0 mg PGF2alpha in consecutive cycles. Two doses of 0.5 mg PGF2alpha 24 h apart caused lysis of the corpus luteum in all mares, whether this was determined from a fall in plasma progesterone concentrations or reproductive tract/behavioural changes; and when 10 mg PGF2, was given, the corpus luteum was lysed in 17 of 22 mares i.e. a lower proportion (P = 0.0485). A single dose of 0.5 mg PGF2a was no more effective than saline in inducing luteolysis.The intensity of side effects of PGF2alpha increased with dose. Although the 0.5 mg dose was no more likely than saline to cause sweating or muscle spasms, it raised plasma cortisol concentrations and prevented the decline in heart rate seen after saline. We conclude that a 2 dose regimen of administration increases the luteolytic efficacy of PGF2alpha and thereby provides a way to minimise adverse side effects.

Threats to the health care safety net.

UNLABELLED: The American health care safety net is threatened due to inadequate funding in the face of increasing demand for services by virtually every segment of our society. The safety net is vital to public safety because it is the sole provider for first-line emergency care, as well as for routine health care of last resort, through hospital emergency departments (ED), emergency medical services providers (EMS), and public/free clinics. Despite the perceived complexity, the causes and solutions for the current crisis reside in simple economics. During the last two decades health care funding has radically changed, yet the fundamental infrastructure of the safety net has change little. In 1986, the Emergency Medical Treatment and Active Labor Act established federally mandated safety net care that inadvertently encouraged reliance on hospital EDs as the principal safety net resource. At the same time, decreasing health care funding from both private and public sources resulted in declining availability of services necessary to support this shift in demand, including hospital inpatient beds, EDs, EMS providers, on-call specialists, hospital-based nurses, and public hospitals/clinics. The result has been ED/hospital crowding and resource shortages that at times limit the ability to provide even true emergency care and threaten the ability of the traditional safety net to protect public health and safety. This paper explores the composition of the American health care safety net, the root causes for its disintegration, and offers short- and long-term solutions. The solutions discussed include restructuring of disproportionate share funding; presumed (deemed) eligibility for Medicaid eligibility; restructuring of funding for emergency care; health care for foreign nationals; the nursing shortage; utilization of a "health care resources commission"; "episodic (periodic)" health care coverage; best practices and health care services coordination; and government and hospital providers' roles. CONCLUSIONS: There is a base amount of funding that must be available to the American health care safety net to maintain its infrastructure and provide appropriate growth, research, development, and expansion of services. Fall below this level and the infrastructure will eventually crumble. America must patch the safety net with short-term funding and repair it with long-term health care policy and environmental changes.

Linear IgA bullous dermatosis responsive to a gluten-free diet.

Dermatitis herpetiformis is associated with a gluten-sensitive enteropathy in >85% of cases. Both the skin lesions and the enteropathy respond to gluten restriction. Linear IgA bullous dermatosis has a much lower prevalence of histological small bowel abnormalities, and lesions are not known to respond to gluten restriction. We report a patient with linear IgA bullous dermatosis and gluten-sensitive enteropathy. This report addresses the issue of whether linear IgA bullous dermatosis can be associated with gluten-sensitive enteropathy. We evaluated the response to gluten restriction and normal diet by following the status of the patient's jejunal biopsies and skin lesions. The patient responded to gluten restriction, as shown by resolution of jejunal abnormalities and skin lesions and subsequently by recurrence of jejunal abnormalities and skin lesions with reinstitution of a gluten-containing diet. This report demonstrates that linear IgA bullous dermatosis can respond to gluten restriction if an underlying gluten-sensitive enteropathy is present.

IgA1 is the major IgA subclass in cutaneous blood vessels in Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is characterized by palpable purpura predominantly involving the lower extremities. On direct immunofluorescence IgA can be seen deposited in the blood vessel walls of the superficial dermis. The subclass distribution of antibodies to this IgA was studied in the biopsies of 28 patients with HSP by direct immunofluorescence using anti-IgA1 and anti-IgA2 specific monoclonal antibodies. All 28 patients' biopsies demonstrated deposition of IgA1 while only one patient had IgA2 deposition. Positive and negative controls stained appropriately. This demonstrates that IgA1 is the dominant IgA subclass found in the skin in Henoch-Schönlein purpura.

IgA antibodies recognizing LABD97 are predominantly IgA1 subclass.

Linear IgA bullous dermatosis is a rare acquired subepidermal blistering disease of the skin. A recognized antigen in linear IgA bullous dermatosis is a 97-kDa basement membrane zone protein termed LABD97. Previous studies, using immunofluorescent techniques, have suggested that the IgA response is restricted to the IgA1 subclass. We studied the IgA antibody subclasses in the sera of 6 patients that contained circulating IgA antibodies reactive with LABD97. The methods used included direct and indirect immunofluorescence and Western immunoblot. All patients tested had IgA1 anti-LABD97 antibodies detected by all 3 methods. Two patients had IgA2 antibodies detected by direct immunofluorescence. Three patients had IgA2 antibodies on indirect immunofluorescence. Two of these also had anti-LABD97 IgA2 antibodies and 1 had secretory component containing anti-LABD IgA antibodies on Western immunoblot. We conclude that the predominant IgA antibody subclass reactive with LABD97 in LABD is IgA1, although the IgA2 subclass may be involved in some cases.

The immunoglobulin A antibody response in clinical subsets of mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is an immunobullous disease. In MMP there is frequently a mixed antibody response with the presence of IgA and/or IgG antibodies directed toward basement membrane zone antigens. The IgG antibody response in MMP has been studied, but the antigens to which the IgA antibodies react have not been studied. OBJECTIVE: To determine the IgA autoantibody reactivity profiles in patients with MMP. METHODS: Patients who had both ocular and oral MMP were compared with patients who had ocular or oral MMP and with patients who had cutaneous linear IgA disease (LABD) by Western immunoblot studies. RESULTS: Five of 15 MMP patients and 1 of 5 LABD patients had IgA antibodies reactive with the 180-kD bullous pemphigoid antigen. Seven of 15 MMP patients had IgA antibodies reactive with the 97-kD LABD antigen. CONCLUSION: Major antigens in IgA MMP are the 180-kD bullous pemphigoid antigen and the 97-kD LABD antigen.

Characterization of the antibody response in oesophageal cicatricial pemphigoid.

Cicatricial pemphigoid (CP) is a subepidermal, autoimmune bullous dermatosis. It is classified as a clinical subset of bullous pemphigoid (BP). However, it differs from BP in some significant ways: (i) in CP mucosal involvement with clinical scarring is prominent; (ii) there is a prominent IgA class antibody response alone or in addition to the IgG class antibody response; and (iii) there is a heterogeneous antibody response in CP, whereas in BP the majority of the antibodies are directed against a 180-kDa hemidesmosomal protein, bullous pemphigoid antigen 2 (BPAg2). Oesophageal involvement in CP is a rare, but often devastating manifestation. In this study we examined the humoral autoimmune response in oesophageal CP, in an attempt to characterize the autoantibody reactivity profile. We used direct and indirect immunofluorescence and Western immunoblotting using normal human skin and oesophagus substrates. We studied patient sera over time in order to search for evidence of epitope spreading in these patients. All patients had positive direct immunofluorescence of perilesional oesophageal epithelium. All patients had positive circulating antibasement membrane zone autoantibody titres. There was a significant IgA class in addition to an IgG class autoantibody response. IgA and IgG antibodies demonstrated significant reactivity with BPAg2 and the 97 kDa linear IgA disease antigen on Western immunoblot suggesting intraprotein epitope spreading. There was no evidence of interprotein epitope spreading over time. Our findings suggest that there is a heterogeneous antibody response in oesophageal CP with the predominant antigen being BPAg2.

Bullous pemphigoid sera that contain antibodies to BPAg2 also contain antibodies to LABD97 that recognize epitopes distal to the NC16A domain.

IgG antibodies from the sera of some patients with bullous pemphigoid (BP) react with a 180 kDa protein termed BPAg2. Antibodies in BP are directed to an extracellular noncollagenous domain of this protein termed NC16A. Our group has recently shown that a portion of the extracellular domain of BPAg2 is identical to LABD97 on the basis of amino acid sequencing. We evaluated sera from 33 patients with BP with circulating IgG antibodies on indirect immunofluorescence, which stained the epidermal side of split skin with titers ranging from 1:40 to 1:640. Immunoblotting was performed against (i) two preparations of proteins from epidermal extract, one containing BPAg2 and one containing LABD97, and (ii) the recombinant NC16A domain of the BPAg2 protein. Twelve sera reacted with the BPAg2 protein. Ten of these also reacted strongly with the NC16A domain. Nine of the 12 sera also reacted with the LABD97 antigen. Bound antibodies were eluted from the 97 kDa band and reapplied to split skin where they bound to the epidermal side. The eluted antibodies also reacted to the BPAg2 protein from the epidermal extract, but did not react with the NC16A domain on immunoblot. We conclude that these nine sera react with an epitope present within BPAg2 and LABD97 but not within the NC16A domain. This epitope is therefore distal to the previously described epitopes in BP. In BP, epitope spreading may occur and antibodies may be produced that recognize the distal portion of the BPAg2 molecule identical to LABD97 but that do not involve the NC16A domain.

The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2.

IgA autoantibodies from the sera of some patients with linear IgA bullous dermatosis (LABD) recognize a 97 kDa antigen (LABD97) located in the lamina lucida of the basement membrane zone. As LABD autoantibodies do not react with the 180 and 230 kDa proteins recognized by bullous pemphigoid autoantibodies, LABD97 has been thought to represent a separate lamina lucida protein. In this study, we purified LABD97 from the extract of human epidermis using a monoclonal antibody immunoaffinity column and analyzed the amino acid sequence of the N terminus of purified LABD97. This revealed a 16 amino acid sequence that was identical to a previously reported sequence of the 180 kDa antigen in bullous pemphigoid (BPAg2). The N terminus was located 41 amino acids downstream from the carboxyl end of the transmembrane domain of BPAg2 and 11 amino acids downstream from the MCW-1 domain, the predominant bullous pemphigoid epitope. Purified LABD97 was subsequently enzymatically digested with endoproteinase Arg C and separated by chromatography, which resulted in multiple peptide fractions. Fourteen of these fractions were subjected to amino acid sequencing. The amino acid sequence of the peptide fractions, totaling 205 amino acids, were identical to sequences contained within the extracellular domain of BPAg2. Whereas the predominant epitope identified with bullous pemphigoid sera is located in the noncollagenous region of this protein, the epitope recognized by LABD sera is either within or adjacent to the collagenous portion. We conclude that LABD97 represents a portion of the extracellular domain of BPAg2 and that the IgA autoantibodies are directed against an epitope within or adjacent to a collagenous domain.

Optimization of the performance of the polymerase chain reaction in silicon-based microstructures.

We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.

Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media.

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics.

IgA antibodies in chronic bullous disease of childhood react with 97 kDa basement membrane zone protein.

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease occurring in prepubertal children. Both CBDC and its adult counter-part, linear IgA bullous dermatosis (LABD), are characterized by linear deposition of IgA along the cutaneous basement membrane zone (BMZ). Circulating IgA antibody in LABD has been found to bind to a 97-kDa BMZ antigen, whereas the antigen in CBDC has not been well characterized. The purpose of this study was to evaluate the immunoreactivity of BMZ IgA antibodies in a series of CBDC patients. We evaluated 12 sera from patients with CBDC with circulating IgA anti-BMZ antibodies on indirect immunofluorescence (IIF), which stained the epidermal side of split skin with titers ranging from 1:20 to 1:640. Immunoblotting was performed against two preparations of BMZ proteins: one enriched with the two bullous pemphigoid antigens (BP230, BP180) and one enriched with the LABD antigen (LABD97). Eight of the twelve sera reacted with a 97-kDa protein that co-migrated with the protein detected in many LABD sera. The intensity of the reaction on immunoblot correlated with serum antibody titers. There was no consistent pattern of reactivity of the IgA anti-BMZ antibodies with either the BP230 or BP180 antigens, although two sera reacted with several higher molecular mass proteins (160-200 kDa). The significance of this reactivity was examined with immunoblotting using BMZ-affinity-purified antibodies, and ELF using nitrocellulose-eluted antibodies. One serum also contained anti-BMZ IgA antibodies that reacted with a 180-kDa protein, corresponding to BP180. We conclude that IgA antibodies in CBDC sera recognize a 97-kDa BMZ antigen present on the epidermal side of BMZ split skin that co-migrates with the antigen previously identified in LABD. These findings suggest that CBDC and LABD are the immunologically related disorders occurring in different age groups.

LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells.

This study characterizes a novel basement membrane component that is the target of autoantibodies in patients with linear IgA bullous dermatosis. Tissue surveys showed that this protein localized to the epidermal side of 1 M NaCl split skin and to basement membranes in cornea, oral mucosa, esophagus, intestine, kidney collecting ducts, ureter, bladder, urethra, and thymus, but was absent in lung, blood vessels, skeletal muscle, and nerve. Monoclonal antibody 123, which recognizes this protein, induced dermal-epidermal separation of human skin in situ, and this protein was found, by immunoelectron microscopy, to localize exclusively to anchoring filaments. This protein was secreted as as a 120-kDa peptide from primary cultures of keratinocytes as determined by radioimmunoprecipitation. Monoclonal antibody 123 recognized this protein as a 120-kDa band from conditioned cell culture medium and a 97-kDa band from human skin extracts as shown by immunoblot. Serum from five patients with the autoimmune blistering disorder linear IgA bullous dermatosis specifically recognized bands of 120 and 97 kDa from culture medium and skin extracts, respectively, that were of identical electrophoretic migration to the bands recognized by monoclonal antibody 123. In summary, this study characterizes a novel anchoring filament protein that is the target of linear IgA bullous dermatosis autoantibodies. Because monoclonal antibody 123 induces blistering of human skin, we hypothesize that this protein functions to maintain dermal-epidermal cohesion and that autoantibodies in this disease are themselves pathogenic. We propose LAD-1 as the name for this protein.