Hydrologic Landscape Regionalisation Using Deductive Classification and Random Forests

Landscape classification and hydrological regionalisation studies are being increasingly used in ecohydrology to aid in the management and research of aquatic resources. We present a methodology for classifying hydrologic landscapes based on spatial environmental variables by employing non-parametric statistics and hybrid image classification. Our approach differed from previous classifications which have required the use of an a priori spatial unit (e.g. a catchment) which necessarily results in the loss of variability that is known to exist within those units. The use of a simple statistical approach to identify an appropriate number of classes eliminated the need for large amounts of post-hoc testing with different number of groups, or the selection and justification of an arbitrary number. Using statistical clustering, we identified 23 distinct groups within our training dataset. The use of a hybrid classification employing random forests extended this statistical clustering to an area of approximately 228,000 km² of south-eastern Australia without the need to rely on catchments, landscape units or stream sections. This extension resulted in a highly accurate regionalisation at both 30-m and 2.5-km resolution, and a less-accurate 10-km classification that would be more appropriate for use at a continental scale. A smaller case study, of an area covering 27,000 km², demonstrated that the method preserved the intra- and inter-catchment variability that is known to exist in local hydrology, based on previous research. Preliminary analysis linking the regionalisation to streamflow indices is promising suggesting that the method could be used to predict streamflow behaviour in ungauged catchments. Our work therefore simplifies current classification frameworks that are becoming more popular in ecohydrology, while better retaining small-scale variability in hydrology, thus enabling future attempts to explain and visualise broad-scale hydrologic trends at the scale of catchments and continents.     

Polyandry and multiple paternities in the threatened Agassiz’s desert tortoise, <Emphasis Type="Italic">Gopherus agassizii</Emphasis>

We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.     

Conditional deletion of focal adhesion kinase leads to defects in ventricular septation and outflow tract alignment

To examine a role for focal adhesion kinase (FAK) in cardiac morphogenesis, we generated a line of mice with a conditional deletion of FAK in nkx2-5-expressing cells (herein termed FAKnk mice). FAKnk mice died shortly after birth, likely resulting from a profound subaortic ventricular septal defect and associated malalignment of the outflow tract. Additional less penetrant phenotypes included persistent truncus arteriosus and thickened valve leaflets. Thus, conditional inactivation of FAK in nkx2-5-expressing cells leads to the most common congenital heart defect that is also a subset of abnormalities associated with tetralogy of Fallot and the DiGeorge syndrome. No significant differences in proliferation or apoptosis between control and FAKnk hearts were observed. However, decreased myocardialization was observed for the conal ridges of the proximal outflow tract in FAKnk hearts. Interestingly, chemotaxis was significantly attenuated in isolated FAK-null cardiomyocytes in comparison to genetic controls, and these effects were concomitant with reduced tyrosine phosphorylation of Crk-associated substrate (CAS). Thus, it is possible that ventricular septation and appropriate outflow tract alignment is dependent, at least in part, upon FAK-dependent CAS activation and subsequent induction of polarized myocyte movement into the conal ridges. Future studies will be necessary to determine the precise contributions of the additional nkx2-5-derived lineages to the phenotypes observed.     

Optimizing Network Connectivity for Mobile Health Technologies in sub-Saharan Africa

Background

Mobile health (mHealth) technologies hold incredible promise to improve healthcare delivery in resource-limited settings. Network reliability across large catchment areas can be a major challenge. We performed an analysis of network failure frequency as part of a study of real-time adherence monitoring in rural Uganda. We hypothesized that the addition of short messaging service (SMS+GPRS) to the standard cellular network modality (GPRS) would reduce network disruptions and improve transmission of data.

Methods

Participants were enrolled in a study of real-time adherence monitoring in southwest Uganda. In June 2011, we began using Wisepill devices that transmit data each time the pill bottle is opened. We defined network failures as medication interruptions of >48 hours duration that were transmitted when network connectivity was re-established. During the course of the study, we upgraded devices from GPRS to GPRS+SMS compatibility. We compared network failure rates between GPRS and GPRS+SMS periods and created geospatial maps to graphically demonstrate patterns of connectivity.

Results

One hundred fifty-seven participants met inclusion criteria of seven days of SMS and seven days of SMS+GPRS observation time. Seventy-three percent were female, median age was 40 years (IQR 33–46), 39% reported >1-hour travel time to clinic and 17% had home electricity. One hundred one had GPS coordinates recorded and were included in the geospatial maps. The median number of network failures per person-month for the GPRS and GPRS+SMS modalities were 1.5 (IQR 1.0–2.2) and 0.3 (IQR 0–0.9) respectively, (mean difference 1.2, 95%CI 1.0–1.3, p-value<0.0001). Improvements in network connectivity were notable throughout the region. Study costs increased by approximately $1USD per person-month.

Conclusions

Addition of SMS to standard GPRS cellular network connectivity can significantly reduce network connection failures for mobile health applications in remote areas. Projects depending on mobile health data in resource-limited settings should consider this upgrade to optimize mHealth applications.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

A Quantitative Diffuse Reflectance Imaging (QDRI) System for Comprehensive Surveillance of the Morphological Landscape in Breast Tumor Margins

In an ongoing effort to address the clear clinical unmet needs surrounding breast conserving surgery (BCS), our group has developed a next-generation multiplexed optical-fiber-based tool to assess breast tumor margin status during initial surgeries. Specifically detailed in this work is the performance and clinical validation of a research-grade intra-operative tool for margin assessment based on diffuse optical spectroscopy. Previous work published by our group has illustrated the proof-of-concept generations of this device; here we incorporate a highly optimized quantitative diffuse reflectance imaging (QDRI) system utilizing a wide-field (imaging area = 17cm²) 49-channel multiplexed fiber optic probe, a custom raster-scanning imaging platform, a custom dual-channel white LED source, and an astronomy grade imaging CCD and spectrograph. The system signal to noise ratio (SNR) was found to be greater than 40dB for all channels. Optical property estimation error was found to be less than 10%, on average, over a wide range of absorption (μ_a = 0–8.9cm^-1) and scattering (μ_s’ = 7.0–9.7cm^-1) coefficients. Very low inter-channel and CCD crosstalk was observed (2% max) when used on turbid media (including breast tissue). A raster-scanning mechanism was developed to achieve sub-pixel resolution and was found to be optimally performed at an upsample factor of 8, affording 0.75mm spatially resolved diffuse reflectance images (λ = 450–600nm) of an entire margin (area = 17cm²) in 13.8 minutes (1.23cm²/min). Moreover, controlled pressure application at the probe-tissue interface afforded by the imaging platform reduces repeated scan variability, providing <1% variation across repeated scans of clinical specimens. We demonstrate the clinical utility of this device through a pilot 20-patient study of high-resolution optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient. An empirical cumulative distribution function (eCDF) analysis is used to reduce optical property maps to quantitative distributions representing the morphological landscape of breast tumor margins. The optimizations presented in this work provide an avenue to rapidly survey large tissue areas on intra-operative time scales with improved sensitivity to regions of focal disease that may otherwise be overlooked.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.