Halophilic microorganisms as a source of novel antibiofilm compounds

Objectives: There is great urgency for alternate sources of antibiotics to be identified. One relatively untapped source of novel bioproducts, including antimicrobials, is organisms derived from extreme environments. Halophiles (which require high salt concentrations) are one such group which is being increasingly explored for their biotechnological potential. The aim of this study was to identify halophilic environmental isolates which possessed in vitro and in vivo antimicrobial and antibiofilm activities. Methods: 73 halophilic bacteria and archaea were isolated from Kilroot salt mine in Northern Ireland. Culture extracts of each isolate were screened for antimicrobial and antibiofilm activity against numerous pathogenic bacteria, including Staphylococcus species and Pseudomonas aeruginosa, both model strains and clinical isolates. The methods used included disc diffusion assays of crude extracts, MIC screening, the MBEC assay, and an in vivo model based on the Greater Wax Moth (Galleria mellonella). Results: The assays indicated >50% of extracts displayed antimicrobial and antibiofilm activity against at least one pathogen, the majority being Staphylococcus species, but also E. coli and P. aeruginosa. Biofilms were either reduced or eradicated by halophile extracts when tested with the MBEC device. Further experiments demonstrated that these effects could be replicated in vivo, with extracts reducing the severity of infections and enhancing the survival of infected G. mellonella. Conclusions: The importance of extremophiles to pharmaceutical research should not be underestimated. While not yet fully characterised, based on the data obtained, the halophiles isolated during this study may provide a promising reservoir of novel antimicrobial and antibiofilm compounds.

Endocrine Surgery: Principles and Practice:adrenal chapter

The paired adrenal (suprarenal) glands are flattened retroperitoneal endocrine glands closely applied to the medial aspect of the superior pole of each kidney. The internal structure of these pale yellow glands are incongruous in that the adrenal gland is composed of two discrete parts, namely an outer cortex enveloping a central medulla. The adrenal cortex and medulla contain distinct endocrine tissues that secrete different hormones and are regulated by separate control systems.

A proposed model membrane and test method for microneedle insertion studies

A commercial polymeric film (Parafilm M (R), a blend of a hydrocarbon wax and a polyolefin) was evaluated as a model membrane for microneedle (MN) insertion studies. Polymeric MN arrays were inserted into Parafilm M (R) (PF) and also into excised neonatal porcine skin. Parafilm M (R) was folded before the insertions to closely approximate thickness of the excised skin. Insertion depths were evaluated using optical coherence tomography (OCT) using either a force applied by a Texture Analyser or by a group of human volunteers. The obtained insertion depths were, in general, slightly lower, especially for higher forces, for PF than for skin. However, this difference was not a large, being less than the 10% of the needle length. Therefore, all these data indicate that this model membrane could be a good alternative to biological tissue for MN insertion studies. As an alternative method to OCT, light microscopy was used to evaluate the insertion depths of MN in the model membrane. This provided a rapid, simple method to compare different MN formulations. The use of Parafilm M (R), in conjunction with a standardised force/time profile applied by a Texture Analyser, could provide the basis for a rapid MN quality control test suitable for in-process use. It could also be used as a comparative test of insertion efficiency between candidate MN formulations. 

BRCA1--conductor of the breast stem cell orchestra:the role of BRCA1 in mammary gland development and identification of cell of origin of BRCA1 mutant breast cancer

Breast cancer treatment has been increasingly successful over the last 20 years due in large part to targeted therapies directed against different subtypes. However, basal-like breast cancers still represent a considerable challenge to clinicians and scientists alike since the pathogenesis underlying the disease and the target cell for transformation of this subtype is still undetermined. The considerable similarities between basal-like and BRCA1 mutant breast cancers led to the hypothesis that these cancers arise from transformation of a basal cell within the normal breast epithelium through BRCA1 dysfunction. Recently, however, a number of studies have called this hypothesis into question. This review summarises the initial findings which implicated the basal cell as the cell of origin of BRCA1 related basal-like breast cancers, as well as the more recent data which identifies the luminal progenitor cells as the likely target of transformation. We compare a number of key studies in this area and identify the differences that could explain some of the contradictory findings. In addition, we highlight the role of BRCA1 in breast cell differentiation and lineage determination by reviewing recent findings in the field and our own observations suggesting a role for BRCA1 in stem cell regulation through activation of the p63 and Notch pathways. We hope that through an increased understanding of the BRCA1 role in breast differentiation and the identification of the cell(s) of origin we can improve treatment options for both BRCA1 mutant and basal-like breast cancer subgroups.

Galleria mellonella as a novel in vivo model for assessment of the toxicity of 1-alkyl-3-methylimidazolium chloride ionic liquids

The larval form of the Greater Wax Moth (Galleria mellonella) was evaluated as a model system for the study of the acute in vivo toxicity of 1-alkyl-3-methylimidazolium chloride ionic liquids. 24-h median lethal dose (LD50) values for nine of these ionic liquids bearing alkyl chain substituents ranging from 2 to 18 carbon atoms were determined. The in vivo toxicity of the ionic liquids was found to correlate directly with the length of the alkyl chain substituent, and the pattern of toxicity observed was in accordance with previous studies of ionic liquid toxicity in other living systems, including a characteristic toxicity ‘cut-off’ effect. However, G. mellonella appeared to be more susceptible to the toxic effects of the ionic liquids tested, possibly as a result of their high body fat content. The results obtained in this study indicate that G. mellonella represents a sensitive, reliable and robust in vivo model organism for the evaluation of ionic liquid toxicity.

The role of secreted frizzled-related protein 2 expression in prostate cancer

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2.

METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients.

CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

Abl expression, tumour grade, and apoptosis in chondrosarcoma

AIMS: To determine whether Abl immunoreactivity correlates with grade and cell kinetics (apoptosis and mitosis) in chondrosarcoma.

METHODS: Sections from 16 chondrosarcomas were stained immunohistochemically using a polyclonal antibody to the c-Abl/Bcr-Abl oncoprotein. Apoptotic indices and mitotic indices were assessed in all tumours. Sections from 24 paraffin wax blocks of human fetal rib (gestational ages, 15-42 weeks) were also stained to determine whether the Abl protein is synthesised consistently throughout endochondral ossification.

RESULTS: Abl staining in immature fetal rib chondrocytes at all stages of development was predominantly nuclear, and 70% of cells showed moderate to strong staining. Abl immunoreactivity was minimal or absent in hypertrophic chondrocytes about to undergo apoptosis at the growth plate. There was strong Abl staining in grade 1 and grade 2 chondrosarcomas but staining was greatly reduced or absent in grade 3 chondrosarcomas. There was a very significant linear correlation between apoptotic index (mean, 0.68%; range, 0-3.2%) and mitotic index (mean, 0.23%; range, 0-0.9%), and both indices were significantly lower in grade 1 than in grade 2 and grade 3 chondrosarcomas.

CONCLUSIONS: These data suggest that abl gene expression is associated with differentiation and apoptosis inhibition in fetal and neoplastic chondrocytes. However, these putative effects cannot be ascribed solely to the Abl protein, because several additional factors contribute to the regulation of both differentiation and apoptosis.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Pre-Radiotherapy Dental Assessment of Head and Neck Oncology Patients: An Audit.

Objectives: Approximately 300 people are diagnosed with Head and Neck cancer annually in Northern Ireland. The management may include treatment by surgery or by chemotherapy and radiotherapy,
or a combination of modalities. Patients whose oral cavity, teeth, salivary glands and jaws that
will be affected by treatment, particularly radiotherapy should have a pre-treatment assessment. This should be done as early as possible to maximise the time available for dental management. However, this can be challenging owing to the complexities of cancer diagnosis, treatment planning and multidisciplinary management. At the Belfast Dental Hospital, a number of patients were referred post- radiotherapy with complications after not having received a pre-treatment assessment. The referrals for pre- treatment dental assessment were also late in patients’ multidisciplinary journey, limiting the time period
for dental input. The purpose of this audit was to examine the time period between dental assessment and commencement of radiotherapy and whether this was an adequate time frame for dental management. This audit will also examine the dental diseases present and the treatments required pre-radiotherapy. Methods: Data for this audit was collected over 4 months in 2012
by analysing the dental charts and referrals of new patients who were referred to and attended the dental head and neck oncology clinic. A standardised referral pro-forma was introduced from September 2013 to improve the referral process.
A re-audit was conducted over 4 months in 2014. Data was collected similarly as previous. The time period between dental assessment and commencement of radiotherapy was examined. The presence of dental disease and subsequent treatments required were also noted.
Results: 63 new patients were examined in the dental head and neck oncology clinic over 4 months in 2012. 48 (76.2%) were examined pre-radiotherapy. The average length of time between dental assessment and radiotherapy commencement was 11 days. A new standardised referral pro-forma was introduced in 2013. In the re-audit, 65 new patients were seen over 4 months in 2014.
60 (92.3%) patients were examined pre-radiotherapy. The average length of time between dental assessment and radiotherapy commencement was 18 days.
Conclusion: Given the high prevalence of pre-existing dental disease amongst head and neck cancer patients, prompt dental assessment and treatment is vital. Efforts aimed at improving the care pathway are on-going through the implementation of a mandatory referral pro-forma and a dedicated assessment clinic.

Shape stabilised phase change materials based on a high melt viscosity HDPE and paraffin waxes

Shape stabilised phase change materials (SSPCMs) based on a high density poly(ethylene)(hv-HDPE) with high (H-PW, Tm = 56–58 °C) and low (L-PW, Tm = 18–23 °C) melting point paraffin waxes were readily prepared using twin-screw extrusion. The thermo-physical properties of these materials were assessed using a combination of techniques and their suitability for latent heat thermal energy storage (LHTES) assessed. The melt processing temperature (160 °C) of the HDPE used was well below the onset of thermal decomposition of H-PW (220 °C), but above that for L-PW (130 °C), although the decomposition process extended over a range of 120 °C and the residence time of L-PW in the extruder was <30 s. The SSPCMs prepared had latent heats up to 89 J/g and the enthalpy values for H-PW in the respective blends decreased with increasing H-PW loading, as a consequence of co-crystallisation of H-PW and hv-HDPE. Static and dynamic mechanical analysis confirmed both waxes have a plasticisation effect on this HDPE. Irrespective of the mode of deformation (tension, flexural, compression) modulus and stress decreased with increased wax loading in the blend, but the H-PW blends were mechanically superior to those with L-PW.

Biomarkers reveal the effects of hydrography on the sources and fate of marine and terrestrial organic matter in the western Irish Sea

A suite of lipid biomarkers were investigated from surface sediments and particulate matter across hydrographically distinct zones associated with the western Irish Sea gyre and the seasonal bloom. The aim was to assess the variation of organic matter (OM) composition, production, distribution and fate associated with coastal and southern mixed regions and also the summer stratified region. Based on the distribution of a suite of diagnostic biomarkers, including phospholipid fatty acids, source-specific sterols, wax esters and C₂₅ highly branched isoprenoids, diatoms, dinoflagellates and green algae were identified as major contributors of marine organic matter (MOM) in this setting. The distribution of cholesterol, wax esters and C₂₀ and C₂₂ polyunsaturated fatty acids indicate that copepod grazing represents an important process for mineralising this primary production. Net tow data from 2010 revealed much greater phytoplankton and zooplankton biomass in well-mixed waters compared to stratified waters. This appears to be largely reflected in MOM input to surface sediments. Terrestrial organic matter (TOM), derived from higher plants, was identified as a major source of OM regionally, but was concentrated in proximity to major riverine input at the Boyne Estuary and Dundalk Bay. Near-bottom residual circulation and the seasonal gyre also likely play a role in the fate of TOM in the western Irish Sea.

Increase in microbial contamination risk with compression of the lid margin in eyes having cataract surgery

Purpose: To assess the bacterial contamination risk in cataract surgery associated with mechanical compression of the lid margin immediately after sterilization of the ocular surface.

Setting: Department of Cataract, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Design: Prospective randomized controlled double-masked trial.

Methods: Patients with age-related cataract were randomly assigned to 1 of 2 groups. In Group A (153 eyes), the lid margin was compressed and scrubbed for 360 degrees 5 times with a dry sterile cotton-tipped applicator immediately after ocular sterilization and before povidone-iodine irrigation of the conjunctival sac. Group B (153 eyes) had identical sterilization but no lid scrubbing. Samples from the lid margin, liquid in the collecting bag, and aqueous humor were collected for bacterial culture. Primary outcome measures included the rate of positive bacterial culture for the above samples. The species of bacteria isolated were recorded.

Results: Group A and Group B each comprised 153 eyes. The positive rate of lid margin cultures was 54.24%. The positive rate of cultures for liquid in the collecting bag was significantly higher in Group A (23.53%) than in Group B (9.80%) (P=.001).The bacterial species cultured from the collecting bag in Group B were the same as those from the lid margin in Group A. The positive culture rate of aqueous humor in both groups was 0%.

Conclusion: Mechanical compression of the lid margin immediately before and during cataract surgery increased the risk for bacterial contamination of the surgical field, perhaps due to secretions from the lid margin glands.

Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

Lake Kumphawapi revisited – The complex climatic and environmental record of a tropical wetland in NE Thailand

Kumphawapi, which is Thailand’s largest natural freshwater lake, contains a >10,000-year-long climatic and environmental archive. New data sets (stratigraphy, chronology, hydrogen isotopes, plant macrofossil and charcoal records) for two sedimentary sequences are here combined with earlier multi-proxy studies to provide a comprehensive reconstruction of past climatic and environmental changes for Northeast Thailand. Gradually higher moisture availability due to a strengthening of the summer monsoon led to the formation of a large shallow lake in the Kumphawapi basin between >10,700 and c. 7000 cal. BP. The marked increase in moisture availability and lower evaporation between c. 7000 and 6400 cal. BP favoured the growth and expansion of vegetation in and around the shallow lake. The increase in biomass led to gradual overgrowing and infilling, to an apparent lake level lowering and to the development of a wetland. Multiple hiatuses are apparent in all investigated sequences between c. 6500 and 1400 cal. BP and are explained by periodic desiccation events of the wetland and erosion due to the subsequent lake level rise. The rise in lake level, which started c. 2000 cal. BP and reached shallower parts c. 1400 cal. BP, is attributed to an increase in effective moisture availability. The timing of hydroclimatic conditions during the past 2000 years cannot be resolved because of chronological limitations.