Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3⁺ T-cells than photo-damaged skin, however, the abundance of CD3⁻CD56⁺ Natural Killer (NK) cells, CD11c⁺HLA-DR⁺ conventional Dendritic Cells (cDC), BDCA-2⁺HLA-DR⁺ plasmacytoid DC (pDC), FoxP3⁺ Regulatory T-cells (T-reg), Vα24⁺Vβ11⁺ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4⁺ T-cells were associated with SCC, yet CD8⁺ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8⁺ T-cells than IEC. We propose that differences in CD8⁺ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4⁺ T-cells to CD8⁺ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.     

Habitat Fragmentation and Species Extirpation in Freshwater Ecosystems; Causes of Range Decline of the Indus River Dolphin (Platanista gangetica minor)

Habitat fragmentation of freshwater ecosystems is increasing rapidly, however the understanding of extinction debt and species decline in riverine habitat fragments lags behind that in other ecosystems. The mighty rivers that drain the Himalaya - the Ganges, Brahmaputra, Indus, Mekong and Yangtze - are amongst the world’s most biodiverse freshwater ecosystems. Many hundreds of dams have been constructed, are under construction, or are planned on these rivers and large hydrological changes and losses of biodiversity have occurred and are expected to continue. This study examines the causes of range decline of the Indus dolphin, which inhabits one of the world’s most modified rivers, to demonstrate how we may expect other vertebrate populations to respond as planned dams and water developments come into operation. The historical range of the Indus dolphin has been fragmented into 17 river sections by diversion dams; dolphin sighting and interview surveys show that river dolphins have been extirpated from ten river sections, they persist in 6, and are of unknown status in one section. Seven potential factors influencing the temporal and spatial pattern of decline were considered in three regression model sets. Low dry-season river discharge, due to water abstraction at irrigation barrages, was the principal factor that explained the dolphin’s range decline, influencing 1) the spatial pattern of persistence, 2) the temporal pattern of subpopulation extirpation, and 3) the speed of extirpation after habitat fragmentation. Dolphins were more likely to persist in the core of the former range because water diversions are concentrated near the range periphery. Habitat fragmentation and degradation of the habitat were inextricably intertwined and in combination caused the catastrophic decline of the Indus dolphin.     

Unintended Consequences of Incentive Provision for Behaviour Change and Maintenance around Childbirth

Financial (positive or negative) and non-financial incentives or rewards are increasingly used in attempts to influence health behaviours. While unintended consequences of incentive provision are discussed in the literature, evidence syntheses did not identify any primary research with the aim of investigating unintended consequences of incentive interventions for lifestyle behaviour change. Our objective was to investigate perceived positive and negative unintended consequences of incentive provision for a shortlist of seven promising incentive strategies for smoking cessation in pregnancy and breastfeeding. A multi-disciplinary, mixed-methods approach included involving two service-user mother and baby groups from disadvantaged areas with experience of the target behaviours as study co-investigators. Systematic reviews informed the shortlist of incentive strategies. Qualitative semi-structured interviews and a web-based survey of health professionals asked open questions on positive and negative consequences of incentives. The participants from three UK regions were a diverse sample with and without direct experience of incentive interventions: 88 pregnant women/recent mothers/partners/family members; 53 service providers; 24 experts/decision makers and interactive discussions with 63 conference attendees. Maternity and early years health professionals (n = 497) including doctors, midwives, health visitors, public health and related staff participated in the survey. Qualitative analysis identified ethical, political, cultural, social and psychological implications of incentive delivery at population and individual levels. Four key themes emerged: how incentives can address or create inequalities; enhance or diminish intrinsic motivation and wellbeing; have a positive or negative effect on relationships with others within personal networks or health providers; and can impact on health systems and resources by raising awareness and directing service delivery, but may be detrimental to other health care areas. Financial incentives are controversial and generated emotive and oppositional responses. The planning, design and delivery of future incentive interventions should evaluate unexpected consequences to inform the evidence for effectiveness, cost-effectiveness and future implementation.     

Protective Ventilation of Preterm Lambs Exposed to Acute Chorioamnionitis Does Not Reduce Ventilation-Induced Lung or Brain Injury

Background

The onset of mechanical ventilation is a critical time for the initiation of cerebral white matter (WM) injury in preterm neonates, particularly if they are inadvertently exposed to high tidal volumes (V_T) in the delivery room. Protective ventilation strategies at birth reduce ventilation-induced lung and brain inflammation and injury, however its efficacy in a compromised newborn is not known. Chorioamnionitis is a common antecedent of preterm birth, and increases the risk and severity of WM injury. We investigated the effects of high V_T ventilation, after chorioamnionitis, on preterm lung and WM inflammation and injury, and whether a protective ventilation strategy could mitigate the response.

Methods

Pregnant ewes (n = 18) received intra-amniotic lipopolysaccharide (LPS) 2 days before delivery, instrumentation and ventilation at 127±1 days gestation. Lambs were either immediately euthanased and used as unventilated controls (LPS_UVC; n = 6), or were ventilated using an injurious high V_T strategy (LPS_INJ; n = 5) or a protective ventilation strategy (LPS_PROT; n = 7) for a total of 90 min. Mean arterial pressure, heart rate and cerebral haemodynamics and oxygenation were measured continuously. Lungs and brains underwent molecular and histological assessment of inflammation and injury.

Results

LPS_INJ lambs had poorer oxygenation than LPS_PROT lambs. Ventilation requirements and cardiopulmonary and systemic haemodynamics were not different between ventilation strategies. Compared to unventilated lambs, LPS_INJ and LPS_PROT lambs had increases in pro-inflammatory cytokine expression within the lungs and brain, and increased astrogliosis (p<0.02) and cell death (p<0.05) in the WM, which were equivalent in magnitude between groups.

Conclusions

Ventilation after acute chorioamnionitis, irrespective of strategy used, increases haemodynamic instability and lung and cerebral inflammation and injury. Mechanical ventilation is a potential contributor to WM injury in infants exposed to chorioamnionitis.     

Interaction of protamine with gram‐negative bacteria membranes: possible alternative mechanisms of internalization in Escherichia coli,Salmonella typhimurium and Pseudomonas aeruginosa

This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram‐negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram‐negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane. By using immunoelectron microscopy of Ptm‐treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation‐selective barrel‐like proteins. Work is currently underway to test this hypothesis. © 2014 The Authors. Journal of Peptide Science published by John Wiley & Sons, Ltd.     

Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.     

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation

We have previously shown that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-β (Aβ) accumulation by affecting both Aβ production and Aβ clearance across the blood-brain barrier (BBB). Nilvadipine consists of a mixture of two enantiomers, (+)-nilvadipine and (−)-nilvadipine, in equal proportion. (+)-Nilvadipine is the active enantiomer responsible for the inhibition of LCC, whereas (−)-nilvadipine is considered inactive. Both nilvadipine enantiomers inhibit Aβ production and improve the clearance of Aβ across the BBB showing that these effects are not related to LCC inhibition. In addition, treatment of P301S mutant human Tau transgenic mice (transgenic Tau P301S) with (−)-nilvadipine reduces Tau hyperphosphorylation at several Alzheimer disease (AD) pertinent epitopes. A search for the mechanism of action of (−)-nilvadipine revealed that this compound inhibits the spleen tyrosine kinase (Syk). We further validated Syk as a target-regulating Aβ by showing that pharmacological inhibition of Syk or down-regulation of Syk expression reduces Aβ production and increases the clearance of Aβ across the BBB mimicking (−)-nilvadipine effects. Moreover, treatment of transgenic mice overexpressing Aβ and transgenic Tau P301S mice with a selective Syk inhibitor respectively decreased brain Aβ accumulation and Tau hyperphosphorylation at multiple AD relevant epitopes. We show that Syk inhibition induces an increased phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase-3β, a primary Tau kinase involved in Tau phosphorylation, by activating protein kinase A, providing a mechanism explaining the reduction of Tau phosphorylation at GSK3β-dependent epitopes following Syk inhibition. Altogether our data highlight Syk as a promising target for preventing both Aβ accumulation and Tau hyperphosphorylation in AD.     

Iron Necessity: The Secret of Wolbachia's Success?

The bacterium Wolbachia (order Rickettsiales) is probably the world''s most successful vertically-transmitted symbiont, distributed among a staggering 40% of terrestrial arthropod species. Wolbachia has great potential in vector control due to its ability to manipulate its hosts'' reproduction and to impede the replication and dissemination of arboviruses and other pathogens within haematophagous arthropods. In addition, the unexpected presence of Wolbachia in filarial nematodes of medical and veterinary importance has provided an opportunity to target the adult worms of Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis with safe drugs such as doxycycline. A striking feature of Wolbachia is its phenotypic plasticity between (and sometimes within) hosts, which may be underpinned by its ability to integrate itself into several key processes within eukaryotic cells: oxidative stress, autophagy, and apoptosis. Importantly, despite significant differences in the genomes of arthropod and filarial Wolbachia strains, these nexuses appear to lie on a continuum in different hosts. Here, we consider how iron metabolism may represent a fundamental aspect of host homeostasis that is impacted by Wolbachia infection, connecting disparate pathways ranging from the provision of haem and ATP to programmed cell death, aging, and the recycling of intracellular resources. Depending on how Wolbachia and host cells interact across networks that depend on iron, the gradient between parasitism and mutualism may shift dynamically in some systems, or alternatively, stabilise on one or the other end of the spectrum.     

Yield Results and Stability Analysis from the Sorghum Regional Biomass Feedstock Trial

Sorghum [Sorghum bicolor (L.) Moench] is one of four herbaceous dedicated bioenergy crops the U.S. Department of Energy identified as critical to annually produce one billion tons of dry biomass. Of these four crops, sorghum is unique as it is a drought-tolerant, annual crop established from seed that is readily tractable to genetic improvement. The purpose of this study was to assess the yield potential and stability of sorghums grown across diverse production environments in the USA. For this study, six sorghum genotypes (one cultivar, five hybrids) were grown in yield trials in seven locations in six states for 5 years (2008–2012). Variation in dry and fresh yield was attributable to not only genotypes, but also to the effects of year, location, and year × location. Even with the highest yielding genotype, environmental conditions were a major factor in determining the yield in a given year. This variability affects the consistency of the biomass supply for ethanol production. In general, the southeastern USA had the highest mean yields for fresh weight and dry weight, indicating that this area may be the most reliable for biomass production. A significant variation was detected among genotypes for fresh weight, dry weight, moisture content, and brix, revealing that sufficient variation within sorghum exists for continued improvement and that certain hybrids are more tractable for biomass/bioenergy production. With dedicated bioenergy sorghum germplasm and proper production environments, sorghum will be a valuable tool in the goal of the sustainable production of one billion tons of dry biomass each year in the USA.