Meta-analysis of apparent ruminal synthesis and postruminal flow of B vitamins in dairy cows.

As milk production has significantly increased over the past decade(s), existing estimates of the B-vitamin needs of the modern dairy cow are currently being reconsidered, as suboptimal B-vitamin supply may affect metabolic efficiency. At the same time, however, "true" (i.e., biologically active forms, excluding nonfunctional analogs) B-vitamin supply also cannot be adequately estimated by dietary intake, as the rumen microbiota has been shown to play a significant role in synthesis and utilization of B vitamins. Given their complex impact on the metabolism of dairy cows, incorporating these key nutrients into the next generation of mathematical models could help to better predict animal production and performance. Therefore, the purpose of this study was to generate hypotheses of regulation in the absence of supplemental B vitamins by creating empirical models, through a meta-analysis, to describe true B-vitamin supply to the cow (postruminal flow, PRF) and apparent ruminal synthesis (ARS). The database used for this meta-analysis consisted of 340 individual cow observations from 15 studies with 16 experiments, where diet and postruminal digesta samples were (post hoc) analyzed for content of B vitamins (B1, B2, B3, B6, B9, B12). Equations of univariate and multivariate linear form were considered. Models describing ARS considered dry matter intake (DMI, kg/d), B-vitamin dietary concentration [mg/kg of dry matter (DM)] and rumen-level variables such as rumen digestible neutral detergent fiber (NDF) and starch (g/kg of DM), total volatile fatty acids (VFA, mM), acetate, propionate, butyrate, and valerate molar proportions (% of VFA), mean pH, and fractional rates of degradation of NDF and starch (%/h). Models describing PRF considered dietary-level driving variables such as DMI, B-vitamin dietary concentration (mg/kg of DM), starch and crude protein (g/kg of DM) and forage NDF (g/kg of DM). Equations developed were required to contain all significant slope parameters and contained no significant collinearity between driving variables. Concordance correlation coefficient was used to evaluate the models on the developmental data set due to data scarcity. Overall, modeling ARS yielded better-performing models compared with modeling PRF, and DMI was included in all prediction equations as a scalar variable. The B-vitamin dietary concentration had a negative effect on the ARS of B1, B2, B3, and B6 but increased the PRF of B2 and B9. The rumen digestible NDF concentration had a negative effect on the ARS of B2, B3, and B6, whereas rumen digestible starch concentration had a negative effect on the ARS of B1 and a positive effect on the ARS of B9. In the best prediction models, the dietary starch increased PRF of B1, B2, and B9 but decreased PRF of B12. The equations developed may be used to better understand the effect of diet and ruminal environment on the true supply of B vitamins to the dairy cow and stimulate the development of better-defined requirements in the future.

Cyanotoxin Analysis and Amino Acid Profiles of Cyanobacterial Food Items from Chad.

In some parts of the world, cyanobacteria are used as a food in the human diet, due to their ready availability. Lake Chad, has long been a traditional site for the collection of Arthrospira fusiformis which is dried and processed at the lake into thin wafers called Dihé for later consumption or is transported to market for sale. However, Dihé purchased from markets in Chad has not been analyzed for known cyanobacterial toxins or assessed for total amino acid content. Since BMAA in traditional foodstuffs of the indigenous Chamorro people of Guam causes neurodegenerative illness, it is important that Dihé from Chad be analyzed for this neurotoxin. BMAA and its isomer AEG were not detected in our analyses, but a further isomer DAB was detected as both a free and bound amino acid, with an increase in the free concentration after acid hydrolysis of this fraction. Microcystins were present in 6 samples at up to 20 µg/g according to UPLC-PDA, although their presence could not be confirmed using PCR for known microcystin synthetic genes. Amino acid analysis of the cyanobacterial material from Chad showed the presence of large amounts of canonical amino acids, suggesting that this may supplement indigenous people on low protein diets, although regular monitoring of the foodstuffs for the presence of cyanotoxins should be performed.

Toxin Analysis of Freshwater Cyanobacterial and Marine Harmful Algal Blooms on the West Coast of Florida and Implications for Estuarine Environments.

Recent marine and freshwater algal and cyanobacterial blooms in Florida have increased public concern and awareness of the risks posed by exposure to these organisms. In 2018, Lake Okeechobee and the Caloosahatchee river, on the west coast of Florida, experienced an extended bloom of Microcystis spp. and a bloom of Karenia brevis in the coastal waters of the Gulf of Mexico that coincided in the Fort Myers area. Samples from the Caloosahatchee at Fort Myers into Pine Island Sound and up to Boca Grande were collected by boat. High concentrations of microcystin-LR were detected in the cyanobacterial bloom along with brevetoxins in the marine samples. Furthermore, ß-N-methylamino-L-alanine (BMAA) and isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) were detected in marine diatoms and dinoflagellates, and cyanobacteria of freshwater origin. High freshwater flows pushed the cyanobacterial bloom to barrier island beaches and Microcystis and microcystins could be detected into the marine environment at a salinity of 41 mS/cm. For comparison, in 2019 collections of Dapis (a new generic segregate from Lyngbya) mats from Sarasota showed high concentrations of BMAA, suggesting the possibility of long-term exposure of residents to BMAA. The findings highlight the potential for multiple, potentially toxic blooms to co-exist and the possible implications for human and animal health.

L-Serine-Mediated Neuroprotection Includes the Upregulation of the ER Stress Chaperone Protein Disulfide Isomerase (PDI).

The unfolded protein response (UPR) is a highly evolutionarily conserved response to endoplasmic reticulum (ER) stress, which functions to return cells to homeostasis or send them into apoptosis, depending on the degree of cellular damage. ß-N-methylamino-L-alanine (L-BMAA) has been shown to induce ER stress in a variety of models and has been linked to several types of neurodegenerative disease including Guamanian amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). L-Serine, an amino acid critical for cellular metabolism and neurological signaling, has been shown to be protective against L-BMAA-induced neurotoxicity in both animal and cell culture models. While the mechanisms of L-BMAA neurotoxicity have been well characterized, less is known about L-serine neuroprotection. We recently reported that L-serine and L-BMAA generate similar differential expression profiles in a human ER stress/UPR array, despite L-serine being neuroprotective and L-BMAA being linked to neurodegenerative disease. Here, we further investigate the mechanism(s) of L-serine-induced UPR dysregulation by examining key genes and proteins in the ER stress/UPR pathways. We report that L-serine selectively increased protein disulfide isomerase (PDI) protein translation, an ER chaperone involved in refolding misfolded proteins, suggesting it may be modulating the UPR to favor recovery from ER stress. This constitutes a new mechanism for L-serine-mediated neuroprotection and has implications for its use as a therapy for neurodegenerative illnesses.

L-Serine: a Naturally-Occurring Amino Acid with Therapeutic Potential.

In human neuroblastoma cell cultures, non-human primates and human beings, L-serine is neuroprotective, acting through a variety of biochemical and molecular mechanisms. Although L-serine is generally classified as a non-essential amino acid, it is probably more appropriate to term it as a "conditional non-essential amino acid" since, under certain circumstances, vertebrates cannot synthesize it in sufficient quantities to meet necessary cellular demands. L-serine is biosynthesized in the mammalian central nervous system from 3-phosphoglycerate and serves as a precursor for the synthesis of the amino acids glycine and cysteine. Physiologically, it has a variety of roles, perhaps most importantly as a phosphorylation site in proteins. Mutations in the metabolic enzymes that synthesize L-serine have been implicated in various human diseases. Dosing of animals with L-serine and human clinical trials investigating the therapeutic effects of L-serine support the FDA's determination that L-serine is generally regarded as safe (GRAS); it also appears to be neuroprotective. We here consider the role of L-serine in neurological disorders and its potential as a therapeutic agent.

Rapid Communication: 16S ribosomal ribonucleic acid characterization of liver abscesses in feedlot cattle from three states in the United States.

Liver abscesses are a major economic burden to beef producers. Although a few causative organisms have been cultured from purulent material, the full polymicrobial diversity of liver abscesses has not been reported. The objective of this study was to characterize purulent material collected from liver abscess in beef cattle produced in different production systems in 3 cattle producing states in the United States using 16S rRNA gene sequencing. Differences between purulent material microbial communities among geographic region of feeding and application of a common antimicrobial were also investigated. Cattle included in the study were fed in California (dairy type) and Colorado and Texas (both beef type). Liver abscesses from a cross section of feedlots, geographic areas, and tylosin phosphate-administered groups were collected at harvest; DNA from 34 liver abscess samples was extracted; and the V4 region of the 16S rRNA gene was amplified and sequenced. Sequences were classified into 5 phyla, 13 classes, and 17 orders in the domain Bacteria. The phyla identified included Bacteroidetes (35.2% of reads), Proteobacteria (28.6%), Fusobacteria (18.2%), Firmicutes (12.4%), and Actinobacteria (5.5%). Sequences matching the genera and , which have previously been identified as causative agents in liver abscesses, were both present in the abscess bacterial communities at a relative abundance of 15.1 and 3.2%, respectively, of the overall relative abundance. Furthermore, 3 of the most common phyla were Gram-negative bacteria. An analysis-of-similarities test was conducted on Euclidean distances to assess differences between cattle treated and not treated with tylosin as well as to assess differences between geographic regions. Geographical region and treatment with tylosin did affect the microbiome ( = 0.002 and = 0.026 respectively); however, a more robust sample scheme is needed to explore these differences. To our knowledge, this is the first publication describing the complex community of liver purulent material using next generation sequencing in cattle. These data provide a framework for research on a more targeted approach to liver abscess prevention and treatment.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

Exogenous essential amino acids stimulate an adaptive unfolded protein response in the mammary glands of lactating cows.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Abrupt weaning reduces postweaning growth and is associated with alterations in gastrointestinal markers of development in dairy calves fed an elevated plane of nutrition during the preweaning period.

The benefits of feeding elevated quantities of milk to dairy calves have been well established. However, there is a reluctance to adopt this method of feeding in commercial dairy production because of concerns around growth, health, and ruminal development during weaning. The objective of this study was to characterize the effect of an abrupt (0 d step-down) or gradual (12 d step-down) feeding scheme when calves are fed an elevated plane of nutrition (offered 1.35 kg of milk replacer/d). For this experiment, a total of 54 calves were randomly assigned to an abrupt or a gradual weaning protocol before weaning at 48 d of life. Calves were housed and sampled in individual pens for the duration of the experiment, and milk, starter, and straw intake were measured on a daily basis. Body weight was measured every 6 d, whereas blood, rumen fluid, and fecal samples were collected on d 36 (pre-step-down), 48 (preweaning), and 54 (postweaning) of the experiment. Although the growth rates of the step-down calves were lower from d 37 to weaning (0.62 ± 0.04 vs. 1.01 ± 0.04 kg/d), the postweaning average daily gain was greater compared with the group that was abruptly weaned (0.83 ± 0.06 vs. 0.22 ± 0.06 kg/d). Total ruminal volatile fatty acid was greater in the step-down group on the day of weaning (d 48; 59.80 ± 2.25 vs. 45.01 ± 2.25 mmol), whereas the fecal starch percentage was lower during postweaning compared with the abruptly weaned calves (d 54; 3.31 ± 0.76 vs. 6.34 ± 0.76%). Analysis of the digestive tract of bull calves on d 55 revealed minimal differences between gross anatomy measurements of gut compartments as well as no morphological differences in rumen papillae development, yet the total mass of rumen when full of contents was larger in the step-down calves (7.83 ± 0.78 vs. 6.02 ± 0.78 kg). Under the conditions of this study, the results showcase the benefits of a step-down feeding strategy from an overall energy balance standpoint, due to increased adaptation of the gastrointestinal tract preweaning.

Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

The influence of age and weaning on permeability of the gastrointestinal tract in Holstein bull calves.

Fourteen Holstein bull calves were used in a randomized complete block design to investigate the effect of calf age and weaning on permeability of the gastrointestinal tract (GIT). Calves were randomly assigned to 1 of 2 treatments: (1) a weaning protocol that was initiated on d 35; WN; n=7), or (2) a control treatment where calves were not weaned (CON; n=7). Calves were bottle-fed milk replacer (150 g/L), in 3 equal portions/d targeting 15% of their body weight (BW) in liquid milk intake [approximately 21.1g/kg of BW/d, dry matter (DM) basis]. On d 35, the amount of milk replacer offered to WN calves was reduced to 7.5% of BW for 7 d before calves were weaned on d 42. On d 14, 28, and 42, calves were orally dosed with 500 mL of Cr-EDTA (179 mM Cr-EDTA solution) and housed in a metabolism crate to enable total urine collection and determination of total urinary Cr recovery as an indicator of total-tract permeability. On d 44, calves were killed and tissues from the rumen, omasum, duodenum, jejunum, ileum, cecum, and proximal and distal colon were collected, rinsed, and transported in buffer solution (pH 7.4 at 38.5°C). Tissues were incubated in Ussing chambers under short-circuit conditions with buffer solutions designed to mimic the mucosal and serosal energy source that would be available in vivo (glucose for tissues from the small intestine and short-chain fatty acids for tissues that would be exposed to fermentation; rumen, omasum, and large intestinal tissues). The serosal to mucosal flux of (14)C-mannitol and (3)H-inulin was measured for each region. Although we detected treatment × period interactions for BW and starter intake, dietary treatments did not differ within a week. Overall, the time that ruminal pH was <5.5 was less before weaning than after weaning. We observed a differential response for the appearance of Cr in urine for WN and CON calves, where the appearance of Cr (mg/48 h) in urine decreased for both treatments from d 14 to 28, but increased from d 28 to 42 for WN, whereas Cr appearance continued to decrease for CON. The flux of mannitol and inulin did not differ between treatments but did differ among region of the GIT, with rumen, duodenum, and jejunum having the greatest permeability. These data suggest that permeability of the GIT decreases with age but weaning may disrupt this process. The rumen, duodenum, and jejunum appear to be the regions with greatest permeability.

Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.

Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5d with saline, 563g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6×6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Bε (eIF2Bε). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Bε abundance suggests that an mTOR-mediated upregulation of eIF2Bε translation occurred. Despite increased mTOR/eIF2Bε signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Bε as a regulator of milk protein yield is warranted.

Technical note: Three-dimensional imaging of rumen tissue for morphometric analysis using micro-computed tomography.

Rumen development in calves has been evaluated by measuring papillae length, width, and density using microscopy for over 50 yr. Although common in the literature, disadvantages to this method exist, such as large variations in rumen papillae size and shape, small numbers of total papillae being measured, and the time required. The objective of this study was to develop a more effective technique for assessing rumen papillae using micro-computed tomography (micro-CT) and to compare this technique with microscopy. Rumen tissue was collected from the ventral sac of 20 postweaned bull calves at 55 d of age, immediately fixed in 10% neutral buffered formalin for 48 h, and stored in 70% ethanol at 4°C before the contrast enhancement. After evaluation of contrast-enhancement protocols, it was determined that mercury chloride provided the most pronounced contrast for accurate micro-CT imaging based on relative density of the papillae. A 1-cm(2) tissue section from the ventral sac of all bull calves was tensioned on a rapid prototyped curved plastic holder and imaged at 4 5 µm resolution for 56 min using a GE Locus Explore micro-CT (General Electric, Milwaukee, WI). MicroView V2.2 software (General Electric) was used to create a 3-dimensional virtual model of the entire sample. The length and width of papillae were measured 3-dimensionally and compared with measurements of papillae under the light microscope taken from the same region. The length and width measurements using micro-CT (2.47 ± 0.12 and 0.55 ± 0.01 mm) compared with light microscope (2.96 ± 0.03 and 0.86 ± 0.01 mm) were significantly smaller. The difference may reflect a more accurate determination in the base of the rumen tissue with micro-CT or the specificity of mercury chloride to bind only to intact rumen tissue. The mean number of papillae per centimeter squared viewed using micro-CT was 128.5 ± 33.9 with a total surface area of 681.8 ± 112.4 mm(2) and volume of 156 mm(3) per sample. Micro-CT data demonstrated that surface area and volume are positively associated and that papillae length was negatively associated with papillae per centimeter squared and positively associated with total volume of tissue section. This study represents the first time that micro-CT has been being used to assess morphology of rumen tissue. Micro-CT has the potential to improve the accuracy and efficiency of rumen tissue measurements; however, more standardization of each factor involved in tissue preparation, imaging, and location of papillae measurements is required.

K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

DNA repair is essential for maintaining genomic stability, and defects in this process significantly increase the risk of cancer. Clear-cell renal cell carcinoma (CCRCC) caused by inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is characterized by high genomic instability. However, the molecular mechanism underlying the association between the loss of VHL and genomic instability remains unclear. Here, we show that suppressor of cytokine signaling 1 (SOCS1) promotes nuclear redistribution and K63-ubiquitylation of VHL in response to DNA double-strand breaks (DSBs). Loss of VHL or VHL mutations that compromise its K63-ubiquitylation attenuates the DNA-damage response (DDR), resulting in decreased homologous recombination repair and persistence of DSBs. These results identify VHL as a component of the DDR network, inactivation of which contributes to the genomic instability associated with CCRCC.

Amino acid neurotoxins in feathers of the Lesser Flamingo, Phoeniconaias minor.

The Lesser Flamingo (Phoeniconaias minor) is known to use cyanobacteria (primarily Arthrospira) as a major food source in the East African Rift Valley lakes. Periodically, mass mortalities have occurred, associated with the cyanobacterial toxins (cyanotoxins), microcystins and anatoxin-a. Deposition of these cyanotoxins into P. minor feathers has been shown to occur, consistent with the presence of cyanotoxins in the livers, stomach and faecal contents after dietary intake. As cyanobacteria have been shown to also produce the neurotoxins ß-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), stored wing feathers, previously recovered from flamingos which had been exposed to microcystins and anatoxin-a and had subsequently died, were analysed for these neurotoxic amino acids. Trace amounts of BMAA were detected in extracts from Lake Nakuru flamingo feathers, with DAB also present at concentrations between 3.5 and 8.5 µg g(-1) dry weight in feathers from both lakes. Toxin recovery by solid-phase extraction of feather digests was tested with spiked deuterated BMAA and showed good recovery when analysed by LC-MS/MS (80-94%). This is the first report of these neurotoxic amino acids in birds. We discuss the origin and significance of DAB, alongside other cyanotoxins of dietary origin, in the feathers of the Lesser Flamingo.

Historical stocking data and 19th century DNA reveal human-induced changes to native diversity and distribution of cutthroat trout.

Many species are threatened with extinction and efforts are underway worldwide to restore imperilled species to their native ranges. Restoration requires knowledge of species' historical diversity and distribution. For some species, many populations were extirpated or individuals moved beyond their native range before native diversity and distribution were documented, resulting in a lack of accurate information for establishing restoration goals. Moreover, traditional taxonomic assessments often failed to accurately capture phylogenetic diversity. We illustrate a general approach for estimating regional native diversity and distribution for cutthroat trout in the Southern Rocky Mountains. We assembled a large archive of historical records documenting human-mediated change in the distribution of cutthroat trout (Oncorhynchus clarkii) and combined these data with phylogenetic analysis of 19th century samples from museums collected prior to trout stocking activities and contemporary DNA samples. Our study of the trout in the Southern Rocky Mountains uncovered six divergent lineages, two of which went extinct, probably in the early 20th century. A third lineage, previously declared extinct, was discovered surviving in a single stream outside of its native range. Comparison of the historical and modern distributions with stocking records revealed that the current distribution of trout largely reflects intensive stocking early in the late 19th and early 20th century from two phylogenetically and geographically distinct sources. Our documentation of recent extinctions, undescribed lineages, errors in taxonomy and dramatic range changes induced by human movement of fish underscores the importance of the historical record when developing and implementing conservation plans for threatened and endangered species.

Cyanotoxins in desert environments may present a risk to human health.

There have been few studies concerning cyanotoxins in desert environments, compared with the multitude of studies of cyanotoxins in aquatic environments. However, cyanobacteria are important primary producers in desert environments, where after seasonal rains they can grow rapidly both stabilising and fertilising arid habitats. Samples of cyanobacteria from wadis - dry, ephemeral river beds - and sabkha - supertidal salt flats - in Qatar were analysed for the presence of microcystins, nodularin, anatoxin-a, cylindrospermopsin and anatoxin-a(S). Microcystins were detected by HPLC-PDA and ELISA at concentrations between 1.5 and 53.7ngg(-1) dry wt of crust. PCR products for the mycD gene for microcystin biosynthesis were detected after amplification of DNA from desert crust samples at two out of three sample sites. The presence of anatoxin-a(S) was also indicated by acetylcholine esterase inhibition assay. As a function of area of desert crust, microcystin concentrations were between 3 and 56µgm(-2). Based on the concentration of microcystins detected in crust, with reference to the published inhalation NOAEL and LOAEL values via nasal spray inhalation of purified microcystin-LR in aqueous solution, and the amount of dust potentially inhaled by a person from these dried crusts, the dose of microcystins could exceed a calculated TDI value of 1-2ngkg(-1)day(-1) for an average adult. The presence of microcystins, and potentially of anatoxin-a(S), in desert crusts has important implications for human health. Further studies are required to monitor desert dust storms for the presence of cyanotoxins. An understanding of the risks of inhaling particles containing cyanotoxins is also warranted.

Nitrogen starvation of cyanobacteria results in the production of ß-N-methylamino-L-alanine.

ß-N-Methylamino-L-alanine, an unusual amino acid implicated in neurodegenerative disease, has been detected in cultures of nearly all genera of environmentally ubiquitous cyanobacteria tested. The compound is present within cyanobacterial cells in free and protein-associated forms, with large variations occurring in the concentration of these pools between species as well as within single strains. With a lack of knowledge and supporting data on the regulation of BMAA production and the role of this compound in cyanobacteria, the association between BMAA and cyanobacteria is still subject to debate. In this study we investigated the biosynthesis of BMAA in axenic non-diazotrophic cyanobacterial cultures using the stable isotope ¹5N. Nitrogen starvation of nutritionally replete cells resulted in an increase in free cellular ¹5N BMAA suggesting that BMAA may be the result of catabolism to provide nitrogen or that BMAA is synthesised to serve a functional role in the cell in response to nitrogen deprivation. The addition of NO3⁻ and NH4⁺ to the culture medium following starvation resulted in a decrease of free cellular BMAA without a corresponding increase in the protein-associated fraction. The use of ammonia as a nitrogen source resulted in a more rapid reduction of BMAA when compared to nitrate. This study provides the first data regarding the regulation of intracellular BMAA concentrations in cyanobacteria with results conclusively showing the production of ¹5N BMAA by an axenic cyanobacterial culture.

Distinguishing the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) from other diamino acids.

ß-N-methylamino-L-alanine (BMAA) is produced by diverse taxa of cyanobacteria, and has been detected by many investigators who have searched for it in cyanobacterial blooms, cultures and collections. Although BMAA is distinguishable from proteinogenic amino acids and its isomer 2,4-DAB using standard chromatographic and mass spectroscopy techniques routinely used for the analysis of amino acids, we studied whether BMAA could be reliably distinguished from other diamino acids, particularly 2,6-diaminopimelic acid which has been isolated from the cell walls of many bacterial species. We used HPLC-FD, UHPLC-UV, UHPLC-MS, and triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) to differentiate BMAA from the diamino acids 2,6-diaminopimelic acid, N-2(amino)ethylglycine, lysine, ornithine, 2,4-diaminosuccinic acid, homocystine, cystine, tryptophan, as well as other amino acids including asparagine, glutamine, and methionine methylsulfonium.

Water administration of the medium-chain fatty acid caprylic acid produced variable efficacy against enteric Campylobacter colonization in broilers.

Campylobacter is one of the most common causes of foodborne illness, and poultry are considered a primary source of Campylobacter infections. Caprylic acid, an 8-carbon fatty acid, has been shown in previous studies to reduce enteric cecal Campylobacter concentrations in poultry when administered in the feed. For greater ease of application for producers, a water-soluble form of caprylic acid, sodium octanoate, was evaluated for efficacy against enteric Campylobacter. The first trial consisted of 70 birds in 7 groups (n = 10 chicks/group): an untreated control and 6 other groups that were challenged with Campylobacter at d 3 and that received 0, 0.175, 0.35, 0.7, 1.4, or 2.8% water-soluble caprylic acid in water 3 d before necropsy at d 14. The second trial consisted of 80 birds in 8 groups (n = 10 chicks/group): an untreated negative control and 7 other groups, all of which were challenged with Campylobacter at d 3 and received 0, 0.044, 0.088, 0.175, 0.35, 0.7, or 1.4% water-soluble caprylic acid for 3 d before necropsy at d 14. In trial 1, only the 0.175% dose caused a reduction in cecal Campylobacter counts in comparison with the positive control (approximately a 3-log reduction). In trial 2, no treatment reduced Campylobacter counts compared with the positive control. Unlike the efficacy of caprylic acid in feed, treatment with caprylic acid in water had an inconsistent effect on intestinal Campylobacter counts.