Global DNA Adenine Methylation in Caenorhabditis elegans after Multigenerational Exposure to Silver Nanoparticles and Silver Nitrate.

Multigenerational and transgenerational reproductive toxicity in a model nematode Caenorhabditis elegans has been shown previously after exposure to silver nanoparticles (Ag-NPs) and silver ions (AgNO3). However, there is a limited understanding on the transfer mechanism of the increased reproductive sensitivity to subsequent generations. This study examines changes in DNA methylation at epigenetic mark N6-methyl-2'-deoxyadenosine (6mdA) after multigenerational exposure of C. elegans to pristine and transformed-via-sulfidation Ag-NPs and AgNO3. Levels of 6mdA were measured as 6mdA/dA ratios prior to C. elegans exposure (F0) after two generations of exposure (F2) and two generations of rescue (F4) using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Although both AgNO3 and Ag-NPs induced multigenerational reproductive toxicity, only AgNO3 exposure caused a significant increase in global 6mdA levels after exposures (F2). However, after two generations of rescue (F4), the 6mdA levels in AgNO3 treatment returned to F0 levels, suggesting other epigenetic modifications may be also involved. No significant changes in global DNA methylation levels were observed after exposure to pristine and sulfidized sAg-NPs. This study demonstrates the involvement of an epigenetic mark in AgNO3 reproductive toxicity and suggests that AgNO3 and Ag-NPs may have different toxicity mechanisms.

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.

KEY MESSAGE: Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

2-Aminobenzothiazoles Inhibit Virulence Gene Expression and Block Polymyxin Resistance in Salmonella enterica.

One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg2+ , a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella's resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.

A Call for Better Integrated Policies to Accelerate the Fertility Decline in Sub-Saharan Africa.

To accelerate their demographic transition, sub-Saharan African (SSA) countries must trigger significant and rapid fertility declines. These fertility declines will open a demographic window of opportunity and enable countries to capture a first demographic dividend. Despite some successes, many programs aimed at decreasing fertility in SSA have yielded disappointing results. This commentary argues that better integrated policies will help to accelerate fertility declines in SSA. Such an approach should harness the synergies between a set of four key policy levers: women's empowerment; female education; family planning, reproductive health services, and universal health coverage; and legal reforms. These policy levers should be coupled with renewed commitment from leaders on the continent and increased investments in both family planning and population institutions. By concomitantly investing in these priorities and harnessing the synergies of these policy levers, countries in SSA have a critical opportunity to accelerate their demographic transition, which could help them reach emerging economy status.

The structure of DcrB, a lipoprotein from Salmonella enterica, reveals flexibility in the N-terminal segment of the Mog1p/PsbP-like fold.

DcrB is an 18 kDa lipoprotein that contains a single domain of unknown function. DcrB is found within Enterobacteriaceae, a family of Gram-negative bacteria which includes pathogens that can cause food-borne illness and hospital-acquired infections. In Salmonella enterica serovar Typhimurium, DcrB is up-regulated by conditions that promote the production of known virulence factors. We determined the structure of a truncated form of DcrB from Salmonella to 1.92 Šresolution by X-ray crystallography. This truncated form, DcrBΔ37, contains the entire domain of unknown function but lacks the lipoprotein signal sequence (residues 1-20) as well as residues 21-37. The DcrBΔ37 monomer contains the Mog1p/PsbP-like fold, which is found in functionally diverse proteins in mammals, yeast, plants, and cyanobacteria. Interestingly, DcrBΔ37 crystallized as a domain-swapped homodimer in which the N-terminal ß-hairpin extends from one protomer to interact with the core of the second protomer. This domain-swapping indicates that the N-terminal portion of the Mog1p/PsbP-like fold likely has conformational flexibility. Overall, our results provide the first example of an enterobacterial protein that contains the Mog1p/PsbP-like fold and expands knowledge of the structural and phylogenetic diversity of Mog1p/PsbP-like proteins.

Inhibition of 5α-reductase alters pregnane metabolism in the late pregnant mare.

In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metabolism (pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol and allopregnanolone), to determine circulating dutasteride concentrations and to assess effects of dutasteride treatment on gestational parameters. Pregnant mares (n = 5) received dutasteride (0.01 mg/kg/day, IM) and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until parturition. Concentrations of dutasteride, pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol, and allopregnanolone were evaluated via liquid chromatography-tandem mass spectrometry. Samples were analyzed as both days post treatment and as days prepartum. No significant treatment effects were detected in pregnenolone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol or allopregnanolone for either analysis; however, progesterone concentrations were increased (P < 0.05) sixfold in dutasteride-treated mares compared to control mares. Dutasteride concentrations increased in the treated mares, with a significant correlation (P < 0.05) between dutasteride concentrations and pregnenolone or progesterone concentrations. Gestational length and neonatal outcomes were not significantly altered in dutasteride-treated mares. Although 5α-reduced metabolites were unchanged, these data suggest an accumulation of precursor progesterone with inhibition of 5α-reductase, indicating the ability of dutasteride to alter progesterone metabolism.

Reaction Decoder Tool (RDT): extracting features from chemical reactions.

UNLABELLED: Extracting chemical features like Atom-Atom Mapping (AAM), Bond Changes (BCs) and Reaction Centres from biochemical reactions helps us understand the chemical composition of enzymatic reactions. Reaction Decoder is a robust command line tool, which performs this task with high accuracy. It supports standard chemical input/output exchange formats i.e. RXN/SMILES, computes AAM, highlights BCs and creates images of the mapped reaction. This aids in the analysis of metabolic pathways and the ability to perform comparative studies of chemical reactions based on these features. AVAILABILITY AND IMPLEMENTATION: This software is implemented in Java, supported on Windows, Linux and Mac OSX, and freely available at https://github.com/asad/ReactionDecoder CONTACT: : asad@ebi.ac.uk or s9asad@gmail.com.

Frequency of satisfaction and dissatisfaction with practice among rural-based, group-employed physicians and non-physician practitioners.

BACKGROUND: Widespread dissatisfaction among United States (U.S.) clinicians could endanger ongoing reforms. Practitioners in rural/underserved areas withstand stressors that are unique to or accentuated in those settings. Medical professionals employed by integrating delivery systems are often distressed by the cacophony of organizational change(s) that such consolidation portends. We investigated the factors associated with dis/satisfaction with rural practice among doctors/non-physician practitioners employed by an integrated healthcare delivery network serving 9 counties of upstate New York, during a time of organizational transition. METHODS: We linked administrative data about practice units with cross-sectional data from a self-administered multi-dimensional questionnaire that contained practitioner demographics plus valid scales assessing autonomy/relatedness needs, risk aversion, tolerance for uncertainty/ambiguity, meaningfulness of patient care, and workload. We targeted medical professionals on the institutional payroll for inclusion. We excluded those who retired, resigned or were fired during the study launch, plus members of the advisory board and research team. Fixed-effects beta regressions were performed to test univariate associations between each factor and the percent of time a provider was dis/satisfied. Factors that manifested significant fixed effects were entered into multivariate, inflated beta regression models of the proportion of time that practitioners were dis/satisfied, incorporating clustering by practice unit as a random effect. RESULTS: Of the 473 eligible participants. 308 (65.1 %) completed the questionnaire. 59.1 % of respondents were doctoral-level; 40.9 % mid-level practitioners. Practitioners with heavier workloads and/or greater uncertainty intolerance were less likely to enjoy top-quintile satisfaction; those deriving greater meaning from practice were more likely. Higher meaningfulness and gratified relational needs increased one's likelihood of being in the lowest quintile of dissatisfaction; heavier workload and greater intolerance of uncertainty reduced that likelihood. Practitioner demographics and most practice unit characteristics did not manifest any independent effect. CONCLUSIONS: Mutable factors, such as workload, work meaningfulness, relational needs, uncertainty/ambiguity tolerance, and risk-taking attitudes displayed the strongest association with practitioner satisfaction/dissatisfaction, independent of demographics and practice unit characteristics. Organizational efforts should be dedicated to a redesign of group-employment models, including more equitable division of clinical labor, building supportive peer networks, and uncertainty/risk tolerance coaching, to improve the quality of work life among rural practitioners.

Community views and public health priority setting: how do health department priorities, community views, and health indicator data compare?

OBJECTIVES: New York, like many other states, provides county-level health statistics for use in local priority settings but does not provide any data on public views about priority health issues. This study assessed whether health department priorities are notably different from community concerns about health, and how both groups' priorities compare with local health statistics. METHOD: Data from a 2009 rural survey on community health concerns were compared to priorities named by the seven area county health departments, and to local health indicator data. RESULTS: Health care/insurance cost (60%), obesity (53%), and prescription cost (41%) were leading community concerns, regardless of age, education, sex, or Internet in the home. Six of seven county health departments selected access to quality health care (which includes health care/insurance cost) as a leading public health priority, but only three identified obesity. The following leading local health issues were suggested by health indicators: Physical activity and nutrition, Smoking, and Unintentional injury. CONCLUSIONS: Health departments diverged from community priorities, from health indicator data, and from one another in choosing priorities. Adding a question about community health priorities to existing state telephone surveys on health behavior and lifestyle would provide an important tool to local health departments.

Conflicting roles for a cell surface modification in Salmonella.

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O-antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram-negative bacteria. We determined that activation of the PmrA/PmrB two-component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA-activated gene wzz(st), which encodes the protein that determines long O-antigen chain length and confers resistance to complement-mediated killing. By contrast, this growth defect did not require the wzz(fepE) gene, which controls production of very long O-antigen, or other PmrA-activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O-antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer-membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O-antigen in its LPS to respond to the different conditions it faces during infection.

Metingear: a development environment for annotating genome-scale metabolic models.

UNLABELLED: Genome-scale metabolic models often lack annotations that would allow them to be used for further analysis. Previous efforts have focused on associating metabolites in the model with a cross reference, but this can be problematic if the reference is not freely available, multiple resources are used or the metabolite is added from a literature review. Associating each metabolite with chemical structure provides unambiguous identification of the components and a more detailed view of the metabolism. We have developed an open-source desktop application that simplifies the process of adding database cross references and chemical structures to genome-scale metabolic models. Annotated models can be exported to the Systems Biology Markup Language open interchange format. AVAILABILITY: Source code, binaries, documentation and tutorials are freely available at http://johnmay.github.com/metingear. The application is implemented in Java with bundles available for MS Windows and Macintosh OS X.

Evaluation of a community-based participatory farmworker eye health intervention in the "black dirt" region of New York state.

BACKGROUND: Eye irritation is a constant hazard for migrant and seasonal farmworkers, but there are few studies of the problem or how to address it. Researchers evaluated the effect of a community-based participatory eye health intervention on farmworker eye symptoms in the Hudson Valley, NY. METHODS: A randomized pre-post intervention with 2, 4-week follow-up periods was implemented with a sample of 97 farmworkers. Five eye symptoms were measured, along with utilization of protective eyewear and eye drops. RESULTS: Leading baseline eye symptoms were redness (49%), blurred vision (43%), itching (43%), and eye pain (29%). Significant reductions in eye pain (P = 0.009), and non-significant reductions in redness were observed for the intervention group while controls experienced increases in both. CONCLUSIONS: The intervention was effective in significantly reducing eye pain, and to a lesser extent, redness. Future eyewear promotion programs should offer a range of eye wear, tailor offerings to local climate and tasks, evaluate eyewear durability, and include eye drops.

Counseling on lifestyle habits in the United States and Sweden: a report comparing primary care health professionals' perspectives on lifestyle counseling in terms of scope, importance and competence.

BACKGROUND: The role of primary care professionals in lifestyle counseling for smoking, alcohol consumption, physical activity, and diet is receiving attention at the national level in many countries. The U. S. and Sweden are two countries currently establishing priorities in these areas. A previously existing international research collaboration provides a unique opportunity to study this issue. METHODS: Data from a national survey in Sweden and a study in rural Upstate New York were compared to contrast the perspectives, attitudes, and practice of primary care professionals in the two countries. Answers to four key questions on counseling for tobacco use, alcohol consumption, physical activity, and eating habits were compared. RESULTS: The response rates were 71% (n=180) and 89% (n=86) in the Sweden and the U.S. respectively. U.S. professionals rated counseling "very important" significantly more frequently than Swedish professionals for tobacco (99% versus 92%, p<.0001), physical activity (90% versus 79%, p=.04), and eating habits (86% versus 69%, p=.003). U.S. professionals also reported giving "very much" counseling more frequently for these same three endpoints than did the Swedish professionals (tobacco 81% versus 38%, p<.0001, physical activity 64% versus 31%, p<.0001, eating 59% versus 34%, p=.0001). Swedish professionals also rated their level of expertise in providing counseling significantly lower than did their U.S. counterparts for all four endpoints. A higher percentage of U.S. professionals expressed a desire to increase levels of counseling "very much", but only significantly so for eating habits (42% versus 28%, p=.037). CONCLUSIONS: The study demonstrates large differences between the extent that Swedish and American primary care professionals report being engaged in counseling on lifestyle issues, how important they perceive counseling to be, and what expertise they possess in this regard. Explanations might be found in inter-professional attitudes, the organization of healthcare, including the method of reimbursement, traditions of preventive healthcare, and cultural differences between the two countries. Further studies are needed to explore these questions, with the aim of facilitating improved lifestyle counseling in primary care.

The healthy start partnership: an approach to obesity prevention in young families.

This article investigates the impact of community-based interventions developed by the Healthy Start Partnership (HSP) to promote healthy body weights in families. Intercept surveys were conducted to monitor community exposure. A nonconcurrent, no treatment control design was used to assess population-level weight outcomes. Control (n = 219) and intervention (n = 276) cohorts of pregnant women were recruited and followed until 6 months postpartum. Data were collected through 2 self-administered questionnaires and medical record audits. Results indicate community residents were exposed to interventions. However, little evidence of positive effects of interventions on weight outcomes was found for mothers or infants.

Levodopa attenuates the feed intake reduction caused by ergot alkaloids in cattle.

Consumption of ergot alkaloids from endophyte-infected tall fescue results in losses to the livestock industry in many countries and a means to mitigate these losses is needed. The objective of this study was to evaluate intra-abomasal infusion of the dopamine precursor, levodopa (L-DOPA), on dopamine metabolism, feed intake, and serum metabolites of steers exposed to ergot alkaloids. Twelve Holstein steers (344.9 ±â€…9.48 kg) fitted with ruminal cannula were housed with a cycle of heat challenge during the daytime (32 °C) and thermoneutral at night (25 °C). The steers received a basal diet of alfalfa cubes containing equal amounts of tall fescue seed composed of a mixture of endophyte-free (E-) or endophyte-infected tall fescue seeds (E+) equivalent to 15 µg ergovaline/kg body weight (BW) for 9 d followed by intra-abomasal infusion of water (L-DOPA-) or levodopa (L-DOPA+; 2 mg/kg BW) for an additional 9 d. Afterward, the steers were pair-fed for 5 d to conduct a glucose tolerance test. The E+ treatment decreased (P = 0.005) prolactin by approximately 50%. However, prolactin increased (P = 0.050) with L-DOPA+. Steers receiving E+ decreased (P < 0.001) dry matter intake (DMI); however, when supplemented with L-DOPA+ the decrease in DMI was less severe (L-DOPA × E, P = 0.003). Also, L-DOPA+ infusion increased eating duration (L-DOPA × E, P = 0.012) when steers were receiving E+. The number of meals, meal duration, and intake rate were not affected (P > 0.05) by E+ or L-DOPA+. The L-DOPA+ infusion increased (P < 0.05) free L-DOPA, free dopamine, total L-DOPA, and total dopamine. Conversely, free epinephrine and free norepinephrine decreased (P < 0.05) with L-DOPA+. Total epinephrine and total norepinephrine were not affected (P > 0.05) by L-DOPA+. Ergot alkaloids did not affect (P > 0.05) circulating free or total L-DOPA, dopamine, or epinephrine. However, free and total norepinephrine decreased (P = 0.046) with E+. Glucose clearance rates at 15 to 30 min after glucose infusion increased with L-DOPA+ (P < 0.001), but not with E+ (P = 0.280). Administration of L-DOPA as an agonist therapy to treat fescue toxicosis provided a moderate increase in DMI and eating time and increased plasma glucose clearance for cattle dosed with E+ seed.

Fescue has become the dominant cool-season perennial grass in the southeastern region of the United States and is also found in other countries. Endophytes from a plant­fungus symbiotic relationship produce toxic alkaloids that have caused significant annual economic losses to the livestock industry. Treatments to alleviate this toxicosis are still demanded. This study evaluates the infusion of the dopamine precursor, levodopa (L-DOPA), to mitigate the toxicosis caused by ergot alkaloids. When L-DOPA was infused, eating duration increased and the decrease in feed intake caused by ergot alkaloids was less severe. Additionally, circulating dopamine and glucose clearance increased with L-DOPA. These results suggest that L-DOPA has the potential to aid in the mitigation of the toxicosis caused by ergot alkaloids.

UDP-galactopyranose mutase in nematodes.

Nematodes represent a diverse phylum of both free living and parasitic species. While the species Caenorhabditis elegans is a valuable model organism, parasitic nematodes or helminths pose a serious threat to human health. Indeed, helminths cause many neglected tropical diseases that afflict humans. Nematode glycoconjugates have been implicated in evasive immunomodulation, a hallmark of nematode infections. One monosaccharide residue present in the glycoconjugates of several human pathogens is galactofuranose (Galf). This five-membered ring isomer of galactose has not been detected in mammals, making Galf metabolic enzymes attractive therapeutic targets. The only known pathway for biosynthetic incorporation of Galf into glycoconjugates depends upon generation of the glycosyl donor UDP-Galf by the flavoenzyme uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM or Glf). A putative UGM encoding gene (glf-1) was recently identified in C. elegans. We sought to assess the catalytic activity of the corresponding gene product (CeUGM). CeUGM catalyzes the isomerization of UDP-Galf and UDP-galactopyranose (UDP-Galp). In the presence of enzyme, substrate, and a hydride source, a galactose-N5-FAD adduct was isolated, suggesting the CeUGM flavin adenine dinucleotide (FAD) cofactor serves as a nucleophile in covalent catalysis. Homology modeling and protein variants indicate that CeUGM possesses an active site similar to that of prokaryotic enzymes, despite the low sequence identity (∼15%) between eukaryotic and prokaryotic UGM proteins. Even with the primary sequence differences, heterocyclic UGM inhibitors developed against prokaryotic proteins also inhibit CeUGM activity. We postulate that inhibitors of CeUGM can serve as chemical probes of Galf in nematodes and as anthelmintic leads. The available data suggest that CeUGM facilitates the biosynthetic incorporation of Galf into nematode glycoconjugates through generation of the glycosyl donor UDP-Galf.

The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

Alterations in Carbohydrate Quantities in Freeze-Dried, Relative to Fresh or Frozen Maize Leaf Disks.

For various reasons, leaves are occasionally lyophilized prior to storage at -80 °C and preparing extracts. Soluble carbohydrate identity and quantity from maize leaf disks were ascertained in two separate years using anion exchange HPLC with pulsed electrochemical detection. Analyses were made from disks after freezing in liquid nitrogen with or without subsequent lyophilization (both years) or directly after removal from plants with or without lyophilization (only in the second year). By adding the lyophilizing step, galactose content consistently increased and, frequently, so did galactoglycerols. The source of the galactose increase with the added lyophilizing step was not due to metabolizing raffinose, as the raffinose synthase (rafs) null mutant leaves, which do not make that trisaccharide, also had a similar increase in galactose content with lyophilization. Apparently, the ester linkages attaching free fatty acids to galactoglycerolipids of the chloroplast are particularly sensitive to cleavage during lyophilization, resulting in increases in galactoglycerols. Regardless of the galactose source, a systematic error is introduced for carbohydrate (and, most likely, also chloroplast mono- or digalactosyldiacylglycerol) amounts when maize leaf samples are lyophilized prior to extraction. The recognition of lyophilization as a source of galactose increase provides a cautionary note for investigators of soluble carbohydrates.

Red clover supplementation modifies rumen fermentation and promotes feed efficiency in ram lambs.

Red clover produces isoflavones, including biochanin A, which have been shown to have microbiological effects on the rumen while also promoting growth in beef cattle. The objective was to determine if supplementation of biochanin A via red clover hay would produce similar effects on the rumen microbiota and improve growth performance of lambs. Twenty-four individually-housed Polypay ram lambs (initial age: 114 ± 1 d; initial weight: 38.1 ± 0.59 kg) were randomly assigned to one of three experimental diets (85:15 concentrate:roughage ratio; N = 8 rams/treatment): CON-control diet in which the roughage component (15.0%, w/w, of the total diet) consisted of orchardgrass hay; 7.5-RC-red clover hay substituted for half (7.5%, w/w, of the total diet) of the roughage component; and 15-RC-the entire roughage component (15.0%, w/w, of the total diet) consisted of red clover hay. Feed intake and weight gain were measured at 14-d intervals for the duration of the 56-d trial, and rumen microbiological measures were assessed on days 0, 28, and 56. Red clover supplementation impacted growth performance of ram lambs. Average daily gains (ADG) were greater in ram lambs supplemented with red clover hay (7.5-RC and 15-RC) than for those fed the CON diet (P < 0.05). Conversely, dry matter intake (DMI) was lower in 7.5-RC and 15-RC than for CON lambs (P = 0.03). Differences in ADG and DMI resulted in greater feed efficiency in ram lambs supplemented with red clover hay (both 7.5-RC and 15-RC) compared to CON (P < 0.01). Rumen microbiota were also altered by red clover supplementation. The total viable number of hyper-ammonia-producing bacteria in 7.5-RC and 15-RC decreased over the course of the experiment and were lower than CON by day 28 (P ≤ 0.04). Amylolytic bacteria were also lower in 15-RC than in CON (P = 0.03), with a trend for lower amylolytic bacteria in 7.5-RC (P = 0.08). In contrast, there was tendency for greater cellulolytic bacteria in red clover supplemented lambs than in CON (P = 0.06). Red clover supplementation also increased fiber utilization, with greater ex vivo dry matter digestibility of hay for both 7.5-RC and 15-RC compared to CON by day 28 (P < 0.03). Results of this study indicate that low levels of red clover hay can elicit production benefits in high-concentrate lamb finishing systems through alteration of the rumen microbiota.

Red clover is rich in the bioactive isoflavone, biochanin A. The goal was to evaluate the impacts of biochanin A supplementation via red clover hay on growth performance of ram lambs as well as the rumen microbiota and fermentation. Low levels of red clover hay inclusion (7.5% and 15.0%, w/w, of the total diet) in high-concentrate finishing diets improved feed efficiency of ram lambs, promoting weight gain while decreasing feed intake. Red clover hay supplementation suppressed ruminal protein-wasting, peptide- and amino-acid degrading and starch-utilizing bacteria compared to control diets without isoflavones. Red clover hay also promoted fiber degrading bacteria and fiber utilization. Lamb growth and microbiological effects of red clover were consistent regardless of supplementation level in the diet. Results of this study indicate that low levels of red clover hay can produce production benefits in lamb finishing systems and demonstrated the efficacy of red clover as a functional feed, or feed with biological activities, in the context of its traditional use as a forage feedstuff.