More efficient mastication allows increasing intake without compromising digestibility or necessitating a larger gut: Comparative feeding trials in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis)

The digestion of plant material in mammalian herbivores basically depends on the chemical and structural composition of the diet, the mean particle size to which the forage is processed, and the ingesta retention time. These different factors can be influenced by the animal, and they can presumably compensate for each other. The pygmy hippopotamus, a non-ruminating foregut fermenter, has longer mean retention times than ruminants; however hippos do not achieve higher (fibre) digestibilities on comparable diets, which could be due to ineffective mastication. We performed feeding trials with six pygmy hippos (Hexaprotodon liberiensis) and six banteng cattle (Bos javanicus) on a grass diet. As predicted, both species achieved similar dry matter, organic matter, crude protein and gross energy digestibilities. However, neutral and acid detergent fibre digestibility was lower in pygmy hippos. Apparently, in these species, fibre digestibility was more influenced by particle size, which was larger in pygmy hippos compared to banteng, than by retention time. In spite of their higher relative food intake, the banteng in this study did not have greater relative gut fills than the hippos. Ruminants traditionally appear intake-limited when compared to equids, because feed particles above a certain size cannot leave the rumen. But when compared to nonruminating foregut fermenters, rumination seems to free foregut fermenters from an intrinsic food intake limitation. The higher energy intakes and metabolic rates in wild cattle compared to hippos could have life-history consequences, such as a higher relative reproductive rate.     

Drosophila Transposon Insertions as Unknowns for Structured Inquiry Recombination Mapping Exercises in an Undergraduate Genetics Course

Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.INQUIRY-BASED learning in which students are engaged in open-ended, student-centered, hands-on activities is an important tool for training undergraduates to think like scientists (Colburn 2000; Handelsman et al. 2004). With this approach, students learn scientific subjects by interpreting and discussing experimental results in a fashion similar to that used by scientific researchers (NRC 2003). There are three main approaches to instruction via inquiry. In open inquiry, students formulate their own problem, as well as the procedures to investigate the problem. In guided inquiry, the instructor provides the problem and necessary materials, but the students devise an experimental procedure to investigate the problem. Finally, in structured inquiry, the instructor provides the problem, the materials, and the procedure, but the students are required to gather and interpret the experimental data independently, coming to their own conclusions (Welch et al. 2006). In each case, the instructor does not provide “the answer” to the problem. In the ideal case, the instructor does not even know what the answer will be prior to the student experiment, forcing the students to grapple with the information themselves. Inquiry-based laboratories can even be extended so that students are participating in novel research as part of their coursework (DebBurman 2002; Buckner et al. 2007), which been shown to improve undergraduate retention and student performance in biology lecture courses (Marcus et al. 2009).The process of inquiry has been identified as central to training students to understand fundamental approaches used in the field of genetics such as the design of controlled crosses and interpretation of experimental data (Cartier and Stewart 2000). Pukkila (2004) discusses effective methods by which inquiry-based learning can be incorporated into undergraduate genetics lecture courses with large enrollments and into recitation sections. However, the implementation of inquiry-based approaches in undergraduate genetics laboratories has not been discussed extensively.Teaching laboratories offer some advantages for inquiry learning because they generally contain small groups of students, facilitating a flexible and intimate learning environment with many interactions between students and the instructor, as well as among classmates. However, teaching laboratories associated with large lecture courses also offer some challenges, in particular how to deliver substantially similar experiences to laboratory sections taught by multiple instructors, as well as how to provide inquiry-based learning in a logistically manageable and cost-effective manner. For these reasons, most inquiry-based genetics laboratory exercises have used the structured inquiry approach, for example, using many Drosophila melanogaster strains with similar mutant phenotypes (e.g., white eyes and black bodies), but a variety of genotypes, in a series of standardized genetic mapping crosses to familiarize students with the collection and interpretation of classical genetic data (MacIntyre 1974; Pye 1980). The difficulty with contrived genetic unknowns carrying well-mapped genetic mutations is that many students become frustrated that their hard work evaluating the crosses over a period of several months is devoted to a fundamentally trivial exercise, as the recombination map locations of the genes are already established in the scientific literature and have been recalculated thousands of times by generations of genetics students.We have expanded upon the structured inquiry approach to genetics to include novel research experiences for the students in our undergraduate genetics laboratories. They conduct mapping crosses with Drosophila strains carrying P-element transposon insertions in genes without documented recombination map positions. The stock centers maintain very large collections of P-element transposon stocks with known insertion sites on the cytological and genome maps (Spradling et al. 1999). However, in spite of the cytology to recombination map equivalence table available in FlyBase (2009), very few of the transposon inserts have been formally placed on the recombination map. By using the eye color phenotypes associated with many transposon inserts as visible markers in genetic crosses (Marcus 2003), it is straightforward to calculate recombination map positions for the interrupted loci. The stock collections contain many stocks with identical transposons inserted at different chromosomal locations, providing a large number of unique, but equivalent genetic unknowns that can be used for recombination mapping exercises. At the same time, this approach provides students with the opportunity to map genes that have never been mapped before, allowing them to make small but useful contributions to the field of Drosophila genetics.     

Drosophila convoluted/dALS Is an Essential Gene Required for Tracheal Tube Morphogenesis and Apical Matrix Organization

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.     

Biogenic Synthesis, Purification, and Chemical Characterization of Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)

Enzymatically oxygenated derivatives of the ω-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197–1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677–14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S™ oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.Enzymatically formed oxygenation products of C-20 and C-22 long chain polyunsaturated fatty acids (LC-PUFAs),⁴ have important biological roles in inflammation, allergies, and blood clotting and are thus believed to have therapeutic potential in several chronic immune diseases (1–10) Several biologically important products of cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA), and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) have been described (4, 11, 12). Proinflammatory oxylipins, such as leukotrienes and some prostaglandins, are derived from ARA, an ω-6 fatty acid. Interestingly, the same fatty acid also serves as a precursor to anti-inflammatory or proresolution molecules like lipoxins (13, 14). Stable analogues of lipoxins are being developed as drugs for asthma and other inflammatory airway diseases (15, 16). Oxylipins derived from ω-3 fatty acids, such as DHA and EPA, known as resolvins, are primarily anti-inflammatory in nature (17). EPA acts as a precursor to the E-series resolvins that have shown potential in the treatment of colitis, arthritis, and periodontitis (18–20). The resolvins of the D-series derived from DHA are useful as neuroprotective agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or neuroprotectin D1 is a resolvin that is formed endogenously in the human brain and eye and is believed to exert its protective effect against cell injury-induced oxidative stress (21–23).The main enzymes responsible for the production of these oxygenated LC-PUFA products are primarily lipoxygenases and, in addition, cyclo-oxygenases and cytochromes P450. These enzymes produce oxylipins via transcellular activity, often involving multiple cell types (24). This activity mainly results in mono-, di-, and tri-hydroxylation products of fatty acids that have varying potencies, depending on the exact structure of the compound. Lipoxygenases are non-heme, iron-containing dioxygenases that catalyze the regioselective and enantioselective oxidation of polyunsaturated fatty acids containing one or more cis,cis-1,4-pentadienoic moieties to give the corresponding hydroperoxy derivatives (25, 26). We thus considered that, in addition to DHA and EPA, other C-22 PUFAs containing such methylene interrupted double bonds may also be substrates for lipoxygenases and that resulting products may have anti-inflammatory activity similar to DHA-derived resolvins. DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present in algal oils, and recent studies have demonstrated that this fatty acid has anti-inflammatory activities in vitro and, in conjunction with DHA, also has anti-inflammatory activity in vivo.⁵ Also, it has been suggested that a combination of DHA and DPAn-6 could be a beneficial natural therapy in neuroinflammatory conditions like Alzheimer disease. Specifically, in a 3×Tg-AD mouse model of Alzheimer disease, DPAn-6 was shown to reduce levels of early stage phospho-Tau epitopes, which in turn correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a putative Tau kinase (27). Although the precise mechanism of action of DPAn-6 in these inflammatory milieus is not known, it suggests a possible role for oxylipin products of DPAn-6 in resolution of inflammation. Also, another LC-PUFA, DPAn-3 (cis-7,10,13,16,19-docosapentaenoic acid) usually present along with DHA and EPA in marine oils is known to be a potent inhibitor of platelet aggregation (28–30). In addition, this LC-PUFA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 (vascular endothelial-cell growth factor receptor 2) expression. Angiogenesis is known to contribute to tumor growth, inflammation, and microangiopathy, again pointing to the possibility that anti-inflammatory activity of DPAn-3 might be mediated through resolvin-like products as in the case of DHA and EPA (31).The purpose of this research was to determine whether oxylipins are formed from the C-22 LC-PUFAs, DPAn-6 and DPAn-3, by lipoxygenase activity; to compare them to products formed from DHA; to chemically characterize products; to purify key oxylipin products from the DPAn-6/15-lipoxygenase reaction; and to test whether these compounds have resolvin-like anti-inflammatory activity. This research also sets the stage for preparation and isolation of a wide range of other C-22 oxylipins that could be evaluated as potential anti-inflammatory compounds.     

Common processes in pathogenesis by fungal and oomycete plant pathogens,described with Gene Ontology terms

Plant diseases caused by fungi and oomycetes result in significant economic losses every year. Although phylogenetically distant, the infection processes by these organisms share many common features. These include dispersal of an infectious particle, host adhesion, recognition, penetration, invasive growth, and lesion development. Previously, many of these common processes did not have corresponding Gene Ontology (GO) terms. For example, no GO terms existed to describe processes related to the appressorium, an important structure for infection by many fungi and oomycetes. In this mini-review, we identify common features of the pathogenic processes of fungi and oomycetes and create a pathogenesis model using 256 newly developed and 38 extant GO terms, with an emphasis on the appressorium and signal transduction. This set of standardized GO terms provides a solid base to further compare and contrast the molecular underpinnings of fungal and oomycete pathogenesis.     

The True Value of HbA1c as a Predictor of Diabetic Complications: Simulations of HbA1c Variables

Aim

The updated mean HbA1c has been used in risk estimates of diabetic complications, but it does not take into account the temporal relationship between HbA1c and diabetic complications. We studied whether the updated mean HbA1c underestimated the risk of diabetic complications.

Method

Continuous HbA1c curves for 10,000 hypothetical diabetes patients were simulated over an average of 7 years. Simulations were based on HbA1c values encountered in clinical practice. We assumed that each short time interval of the continuous HbA1c curves had a long-lasting effect on diabetic complications, as evidenced by earlier studies. We tested several different HbA1c variables including various profiles, e.g. different duration, of such a long-lasting effect. The predictive power of these variables was compared with that of the updated mean HbA1c.

Results

The predictive power of the constructed HbA1c variables differed considerably compared to that of the updated mean HbA1c. The risk increase per standard deviation could be almost 100% higher for a constructed predictor than the updated mean HbA1c.

Conclusions

The importance of good glycemic control in preventing diabetic complications could have been underestimated in earlier hallmark studies by not taking the time-dependent effect of HbA1c into account.     

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na⁺ and K⁺, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K⁺ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K⁺ milieu and in vivo when the endocochlear K⁺-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K⁺-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.