Single molecule delivery into living cells.

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

Single-cell nanobiopsy enables multigenerational longitudinal transcriptomics of cancer cells.

Single-cell RNA sequencing has revolutionized our understanding of cellular heterogeneity, but routine methods require cell lysis and fail to probe the dynamic trajectories responsible for cellular state transitions, which can only be inferred. Here, we present a nanobiopsy platform that enables the injection of exogenous molecules and multigenerational longitudinal cytoplasmic sampling from a single cell and its progeny. The technique is based on scanning ion conductance microscopy (SICM) and, as a proof of concept, was applied to longitudinally profile the transcriptome of single glioblastoma (GBM) brain tumor cells in vitro over 72 hours. The GBM cells were biopsied before and after exposure to chemotherapy and radiotherapy, and our results suggest that treatment either induces or selects for more transcriptionally stable cells. We envision the nanobiopsy will contribute to transforming standard single-cell transcriptomics from a static analysis into a dynamic assay.

Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer-Electrolyte Nanopore.

Solid-state nanopores have been widely employed in the detection of biomolecules, but low signal-to-noise ratios still represent a major obstacle in the discrimination of nucleic acid and protein sequences substantially smaller than the nanopore diameter. The addition of 50% poly(ethylene) glycol (PEG) to the external solution is a simple way to enhance the detection of such biomolecules. Here, we demonstrate with finite-element modeling and experiments that the addition of PEG to the external solution introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the current response of the nanopore. We further show that the strong asymmetric current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across its aperture. We provide evidence that a combination of the decreased/increased diffusion coefficients of cations/anions in the bath outside the nanopore and the interaction between a translocating molecule and the nanopore-bath interface is responsible for the increase in the translocation signals. We expect this new mechanism to contribute to further developments in nanopore sensing by suggesting that tuning the diffusion coefficients of ions could enhance the sensitivity of the system.

The anatomy and function of a bilateral accessory flexor digitorum superficialis muscle: A case report and review of literature.

Anatomical variations of the flexor digitorum superficialis (FDS) include variations of the muscle belly and/or tendon. We present a case of a bilateral accessory FDS muscle and tendon. Dissection was performed in a male specimen (56 years), where an unusual variation was found: a bilateral accessory FDS muscle and tendon. The accessory FDS muscle belly originated at the medial epicondyle, between the tendinous portions of the FDS and flexor carpi radialis, with average dimensions of 70mm in length and 23mm in width. The accessory FDS muscle belly was innervated by the median nerve. After developing a strong tendon, with a length of 51mm (right) and 85mm (left) and width of approximately 9.5mm, the accessory FDS inserted into the dorsal muscle belly of the main FDS. To further analyze the variation, the left accessory FDS muscle and tendon were separated from the adjacent tissues at the elbow and kept attached at the wrist. On a custom jig, 10N were applied sequentially to the main and accessory FDS separately and then together to access their function. Loading of the main FDS muscle resulted in flexion of the fingers with accentuation of the ulnar rays. Loading of the accessory FDS caused flexion of all four long fingers. We described a rare case of an accessory flexor digitorum superficialis, which may assist surgeons with clinical decisions. Surgeons should be aware of variability in forearm anatomy.

Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore.

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (ß-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein ß-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.

Randomized open-label trial comparing topical prescription triamcinolone to over-the-counter hydrocortisone for the treatment of phimosis.

BACKGROUND: Phimosis is a common condition affecting most infant boys and generally resolves over time without symptoms. Severe cases of phimosis can lead to balanoposthitis, urinary tract infections, and urinary retention. Medical treatment for symptomatic phimosis includes topical corticosteroids with manual foreskin retraction. OBJECTIVE: Over-the-counter hydrocortisone 1% cream was compared in a randomized controlled fashion with prescription triamcinolone 0.1% cream for the medical management of symptomatic phimosis. METHODS: The study institution conducted a randomized open-label trial for the treatment of grades 4-5 phimosis (phimosis grade scale 0-5). Boys aged 3-13 years were randomized to hydrocortisone 1% cream or triamcinolone 0.1% cream dosed at least twice daily for a course of 12 weeks. Instructions were provided for appropriate application and manual retraction of the foreskin. Evaluations were performed at 4, 8, and 12 weeks. Successful completion of the study was determined by reaching phimosis grade 2 or less or after completing 12 weeks of treatment. RESULTS: A total of 52 boys enrolled in the trial, with a total of 32 boys completing the 12-week duration. Of the 13 boys in the hydrocortisone arm, there was a 30.8% success rate at 4 weeks, 53.8% success rate at 8 weeks, and 61.5% success rate at 12 weeks. Of the 19 boys in the triamcinolone arm, there was a 31.6% success rate at 4 weeks, 52.6% success rate at 8 weeks, and 68.4% success rate at 12 weeks. There was no statistical difference between the two arms at each interval. DISCUSSION: To the study authors' knowledge, this is the first open-label trial with direct comparison of hydrocortisone 1% cream with triamcinolone 0.1% cream. The study results support those reported in other studies when each topical steroid was compared with placebo. Limitations of the study include loss to follow-up, unblinded treatment allocation, and reduced power to detect differences by treatment frequency and duration. CONCLUSION: Over-the-counter hydrocortisone 1% cream is not inferior to triamcinolone 0.1% cream when paired with manual retraction for the treatment of grade 4-5 phimosis. Successful treatment response may be seen up to 12 weeks.

Dynamical Density Functional Theory for the Evaporation of Droplets of Nanoparticle Suspension.

We develop a lattice gas model for the drying of droplets of a nanoparticle suspension on a planar surface, using dynamical density functional theory (DDFT) to describe the time evolution of the solvent and nanoparticle density profiles. The DDFT assumes a diffusive dynamics but does not include the advective hydrodynamics of the solvent, so the model is relevant to highly viscous or near to equilibrium systems. Nonetheless, we see an equivalent of the coffee-ring stain effect, but in the present model it occurs for thermodynamic rather the fluid-mechanical reasons. The model incorporates the effect of phase separation and vertical density variations within the droplet and the consequence of these on the nanoparticle deposition pattern on the surface. We show how to include the effect of slip or no-slip at the surface and how this is related to the receding contact angle. We also determine how the equilibrium contact angle depends on the microscopic interaction parameters.

Modelling the evaporation of nanoparticle suspensions from heterogeneous surfaces.

We present a Monte Carlo (MC) grid-based model for the drying of drops of a nanoparticle suspension upon a heterogeneous surface. The model consists of a generalised lattice-gas in which the interaction parameters in the Hamiltonian can be varied to model different properties of the materials involved. We show how to correctly choose the interactions, to minimise the effects of the underlying grid so that hemispherical droplets form. We also include the effects of surface roughness to examine the effects of contact-line pinning on the dynamics. When there is a 'lid' above the system, which prevents evaporation, equilibrium drops form on the surface, which we use to determine the contact angle and how it varies as the parameters of the model are changed. This enables us to relate the interaction parameters to the materials used in applications. The model has also been applied to drying on heterogeneous surfaces, in particular to the case where the suspension is deposited on a surface consisting of a pair of hydrophilic conducting metal surfaces that are either side of a band of hydrophobic insulating polymer. This situation occurs when using inkjet printing to manufacture electrical connections between the metallic parts of the surface. The process is not always without problems, since the liquid can dewet from the hydrophobic part of the surface, breaking the bridge before the drying process is complete. The MC model reproduces the observed dewetting, allowing the parameters to be varied so that the conditions for the best connection can be established. We show that if the hydrophobic portion of the surface is located at a step below the height of the neighbouring metal, the chance of dewetting of the liquid during the drying process is significantly reduced.

Caring for continence in stroke care settings: a qualitative study of patients' and staff perspectives on the implementation of a new continence care intervention.

OBJECTIVES: Investigate the perspectives of patients and nursing staff on the implementation of an augmented continence care intervention after stroke. DESIGN: Qualitative data were elicited during semi-structured interviews with patients (n = 15) and staff (14 nurses; nine nursing assistants) and analysed using thematic analysis. SETTING: Mixed acute and rehabilitation stroke ward. PARTICIPANTS: Stroke patients and nursing staff that experienced an enhanced continence care intervention. RESULTS: Four themes emerged from patients' interviews describing: (a) challenges communicating about continence (initiating conversations and information exchange); (b) mixed perceptions of continence care; (c) ambiguity of focus between mobility and continence issues; and (d) inconsistent involvement in continence care decision making. Patients' perceptions reflected the severity of their urinary incontinence. Staff described changes in: (i) knowledge as a consequence of specialist training; (ii) continence interventions (including the development of nurse-led initiatives to reduce the incidence of unnecessary catheterisation among patients admitted to their ward); (iii) changes in attitude towards continence from containment approaches to continence rehabilitation; and (iv) the challenges of providing continence care within a stroke care context including limitations in access to continence care equipment or products, and institutional attitudes towards continence. CONCLUSION: Patients (particularly those with severe urinary incontinence) described challenges communicating about and involvement in continence care decisions. In contrast, nurses described improved continence knowledge, attitudes and confidence alongside a shift from containment to rehabilitative approaches. Contextual components including care from point of hospital admission, equipment accessibility and interdisciplinary approaches were perceived as important factors to enhancing continence care.

A review of infection control in community healthcare: new challenges but old foes.

The demographics of the healthcare population are changing, with an ever-greater proportion of people being treated outside the traditional hospital setting through community healthcare. This shift in the way that healthcare is delivered raises new concerns over community healthcare-associated infections (HCAIs). A literature search between 2000 and December 2013 was conducted in databases including PubMed, SciVerse ScienceDirect and Google Scholar. National and international guideline and policy documents were searched using Google. Many terms were used in the literature searches, including 'nosocomial', 'healthcare infection', 'community' and 'nursing home'. The rates of HCAI in community healthcare are similar to the rates found in the acute hospital setting, but the types of infection differ, with a greater focus on urinary tract infections (UTIs) in the community and ventilator-associated pneumonias in the hospital setting. Patients who acquire a community HCAI are more likely to exhibit reduced physical condition, have increased levels of morbidity and have higher mortality rates than individuals without infection. Infection control programmes have been developed worldwide to reduce the rates of hospital HCAIs. Such interventions are equally as valid in the community, but how best to implement them and their subsequent impact are much less well understood. The future is clear: HCAIs in the community are going to become an ever-increasing burden and it is critical that our approach to these infections is brought quickly in line with present hospital sector standards.

Breast reconstruction in the high risk patient with systemic connective tissue disease: a case series.

INTRODUCTION: The presence of severe underlying connective tissue disease may restrict the reconstructive options offered to a woman in the event of mastectomy. Putative concerns about reconstructive surgery include the effects of connective tissue disease and immunosuppression on wound healing and donor site morbidity, and increased risks of deranged clotting and thrombophilia after free tissue transfer. There is also the possibility of an unpredictable tissue reaction after oncological resection surgery and adjuvant radiotherapy. METHODOLOGY: Here we present a review of the current sparse evidence regarding reconstructive breast surgery in this challenging group of patients. In addition we present a series of six consecutive patients with a spectrum of connective tissue disorders including combinations of longstanding Systemic Lupus Erythematosis (SLE), Rheumatoid arthritis and Raynaud's Disease who underwent successful post-mastectomy reconstruction with an extended autologous latissimus dorsi flap, along with subsequent successful correction of asymmetry and/or nipple reconstruction. RESULTS: There is a paucity of literature on this subject perhaps suggesting that surgeons are reluctant to offer reconstruction or that uptake is poor in this group. Complications related to radiotherapy and free tissue transfer in patients with severe CTD is less than may be expected. The most common complications experienced by our patients with CTD after extended ALD breast reconstruction were persistent donor site seroma, wound dehiscence and delayed haematoma formation, reflecting the abnormal inflammatory response and deranged haemostatic cascade common to connective tissue disease. However, all six patients made a full recovery from surgery without residual donor site morbidity and with an acceptable aesthetic breast reconstruction. CONCLUSION: Careful peri-operative management is crucial in this group of patients, but good outcomes are possible using a variety of reconstructive techniques. This is the first reported series of patients with severe connective tissue disease who have been managed with extended ALD breast reconstruction. The majority of complications relate to the donor site but the favourable outcomes demonstrate that the extended ALD flap remains a reliable reconstructive option for this group.

Epidemiology and management of candidaemia--a retrospective, multicentre study in five hospitals in the UK.

Candidaemia is associated with high mortality. Despite the fact that Candida species account for close to 10% of all nosocomial bloodstream infections, relatively few studies have investigated the management of candidaemia in hospitals. Our objective was to find out how candidaemia is managed in hospitals. Data relating to all episodes of candidaemia for the year 2008 were retrospectively collected in five centres in Scotland and Wales. A total of 96 candidaemic episodes were recorded in the year 2008, yielding 103 isolates of Candida. Fifty candidaemic episodes were caused by Candida albicans. Fluconazole was the most common agent prescribed for the treatment of candidaemia. There was great variation in the prescribed dose of fluconazole. Forty per cent of patients who survived received <2 weeks of systemic antifungal therapy. Central venous catheters (CVC) were removed in 57% of patients. CVC removal was not associated with better survival. The overall mortality was 40.4%. Management of candidaemia varies between the UK centres and is often inadequate. There is need to have consensus on the dosages of antifungal agents and the duration of therapy. The current guidance on removal of CVC in all cases of candidaemia should be reviewed.

Management of fungal infections in the intensive care unit: a survey of UK practice.

BACKGROUND: Candida species are a common cause of nosocomial bloodstream infection. Such infections commonly affect patients in the intensive care unit (ICU) and carry a high mortality. There are published guidelines for the management of fungal infections, but there are no data on the usual management of invasive Candida infections in UK ICUs. METHODS: An electronic survey was sent by email to a representative clinician in 236 ICUs, over 90% of units in the UK. Questions related to the institution of empirical therapy and to the management of proven candidaemia. RESULTS: There were 72 responses. A minority of units follow a policy regarding the management of these infections but the involvement of microbiologists is usual. Empirical therapy is used in 85.9% of units, often for patients perceived to be at high risk. Fluconazole is the most commonly used antifungal agent, both for empirical therapy and for the treatment of proven candidaemia. For candidaemic patients, 73.9% of ICUs frequently or always remove central venous catheters within 48 h, while 15.1% frequently or always arrange ophthalmology review. CONCLUSIONS: Management of fungal infections is relatively consistent among responding units. However, recent developments in the field have not yet been incorporated into standard practice. Adherence to published guidelines could be improved, potentially reducing morbidity and mortality from these common infections.

Immunotoxicity and disease resistance in Japanese quail (Corturnix coturnix japonica) exposed to malathion.

The purpose of this study was to evaluate the impact of malathion on the immune system of wild birds, using Japanese quail (Coturnix coturnix japonica) as a model. Quail were exposed to malathion in drinking water at environmentally realistic concentrations (0 ppm, 1 ppm, and 10 ppm). In the fifth week, several arms of the immune response were tested using the T-cell based phytohemagglutinin (PHA) skin test, the B-cell mediated antibody response, and the chemiluminescence assay measuring innate immunity. After the sixth week of malathion exposure, quail were challenged with E. coli O2. The bursa of Fabricius and the spleen were assessed for histopathology. No clinical signs of malathion toxicity were observed. Morbidity or mortality subsequent to E. coli exposure tended (P = 0.08) to be higher in the high exposure group (50.0%) compared to the control (22.2%) group. There was no difference in the innate immune response in the malathion exposed birds, however, humoral immunity was suppressed (P = 0.03) with the higher malathion exposure. Histopathological evaluation revealed an immunosuppressive effect of malathion on the bursa of Fabricius; bursal atrophy, decreased B-cell density and increased apoptosis in the medulla, and increased connective tissue thickness of the follicular epithelium. Antibody suppression was correlated with bursal changes and peripheral blood lymphocyte count, the organ and cells involved in antibody production. Following the same pattern as other immunotoxicity tests, the PHA T-cell proliferative response also tended to be suppressed in the high exposure group. This study provides evidence that subchronic, moderate malathion exposure is immunotoxic to quail and that testing integrated, functional immunity using an infectious challenge is a better predictor of immunotoxicity than individual responses to immunotoxicity tests. The secondary antibody response, circulating lymphocyte populations, and bursal histopathology were the most sensitive indicators of immune status, as these predicted decreased disease resistance with malathion exposure.

Necrotising fasciitis complicating Haemophilus influenzae type b epiglottitis in an adult.

OBJECTIVE: To present the first reported case of Haemophilus influenzae type b epiglottitis leading to necrotising fasciitis. METHOD: Case report and review of the literature regarding the association of necrotising fasciitis with Haemophilus influenzae infection and with epiglottitis. CASE REPORT: A previously well, 64-year-old woman presented with epiglottitis, and subsequently developed necrotising fasciitis of her chest wall. The cause of both infections was Haemophilus influenzae serotype b. This organism has frequently been implicated in epiglottitis, but has not previously been reported to cause simultaneous necrotising fasciitis. The patient became critically ill, requiring intensive care management, but following surgical debridement and antibiotic treatment she made a full recovery. CONCLUSION: Although increasingly uncommon, clinicians must continue to be proficient in the diagnosis and management of epiglottitis, and to be aware of its full range of possible complications. This case report highlights a previously unknown and potentially fatal complication of Haemophilus influenzae type b epiglottitis.

Can patients' preferences for involvement in decision-making regarding the use of medicines be predicted?

OBJECTIVE: The current study aimed to develop a model of patients' preferences for involvement in decision-making concerning the use of medicines for chronic conditions in the UK and test it in a large representative sample of patients with one of two clinical conditions. METHODS: Following a structured literature review, an instrument was developed which measured the variables that had been identified as predictors of patients' preferences for involvement in decision making in previous research. Five hundred and sixteen patients with rheumatoid arthritis or type 2 diabetes were recruited from outpatient and primary care clinics and asked to complete the instrument. RESULTS: Multivariate analysis revealed that age, social class and clinical condition were associated with preferences for involvement in decision-making concerning the use of medicines for chronic illness but gender, ethnic group, concerns about medicines, beliefs about necessity of medicines, health status, quality of life and time since diagnosis were not. In total, the fitted model explained only 14% of the variance. CONCLUSION: This study has demonstrated that current research does not provide a basis for predicting patients' preferences for involvement in decision-making. PRACTICE IMPLICATIONS: Building concordant relationships may depend on practitioners developing strategies to establish individuals' preferences for involvement in decision-making as part of the ongoing prescriber-patient relationship.

The influence of invasive growth pattern and microvessel density on prognosis in colorectal cancer and colorectal liver metastases.

The nature of the invasive growth pattern and microvessel density (MVD) have been suggested to be predictors of prognosis in primary colorectal cancer (CRC) and colorectal liver metastases. The purpose of the present study was to determine whether these two histological features were interrelated and to assess their relative influence on disease recurrence and survival following surgical resection. Archival tissue was retrieved from 55 patients who had undergone surgical resection for primary CRC and matching liver metastases. The nature of the invasive margin was determined by haematoxylin and eosin (H&E) histochemistry. Microvessel density was visualised using immunohistochemical detection of CD31 antigen and quantified using image capture computer software. Clinical details and outcome data were retrieved by case note review and collated with invasive margin and MVD data in a statistical database. Primary CRCs with a pushing margin tended to form capsulated liver metastases (P<0.001) and had a significantly better disease-free survival than the infiltrative margin tumours (log rank P=0.01). Primary cancers with a high MVD tended to form high MVD liver metastases (P=0.007). Microvessel density was a significant predictor of disease recurrence in primary CRCs (P=0.006), but not liver metastases. These results suggest that primary CRCs and their liver metastases show common histological features. This may reflect common mechanisms underlying the tumour-host interaction.

The role of (99m)technetium-labelled hepato imino diacetic acid (HIDA) scan in the management of biliary pain.

OBJECTIVE: To assess the outcome of laparoscopic cholecystectomy on the basis of an abnormal provocative (99m)technetium-labelled hepato imino diacetic acid (HIDA) scan for patients with typical biliary pain and normal trans-abdominal ultrasound (TUS) scan. PATIENTS AND METHODS: Prospective data were collected for 1201 consecutive patients with typical biliary symptoms. Patients who were found to have a normal TUS and upper GI endoscopy subsequently underwent cholescintigraphy (HIDA scan). Patients with an abnormal HIDA scan, i.e.<40% ejection fraction with Sincalide (cholecystokinin octapeptide)--were offered cholecystectomy. Symptoms and histology were reviewed postoperatively. RESULTS: In all, 48/1201 (4%) patients with typical biliary symptoms had a normal ultrasound and endoscopy; 35/48 patients had an abnormal provocative HIDA scan and all underwent laparoscopic cholecystectomy. Histology in all cases revealed chronic cholecystitis and 18 patients had sludge or microlithiasis within the gallbladder. At 6-week follow-up, 31 of the 35 patients were completely asymptomatic or improved. Furthermore, 79% of patients remained symptom-free or improved at a median follow-up of 28.5 months (range 4-70). CONCLUSIONS: HIDA scan is a useful clinical tool as an adjunct to the diagnosis and management of patients who present with typical biliary pain and a normal TUS scan.