Recent structural advances towards understanding of the bacterial type III secretion injectisome.

The bacterial injectisome is a structurally conserved, syringe-shaped nanomachine that spans the Gram-negative envelope and forms a continuous channel for type III secretion of protein effectors. The injectisome, and the host-modulating effectors it secretes, are essential for the pathogenesis of several Gram-negative bacterial species, and it is a key virulence factor associated with the progression of many clinical and community-based infectious diseases. The molecular structure of the injectisome has been the focus of intense research efforts over the past 30 years, and during this time significant progress has been made in determining the molecular structures of many components. In this review we present major advances in our structural and mechanistic understanding of the injectisome, as facilitated by cryoelectron microscopy approaches.

Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins.

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an "open" conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

Cryo-EM analysis of the T3S injectisome reveals the structure of the needle and open secretin.

The bacterial type III secretion system, or injectisome, is a syringe shaped nanomachine essential for the virulence of many disease causing Gram-negative bacteria. At the core of the injectisome structure is the needle complex, a continuous channel formed by the highly oligomerized inner and outer membrane hollow rings and a polymerized helical needle filament which spans through and projects into the infected host cell. Here we present the near-atomic resolution structure of a needle complex from the prototypical Salmonella Typhimurium SPI-1 type III secretion system, with local masking protocols allowing for model building and refinement of the major membrane spanning components of the needle complex base in addition to an isolated needle filament. This work provides significant insight into injectisome structure and assembly and importantly captures the molecular basis for substrate induced gating in the giant outer membrane secretin portal family.

Structural and biochemical characterization of SpoIIIAF, a component of a sporulation-essential channel in Bacillus subtilis.

Environmental stress factors initiate the developmental process of sporulation in some Gram-positive bacteria including Bacillus subtilis. Upon sporulation initiation the bacterial cell undergoes a series of morphological transformations that result in the creation of a single dormant spore. Early in sporulation, an asymmetric cell division produces a larger mother cell and smaller forespore. Next, the mother cell septal membrane engulfs the forespore, and an essential channel, the so-called feeding-tube apparatus, is formed. This assembled channel is thought to form a transenvelope secretion complex that crosses both mother cell and forespore membranes. At least nine proteins are essential for channel formation including SpoIIQ under forespore control and the eight SpoIIIA proteins (SpoIIIAA-AH) under mother cell control. Several of these proteins share similarity with components of Gram-negative bacterial secretion systems and the flagellum. Here we report the X-ray crystallographic structure of the soluble domain of SpoIIIAF to 2.7 Šresolution. Like the channel components SpoIIIAG and SpoIIIAH, SpoIIIAF adopts a conserved ring-building motif (RBM) fold found in proteins from numerous dual membrane secretion systems of distinct function. The SpoIIIAF RBM fold contains two unique features: an extended N-terminal helix, associated with multimerization, and an insertion at a loop region that can adopt two distinct conformations. The ability of the same primary sequence to adopt different secondary structure conformations is associated with protein regulation, suggesting a dual structural and regulatory role for the SpoIIIAF RBM. We further analyzed potential interaction interfaces by structure-guided mutagenesis in vivo. Collectively, our data provide new insight into the possible roles of SpoIIIAF within the secretion-like apparatus during sporulation.

Structural characterization of SpoIIIAB sporulation-essential protein in Bacillus subtilis.

Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Šresolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.

Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.

The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.

In situ monitoring of 3D in vitro cell aggregation using an optical imaging system.

Bioreactor systems that maintain cells and tissues in suspension are increasingly popular for culturing 3D constructs to avoid the loss of in vivo cell function associated with traditional 2D culture methods. There is a need for the online monitoring of such systems to provide better understanding and control of the processes involved and to prevent the disruption of these processes caused by offline sampling and endpoint analysis. We describe a system for the imaging and analysis of cell aggregation, over long periods, within a high aspect rotating vessel (HARV). The system exploits side illumination, using an adjustable beam pattern, to restrict the detected light to that scattered by the cell aggregates, thus eliminating the need for the fluorescent labeling of the cells. The in situ aggregation of mammalian cells (MCF-7 breast carcinoma cells) was monitored over an 8 h period and image sequences showing the growth and motion of the aggregates within the bioreactor were obtained. Detailed size and population data have been derived characterizing the development of the aggregates during this time. We show how the number of resolvable aggregates increases to reach a peak and then declines as these aggregates merge. Once formed, remaining aggregates are found to consolidate to form more tightly packed bodies, typically reducing in cross-sectional area by one third. These results provide the basis for the development of an automated feedback system to control the growth of 3D cell cultures for repeatable, reliable, and quality controlled experimentation.

Cellular adaptations to hypoxia and acidosis during somatic evolution of breast cancer.

Conceptual models of carcinogenesis typically consist of an evolutionary sequence of heritable changes in genes controlling proliferation, apoptosis, and senescence. We propose that these steps are necessary but not sufficient to produce invasive breast cancer because intraductal tumour growth is also constrained by hypoxia and acidosis that develop as cells proliferate into the lumen and away from the underlying vessels. This requires evolution of glycolytic and acid-resistant phenotypes that, we hypothesise, is critical for emergence of invasive cancer. Mathematical models demonstrate severe hypoxia and acidosis in regions of intraductal tumours more than 100 microm from the basement membrane. Subsequent evolution of glycolytic and acid-resistant phenotypes leads to invasive proliferation. Multicellular spheroids recapitulating ductal carcinoma in situ (DCIS) microenvironmental conditions demonstrate upregulated glucose transporter 1 (GLUT1) as adaptation to hypoxia followed by growth into normoxic regions in qualitative agreement with model predictions. Clinical specimens of DCIS exhibit periluminal distribution of GLUT-1 and Na(+)/H(+) exchanger (NHE) indicating transcriptional activation by hypoxia and clusters of the same phenotype in the peripheral, presumably normoxic regions similar to the pattern predicted by the models and observed in spheroids. Upregulated GLUT-1 and NHE-1 were observed in microinvasive foci and adjacent intraductal cells. Adaptation to hypoxia and acidosis may represent key events in transition from in situ to invasive cancer.

An Internet training intervention for people with traumatic brain injury: barriers and outcomes.

PRIMARY OBJECTIVE: To test whether people with cognitive-linguistic impairments following traumatic brain injury could learn to use the Internet using specialized training materials. RESEARCH DESIGN: Pre-post test design. METHODS AND PROCEDURES: Seven participants were each matched with a volunteer tutor. Basic Internet skills were taught over six lessons using a tutor's manual and a student manual. Instructions used simple text and graphics based on Microsoft Internet Explorer 5.5. Students underwent Internet skills assessments and interviews pre- and post-training. Tutors completed a post-training questionnaire. MAIN OUTCOMES AND RESULTS: Six of seven participants reached moderate-to-high degrees of independence. Literacy impairment was an expected training barrier; however, cognitive impairments affecting concentration, memory and motivation were more significant. CONCLUSIONS: Findings suggest that people with cognitive-linguistic impairments can learn Internet skills using specialized training materials. Participants and their carers also reported positive outcomes beyond the acquisition of Internet skills.

Stakeholder opinion of functional communication activities following traumatic brain injury.

PRIMARY OBJECTIVE: To establish a process whereby assessment of functional communication reflects the authentic communication of the target population. The major functional communication assessments available from the USA may not be as relevant to those who reside elsewhere, nor assessments developed primarily for persons who have had a stroke as relevant for traumatic brain injury rehabilitation. RESEARCH DESIGN: The investigation used the Nominal Group Technique to elicit free opinion and support individuals who have compromised communication ability. A survey mailed out sampled a larger number of stakeholders to test out differences among groups. METHODS AND PROCEDURES: Five stakeholder groups generated items and the survey determined relative 'importance'. The stakeholder groups in both studies comprised individuals with traumatic brain injury and their families, health professionals, third-party payers, employers, and Maori, the indigenous population of New Zealand. MAIN OUTCOMES AND RESULTS: There was no statistically significant difference found between groups for 19 of the 31 items. Only half of the items explicitly appear on a well-known USA functional communication assessment. CONCLUSIONS: The present study has implications for whether functional communication assessments are valid across cultures and the type of impairment.

Why do asthmatic subjects respond so strongly to inhaled adenosine?

Bronchospasm induced by adenosine is blocked by representatives of all the major classes of drugs used in the treatment of asthma. Understanding the mechanism of this bronchospasm may help understand the way these drugs work. Clinical studies have suggested involvement of neural pathways, mast-like cells and mediators such as histamine, serotonin and lipoxygenase products. There is a strong link between responsiveness to adenosine and eosinophilia. In different animal models A1, A2b and A3 adenosine receptor subclasses have all been implicated in inducing bronchospasm. whilst occupation of the A2a receptor generally has no, or the opposite effect. At least two different mechanisms, both involving neural pathways, exist. One, involving the adenosine A1 receptor, functions in mast cell depleted animals; the other requires interaction with a population of mast-like cells activated over A2b or A3 receptors. Not only histamine but also serotonin and lipoxygenase products released from the mast-like cells are potential mediators. In animal models good reactivity to adenosine receptor agonists is generally only found when the animals are first sensitized and exposed to allergen in ways likely to induce an allergic inflammation. An exception is the BDE rat, which reacts to adenosine receptor agonists such as APNEA or NECA even without allergen exposure. This rat strain does however show evidence of spontaneous eosinophilic inflammation in the lung even without immunization. As mast cells both release adenosine and respond to adenosine, adenosine provides a non-specific method of amplifying specific signals resulting from IgE/antigen interaction. This mechanism may not only have a pathological significance in asthma; it may be part of a normal bodily defense response that in asthmatic subjects is inappropriately activated.

Feeding problems in infants with gastro-oesophageal reflux disease: a controlled study.

OBJECTIVE: Gastro-oesophageal reflux disease (GORD) in infants is commonly associated with feeding problems but has not been subject to systematic controlled study. We evaluated feeding, dietary, behavioural data obtained from systematic objective studies of six-month old infants with and without GORD. METHODS: Infants with GORD (defined by 24-h pH monitoring, n = 20), and age, gender, gestation, and socio-economic matched healthy infants (n = 20) had standardised assessments of dietary intake, oromotor function by videoanalysis (Feeding Assessment Schedule, FAS), and infant feeding behaviour by Testers and Maternal Ratings (TRIB and MRIB). Videofluoroscopic analyses of swallowing was undertaken in 11/20 GORD infants and analysed by standardised paediatric check list. RESULTS: Compared with control data: GORD infants had significantly lower energy intakes; the FAS showed GORD infants to have significantly fewer adaptive skills and readiness behaviour for solids, significantly more food refusal and food loss; the TRIB showed GORD infants to be significantly more demanding and difficult with feeds; and the MRIB revealed that mothers of GORD infants had significantly more negative feelings, significantly less enjoyment of feeds, and reported significantly more crying behaviour. On videofluoroscopy, oral-preparatory and oral phase problems predominated, particularly with solids, silent aspiration occurred during the pharyngeal phase in 2/11, and delayed oesophageal transit occurred in 4/11. CONCLUSIONS: Feeding problems affecting behaviour, swallowing, food intake, and mother-child interaction occur in infants with GORD, who displayed a lack of development of age-appropriate feeding skills. The contribution of feeding problems to morbidity in GORD in infants has been underestimated in the past.

Recognizing and managing anemia.

Anemia is not an uncommon problem. Knowledge of a few common laboratory tests can be helpful when caring for patients with anemia. By reviewing approaches to classifying, evaluating, and managing patients with common anemias, nurses will be prepared to care for patients with an anemia more confidently.

The effect of hearing impairment on the vocal characteristics of older people.

Measures of vocal intensity, frequency and harshness were compared for 19 hearing-impaired and 21 normal-hearing people over 60 years of age. Significantly greater comfortable intensity levels were found in the hearing-impaired group, but the other measures of frequency and harshness were not significantly different. A large proportion of the subjects in both groups reported a history of gastro-oesophageal reflux (GER), a condition associated with vocal fold pathology and hoarseness. Comparison of the GER and non-GER subjects on the measures of vocal function showed that the female GER speakers exhibited lower frequency on the vowel /u/ than the non-GER subjects. Clinicians need to be aware of the effect of highly prevalent disorders such as hearing impairment and GER on the voices of elderly speakers.

Communication difficulties of nursing home residents. How can staff help?

Recent studies have found a high prevalence of communication disorders in elderly residents of nursing homes. This study was undertaken to investigate staff attitudes to the communication problems and communicative needs of residents in their care. A questionnaire was administered to 32 staff members from one nursing home. Responses indicate little evidence of the negative staff attitudes previously reported in the literature. Staff wanted more time to spend communicating with residents and wanted to learn more about the residents' communication problems. Implications of the questionnaire responses for staff training are presented.

Oncology Nursing Society: development and implementation of practice guidelines.

With more than 25,000 professional nurse members, the Oncology Nursing Society (ONS) is the largest oncology membership organization in the world. Now in its 20th year, one way that ONS supports its mission of promoting excellence in oncology nursing is through the development and dissemination of patient care standards and guidelines. The first guidelines to be developed by ONS were the Cancer Chemotherapy Guidelines. Now in their third edition, the historical development and revision of these guidelines is detailed in this article, along with the evaluation and refinement of the process utilized by ONS.

Hearing impairment, disability and handicap in elderly people living in residential care and in the community.

This study aimed to compare the prevalence of hearing impairment, disability and handicap in the elderly living in residential care with those living in the community, and to examine the relationship between impairment, disability and handicap in both groups. Fifty community-based and 129 residential subjects were assessed using pure-tone audiometry as a measure of impairment, and a self-assessment questionnaire as a measure of disability and handicap. Community-based subjects were also assessed using a test of speech discrimination as an objective measure of disability. Results indicated 95% of residential subjects and 70% of community-based subjects were hearing-impaired, while 27% of residential subjects and 42% of community-based subjects demonstrated significant disability/handicap. Significant correlations were obtained between impairment, disability and handicap in both subject groups. The implications of the findings for the aural rehabilitation of the elderly are discussed.

The evaluation of intervention programmes for communicatively impaired elderly people.

A study is described which examined the efficacy of group intervention programmes using trained volunteers as agents of intervention with elderly nursing home residents with communication impairments. A series of intervention programmes, which consisted of six weekly sessions, was designed. Sessions were a combination of information giving, discussion and practical components where residents could practise newly acquired skills. Sessions covered a number of core components--the communication process, comprehension, expression and pragmatics, as well as information that was specific to the communication impairment. Volunteers were trained to administer the programme. Four groups of communicatively impaired elderly people were included in the study--a group of hearing-impaired residents, those with communication impairment subsequent to Parkinson's disease, those with communication impairment subsequent to cerebrovascular accident and those in varying stages of dementia. Results indicated that, although the residents studied responded very individually to the intervention strategies, with several subjects failing to demonstrate any gains in communicative competence from pre- to post-intervention, positive gains in communication skills and self-management of communicative impairment were observed for a number of subjects. Complicating factors such as general health and well-being are considered as uncontrollable variables in the measurement of performance in the elderly.