Conventional myosins – unconventional functions

While the discovery of unconventional myosins raised expectations that their actions were responsible for most aspects of actin-based cell motility, few anticipated the wide range of cellular functions that would remain the purview of conventional two-headed myosins. The three nonsarcomeric, cellular myosins—M2A, M2B and M2C—participate in diverse roles including, but not limited to: neuronal dynamics, axon guidance and synaptic transmission; endothelial cell migration; cell adhesion, polarity, fusion and cytokinesis; vesicle trafficking and viral egress. These three conventional myosins each take on specific, differing functional roles during development and maturity, characteristic of each cell lineage; exact roles depend on the developmental stage of the cell, cellular location, upstream regulatory controls, relative isoform expression, orientation and associated state of the actin cytoscaffolds in which these myosins operate. Here, we discuss the separate yet related roles that characterise the actions of M2A, M2B and M2C in various cell types and show that these conventional myosins are responsible for functions as unconventional as any performed by unconventional myosins.     

Natural Products: An Alternative to Conventional Therapy for Dermatophytosis?

The increased incidence of fungal infections, associated with the widespread use of antifungal drugs, has resulted in the development of resistance, making it necessary to discover new therapeutic alternatives. Among fungal infections, dermatophytoses constitute a serious public health problem, affecting 20–25 % of the world population. Medicinal plants represent an endless source of bioactive molecules, and their volatile and non-volatile extracts are clearly recognized for being the historical basis of therapeutic health care. Because of this, the research on natural products with antifungal activity against dermatophytes has considerably increased in recent years. However, despite the recognized anti-dermatophytic potential of natural products, often advantageous face to commercial drugs, there is still a long way to go until their use in therapeutics. This review attempts to summarize the current status of anti-dermatophytic natural products, focusing on their mechanism of action, the developed pharmaceutical formulations and their effectiveness in human and animal models of infection.     

Conventional culture for water quality assessment: is there a future?

Conventional culture for the detection, enumeration and identification of micro-organisms has been the traditional tool of the microbiologist. It is, however, time-consuming and labour-intensive and confirmed results often require several days of analysis. Culture may not grow the organisms being sought and for enumeration may only detect a small proportion of the total population. However, it does have the advantage of being simple to use and relatively inexpensive. It is also a direct means of assessing cell viability. Novel fluorogenic dyes and fluorgenic and chromogenic substrates have overcome some of these problems by providing a means of rapid and specific detection and enumeration whilst removing the need for subculture and confirmation tests. Immunological tests such as ELISA have significantly reduced analysis time by providing specific target organism detection. Molecular techniques have removed the need for culture. Improvements in sensitivity, and removal of the inhibitory nature of sample matrices, have allowed analysts to detect low levels of micro-organisms but the questions of viability and comparability with cultural techniques still remain. Are we about to see a change of culture in water quality assessment, or can cultural techniques be developed that reduce analysis time to a few hours and can rapid methods be used for detecting the presence and viability of organisms?     

Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.     

Mimotopes for Alloreactive and Conventional T Cells in a Peptide–MHC Display Library

The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins.     

Conventional and novel Gγ protein families constitute the heterotrimeric G-protein signaling network in soybean

Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ proteins are important signal transducers in all eukaryotes. The Gγ protein of the G-protein heterotrimer is crucial for its proper targeting at the plasma membrane and correct functioning. Gγ proteins are significantly smaller and more diverse than the Gα and Gβ proteins. In model plants Arabidopsis and rice that have a single Gα and Gβ protein, the presence of two canonical Gγ proteins provide some diversity to the possible heterotrimeric combinations. Our recent analysis of the latest version of the soybean genome has identified ten Gγ proteins which belong to three distinct families based on their C-termini. We amplified the full length cDNAs, analyzed their detailed expression profile by quantitative PCR, assessed their localization and performed yeast-based interaction analysis to evaluate interaction specificity with different Gβ proteins. Our results show that ten Gγ genes are retained in the soybean genome and have interesting expression profiles across different developmental stages. Six of the newly identified proteins belong to two plant-specific Gγ protein families. Yeast-based interaction analyses predict some degree of interaction specificity between different Gβ and Gγ proteins. This research thus identifies a highly diverse G-protein network from a plant species. Homologs of these novel proteins have been previously identified as QTLs for grain size and yield in rice.     

Retained Cell�CCell Adhesion in Serrated Neoplastic Pathway as Opposed to Conventional Colorectal Adenomas

The molecular features of serrated polyps of colorectum remain to be elucidated. The expression pattern of adhesive molecules (E-cadherin, α-catenin, and β-catenin) has not been examined in serrated neoplastic pathway. The expression of E-cadherin, α-catenin, and β-catenin were analyzed by immunohistochemistry in 32 hyperplastic polyps (HPs), 28 sessile serrated adenomas (SSAs), 37 traditional serrated adenomas (TSAs), 51 traditional adenomas (TAs), and 10 normal colonic tissues (NCs). Retained membranous expression for E-cadherin, α-catenin, and β-catenin was more frequent in HPs, SSAs, and TSAs than that in TAs (p < 0.001). Nuclear labeling of β-catenin was detected in 19.6% of TAs, but in none of HPs, SSAs, and TSAs (p < 0.001). Cytoplasmic accumulation of β-catenin was found in 3.1% of HPs, 3.6% of SSAs, and 21.6% of TSAs, significantly lower than that in TAs (60.8%, p < 0.001). The membranous co-expression of E-cadherin, α-catenin, and β-catenin was more frequent in HPs (68.8%), SSAs (60.7%), and TSAs (37.8%) than that in TAs (7.8%, p < 0.001). Cell adhesion function is retained in serrated neoplastic pathway. Wnt signaling pathway plays a less active role in the development of colorectal serrated polys than in TAs.     

Relative Bioavailability of Manganese Proteinate for Broilers Fed a Conventional Corn–Soybean Meal Diet

An experiment was conducted to investigate the bioavailability of organic manganese proteinate (Mn) relative to inorganic Mn sulfate for broilers fed a conventional corn–soybean meal basal diet. A total of 448-day-old Arbor Acres commercial male chicks were fed the Mn-unsupplemented basal diet (control) or basal diet supplemented with 60, 120, or 180 mg Mn/kg from each Mn source. At 21 days of age, heart tissue was excised for testing DM, Mn concentration, manganese superoxide dismutase (MnSOD) activity, and MnSOD mRNA level. The Mn concentration, MnSOD activity, and MnSOD mRNA level in heart tissue increased (P < 0.01) linearly as dietary manganese concentration increased. Based on slope ratios from multiple linear regressions of the above three indices on added Mn level, there was no significant difference (P > 0.21) in bioavailability between Mn proteinate and Mn sulfate for broilers in this experiment.     

Formation of β-Lactoglobulin Nanofibrils by Microwave Heating Gives a Peptide Composition Different from Conventional Heating

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of β-lactoglobulin (β-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of β-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact β-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of β-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of β-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.     

Is There Still a Place for Conventional Amphotericin B in the Treatment of Neonatal Fungal Infections?

Neonatal invasive fungal infections (IFIs) remain an increasing problem associated with high rates of morbidity and mortality, as well as late-onset neurodevelopmental implications. Invasive candidiasis remains the leading neonatal IFI. Candida albicans is the fungal species most often affecting this population, although a changing epidemiologic incidence to non-albicans Candida species is reported in some neonatal intensive care units. Many treatment recommendations are extrapolated from adult populations, emphasizing the need to establish the optimal antifungal agent, dosage, and duration of therapy in neonates. Historically, conventional amphotericin B has been considered an efficient and safe treatment approach for most neonatal IFIs. More recently, lipid formulations of amphotericin B have been studied, used alone or in combination with other antifungal agents such as azoles or echinocandins. The aim of this article is to review the published experience in the use of amphotericin B formulations to treat neonatal IFIs.     

Conventional protein kinase C-α (PKC-α) and PKC-β negatively regulate RIG-I antiviral signal transduction

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K₆₃-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I''s ability to induce an antiviral IFN response, phosphorylation of RIG-I at S₈ or T₁₇₀ suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S₈ and T₁₇₀ phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-α (PKC-α) and PKC-β as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S₈ or T₁₇₀ potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-α/β are the primary kinases responsible for RIG-I S₈ and T₁₇₀ phosphorylation. Coimmunoprecipitation showed that PKC-α/β interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-α/β double-knockdown cells exhibited markedly decreased S₈/T₁₇₀ phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/β-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.     

Misfolded Amyloid Ion Channels Present Mobile β-Sheet Subunits in Contrast to Conventional Ion Channels

In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Aβ channels consist of the solid-state NMR-based U-shaped β-strand-turn-β-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that β-sheet channels break into loosely associated mobile β-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for β-sheet channels formed by cytolytic PG-1 β-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting α-helices that robustly prevent ion leakage, rather than hydrogen-bonded β-strands. The simulations suggest why conventional gated channels evolved to consist of interacting α-helices rather than hydrogen-bonded β-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.     

Yield and Economic Performance of Organic and Conventional Cotton-Based Farming Systems – Results from a Field Trial in India

The debate on the relative benefits of conventional and organic farming systems has in recent time gained significant interest. So far, global agricultural development has focused on increased productivity rather than on a holistic natural resource management for food security. Thus, developing more sustainable farming practices on a large scale is of utmost importance. However, information concerning the performance of farming systems under organic and conventional management in tropical and subtropical regions is scarce. This study presents agronomic and economic data from the conversion phase (2007–2010) of a farming systems comparison trial on a Vertisol soil in Madhya Pradesh, central India. A cotton-soybean-wheat crop rotation under biodynamic, organic and conventional (with and without Bt cotton) management was investigated. We observed a significant yield gap between organic and conventional farming systems in the 1^st crop cycle (cycle 1: 2007–2008) for cotton (−29%) and wheat (−27%), whereas in the 2^nd crop cycle (cycle 2: 2009–2010) cotton and wheat yields were similar in all farming systems due to lower yields in the conventional systems. In contrast, organic soybean (a nitrogen fixing leguminous plant) yields were marginally lower than conventional yields (−1% in cycle 1, −11% in cycle 2). Averaged across all crops, conventional farming systems achieved significantly higher gross margins in cycle 1 (+29%), whereas in cycle 2 gross margins in organic farming systems were significantly higher (+25%) due to lower variable production costs but similar yields. Soybean gross margin was significantly higher in the organic system (+11%) across the four harvest years compared to the conventional systems. Our results suggest that organic soybean production is a viable option for smallholder farmers under the prevailing semi-arid conditions in India. Future research needs to elucidate the long-term productivity and profitability, particularly of cotton and wheat, and the ecological impact of the different farming systems.     

Conventional and novel modes of exine patterning in members of the Araceae – the consequence of ecological paradigm shifts?

Summary. In the family Araceae, the members of all subfamilies except Aroideae follow the conventional mode of exine formation pattern, which conforms with the textbook view of sporoderm stratification and chemistry (sporopollenin ektexine formed before the endexine). Only members of the subfamily Aroideae show a quite uncommon mode of exine formation pattern, with an endexine formed prior to the nonsporopollenin, polysaccharidic outer exine layer. The intine is formed simultaneously with this non-sporopollenin layer. From the differing timetable and especially from the different origin it is concluded that this outer exine layer is not homologous to the angiosperm ektexine. The fundamental question, why members of the Aroideae lack an elaborated sporopollenin ektexine, is discussed in terms of functionality of the nonsporopollenin outer exine layer. It seems that a major change in aroid evolution took place at the point when the family phylogenetically and ecologically shifted from bisexual (most subfamilies) to unisexual flowers (Aroideae only). The hypothesis is that ephemeral spathes and the absence of sporopollenin are the consequence of an adaptive syndrome for a short pollination time window in many members of the Aroideae, with short-lived pollen, an energetically not costly pollen wall, rapid germination of pollen tube, and brief receptivity of stigma. Correspondence and reprints: Institute of Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria.     

Incommensurable Worldviews? Is Public Use of Complementary and Alternative Medicines Incompatible with Support for Science and Conventional Medicine?

Proponents of controversial Complementary and Alternative Medicines, such as homeopathy, argue that these treatments can be used with great effect in addition to, and sometimes instead of, ‘conventional’ medicine. In doing so, they accept the idea that the scientific approach to the evaluation of treatment does not undermine use of and support for some of the more controversial CAM treatments. For those adhering to the scientific canon, however, such efficacy claims lack the requisite evidential basis from randomised controlled trials. It is not clear, however, whether such opposition characterises the views of the general public. In this paper we use data from the 2009 Wellcome Monitor survey to investigate public use of and beliefs about the efficacy of a prominent and controversial CAM within the United Kingdom, homeopathy. We proceed by using Latent Class Analysis to assess whether it is possible to identify a sub-group of the population who are at ease in combining support for science and conventional medicine with use of CAM treatments, and belief in the efficacy of homeopathy. Our results suggest that over 40% of the British public maintain positive evaluations of both homeopathy and conventional medicine simultaneously. Explanatory analyses reveal that simultaneous support for a controversial CAM treatment and conventional medicine is, in part, explained by a lack of scientific knowledge as well as concerns about the regulation of medical research.     

Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Na?ve Conventional Neonatal and Weaned Pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID₅₀/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID₅₀/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID₅₀/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID₅₀/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID₅₀/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.     

Direct Contra Na?ve-Indirect Comparison of Clinical Failure Rates between High-Viscosity GIC and Conventional Amalgam Restorations: An Empirical Study

Background

Naïve-indirect comparisons are comparisons between competing clinical interventions’ evidence from separate (uncontrolled) trials. Direct comparisons are comparisons within randomised control trials (RCTs). The objective of this empirical study is to test the null-hypothesis that trends and performance differences inferred from naïve-indirect comparisons and from direct comparisons/RCTs regarding the failure rates of amalgam and direct high-viscosity glass-ionomer cement (HVGIC) restorations in permanent posterior teeth have similar direction and magnitude.

Methods

A total of 896 citations were identified through systematic literature search. From these, ten and two uncontrolled clinical longitudinal studies for HVGIC and amalgam, respectively, were included for naïve-indirect comparison and could be matched with three out twenty RCTs. Summary effects sizes were computed as Odds ratios (OR; 95% Confidence intervals) and compared with those from RCTs. Trend directions were inferred from 95% Confidence interval overlaps and direction of point estimates; magnitudes of performance differences were inferred from the median point estimates (OR) with 25% and 75% percentile range, for both types of comparison. Mann-Whitney U test was applied to test for statistically significant differences between point estimates of both comparison types.

Results

Trends and performance differences inferred from naïve-indirect comparison based on evidence from uncontrolled clinical longitudinal studies and from direct comparisons based on RCT evidence are not the same. The distributions of the point estimates differed significantly for both comparison types (Mann–Whitney U  =  25, n_indirect  =  26; n_direct  =  8; p  =  0.0013, two-tailed).

Conclusion

The null-hypothesis was rejected. Trends and performance differences inferred from either comparison between HVGIC and amalgam restorations failure rates in permanent posterior teeth are not the same. It is recommended that clinical practice guidance regarding HVGICs should rest on direct comparisons via RCTs and not on naïve-indirect comparisons based on uncontrolled longitudinal studies in order to avoid inflation of effect estimates.