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AbstractSheep production in Ethiopia is an important activity for smallholder farmers, but the sector has been challenged by many factors among which feed shortage is the major one. To solve this challenge, farmers traditionally supplement sheep with coffee leaf. This study was conducted at Areka Agricultural Research Center, Wolaita zone, Southern Ethiopia, with the objectives of evaluating carcass yield, composition and economic profitability of supplementing powdered coffee leaves to indigenous Wolaita highland sheep fed hay-based diets. This experiment followed the layout of Completely Randomized Design (CRD) on twenty male intact sheep with mean initial body weight 17.4 ± 0.1 kg and five treatments. Treatments were T1, T2, T3, T4, and T5 with powdered coffee leave meal inclusion (CLPMI) proportion of 0, 5, 10, 15, and 20%, respectively with four replications per treatment. Concentrate mix (CM) of 300 gm on dry matter base/head/day was provided for the lambs. The trial was conducted for 90 days followed by digestibility trial of 7 days. Significant difference (P

Malt Barley has a great contribution to food security and farm income in Debark district. Despite the contribution, there was a loss in Malt Barley production due to improper post-harvest handling at the farm level. Thus, this study assessed the extent of post-harvest loss in Malt Barley at different stages at the farm level and the factor affecting post-harvest loss of Malt Barley in Debark district. Three-stage purposive and simple random sampling techniques were applied to draw 223 samples. Tobit model was used to analyse factors affecting post-harvest loss at the farm level. The study result shows that post-harvest loss at the farm level was estimated to be 12.68%. The result from the Tobit model revealed that post-harvest loss was significantly influenced by variables such as family labour, education level, land allocated for Malt Barley, access to frequency of extension contact, storage facility, and volume of Malt Barley production. Therefore, expansion of adult and basic education for farmers, increasing the frequency of extension contact, the introduction of a harvesting machine, and proper storage facilities are expected to minimize post-harvest losses of Malt Barley at the farm level.

Abstract Background Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. Methods To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. Results We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Conclusions Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Almonds are susceptible to infestation by Aspergillus flavus , an aflatoxin-producing fungus. The objective of this study was to investigate the effects of kernel type (inshell, shelled and split almonds) on the ability of A. flavus to grow and produce aflatoxins at different combinations of temperature (20, 27 and 35 °C), water activity (0.85, 0.92, 0.95 and 0.98 a w ) and incubation period (10, 20 and 30 days). There was no fungal growth at 0.85 a w on any of the kernel types. At 0.92 a w , only the split kernels supported growth and aflatoxin synthesis. The fungus was able to grow and produce aflatoxins on all three kernels at 0.95-0.98 a w and 20-35 °C. At 0.98 a w , high total aflatoxin concentrations (>300 µg/kg) were found on the shelled and split kernels at all temperatures. On the inshell nuts, the fungus produced up to 372 µg/kg of total aflatoxins at 0.98 a w and 27 °C. Regression analysis showed that significantly higher levels of aflatoxins were produced at 27 °C (as compared to at 20 and 35 °C) on shelled and split almonds. Incubation time was also a significant predictor of aflatoxin accumulation. The results of this study indicated that shipping almonds below 0.85 a w and reducing storage time would significantly decrease the risk of infestation and aflatoxin production by A. flavus .

Abstract: Flax seed has become an increasingly popular food ingredient because of its nutrient richness as well as potential health benefits. Flax seeds are often ground before consumption, and flax seed cakes are used as animal feed. Aflatoxin production may occur subsequently when the ground seeds are stored in an environment that supports fungal growth. The objectives of this study were to determine the growth of two toxigenic fungi, Aspergillus flavus and A. parasiticus, and to quantify the concentrations of four major aflatoxins (AFB1, AFG1, AGB2, and AFG2) produced by the two fungi on ground flax seeds with water activities (aws) of 0.82, 0.86, 0.90, 0.94, and 0.98, incubated for 30 days at 20, 27, and 35°C. Results of the study showed that A. flavus was able to grow on ground seeds with aw from 0.86 to 0.98 at all three temperatures, and the most rapid growth occurred at aws 0.90 and 0.94 at 27°C. In comparison, A. parasiticus grew on seeds with aw from 0.86 to 0.98 at 27 and 35°C as well as on seeds with aw from 0.86 to 0.90 at 20°C, and the most favorable growth condition was aw from 0.90 to 0.94 at 35°C. A. flavus produced aflatoxins on seeds with aw from 0.90 to 0.94 at 27°C as well as on seeds with aw from 0.86 to 0.98 at 35°C, and the maximum total aflatoxin (298 μg/kg), AFB1 (247 μg/kg), and AFG1 (51 μg/kg) were found on seeds with aw 0.90 at 35°C. In comparison, A. parasiticus produced aflatoxins under a wider range of conditions, which included aw 0.86 at 27 and 35°C, aw 0.90 at 20 and 27°C, aw 0.94 at 27°C, and aw 0.98 at 35°C. The maximum total aflatoxin (364 μg/kg) and maximum AFB1 (324 μg/kg) along with 34 μg/kg AFG1 and 6 μg/kg AFB2 were produced by A. parasiticus on seeds with aw 0.98 incubated at 35°C for 30 days. Linear regression models also indicated that high incubation temperature (35°C) was optimal for overall fungal growth and for formation of high levels of aflatoxin by both fungi. Future studies should also address aflatoxin contamination in flax seed oil., (Copyright ©, International Association for Food Protection.)

Nyjer oil seed cake supports high levels of aflatoxin B1 (AFB1) production. AFB1 is a secondary metabolite of Aspergillus flavus and A. parasiticus, classified as a Class 1A carcinogen. The aim of this study was to determine the effects of temperature (20, 27, and 35 °C) and water activity (0.82, 0.86, 0.90, 0.94, and 0.98 a w ) on fungal growth and AFB1 production of A. flavus and A. parasiticus on ground Nyjer seeds over a 30-day incubation period. Linear regression models indicated that both fungal growth and AFB1 production were significantly influenced by water activity of Nyjer seeds and incubation temperature. The two fungi did not grow on Nyjer seeds at 0.82 a w at the three incubation temperatures. The most favorable growth conditions for both fungi were 0.90-0.98 a w at 27 °C or 0.90-0.94 a w at 35 °C. The optimum temperature for AFB1 production was 27 °C for both A. flavus and A. parasiticus (with regression coefficients of 6.01 and 9.11, respectively). Both fungi were likely to produce high levels of AFB1 at 0.90 a w (with regression coefficients of 3.56 for A. flavus and 7.17 for A. parasiticus). Aspergillus flavus only produced AFB1 on seeds with 0.90-0.98 a w at 27 °C (in the range of 203-282 μg/kg) and on seeds with 0.90 a w at 35 °C (212 μg/kg). No detectable AFB1 was produced by this fungus in any other culture conditions that were studied. Aspergillus parasiticus, in contrast, was able to produce AFB1 under all of the growth conditions. At 20 °C, this fungus produced the highest level of AFB1 (212 μg/kg) at high water activity (0.98 a w ). At 27 °C, A. parasiticus produced high levels of AFB1 (in the range of 209-265 μg/kg) at a wide range of water activities (0.86-0.98 a w ). In the entire study, the highest AFB1 concertation for A. parasiticus was detected on seeds incubated at high temperature (35 °C) and low water activity (0.86 a w ). The findings of this study could help optimize the storage conditions of Nyjer oil seeds to reduce aflatoxin contamination., (Copyright © 2019. Published by Elsevier B.V.)

Aspergillus fresenii and Aspergillus sulphureus produce ochratoxin A (OTA), which is a secondary metabolite of Aspergillus and Penicillium species, with nephrotoxic effects and potential carcinogenic activity. The aim of this study was to determine the effects of temperature (20, 30, and 37 °C), water activity (0.82, 0.86, 0.90, 0.94, and 0.98 a w ), incubation period (5, 10, 15, and 30 days) on fungal growth, and OTA production by A. fresenii and A. sulphureus on Nyjer oil seeds. There was no fungal growth at 0.82 a w . The two fungal species were able to produce OTA from the fifth day of incubation from 0.86 to 0.98 a w and temperature 20 to 37 °C. Aspergillus fresenii produced the highest concentration of OTA (643 μg/kg) at 0.90 a w and 37 °C within 15 days, while A. sulphureus produced the highest level of OTA (724 μg/kg) at 0.98 a w and 20 °C within 10 days. The optimum water activity and temperature for the growth of both fungi were similar at 0.94 a w and at 30 °C. There was statistically significant difference between the levels of OTA production among the two fungi. Overall, A. sulphureus produced significantly higher levels of OTA (p < 0.05). Higher temperature (37 °C) and 0.90-0.94 a w were optimal for OTA production by A. fresenii. Our results show that Nyjer seeds can support the growth of A. fresenii and A. sulphureus and OTA production, and the two species had similar temperature and water activity requirements for growth but different requirements for OTA production on Nyjer seeds.

Background: Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems., Methods: To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis., Results: We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A)., Conclusions: Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

Protein-protein interactions control signaling, specific adhesion, and many other biological functions. The three-dimensional structures of the interfaces and bound ligand can be approached with transferred nuclear Overhauser effect spectroscopy NMR, which can be applied to much larger proteins than conventional NMR and requires less concentrated protein. However, it is not clear how accurately the structures of protein-bound peptides can be determined by transferred nuclear Overhauser effect spectroscopy. We studied the structure of a biotin mimetic peptide (FSHPQNT) bound to streptavidin, because the X-ray structure of the complex is available to 1.74 Å resolution, and we found that conditions could be adjusted so that the off-rates were fast enough for transferred nuclear Overhauser effect spectroscopy NMR. The off-rate was determined with (19)F NMR, using a para-fluoro-phenylalanine analog of the peptide. A new criterion for a lower limit on kinetic off-rate was found, which allowed accurate structure determination at a slower off-rate. Non-specific binding of the peptide to streptavidin was not significant, because biotin blocked the peptide transferred nuclear Overhauser effect spectroscopy. Protein mediation for the long-range peptide transferred nuclear Overhauser effect spectroscopy cross-peaks was corrected by a transferred nuclear Overhauser effect spectroscopy/ROESY averaging procedure. The protein-bound structure of the peptide was determined by transferred nuclear Overhauser effect spectroscopy constrained and simulated annealing. The structure deduced from the NMR was close to the X-ray structure., (© 2011 John Wiley & Sons A/S.)

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.

Cdc42Hs is a member of the Ras superfamily of GTPases which, when active, initiates a cascade beginning with the activation of several kinases, including P(21)-activated kinase (PAK). We previously determined the structure of a complex between a 46 amino acid fragment peptide derived from the PAK binding domain (PBD46) and Cdc42Hs.GMPPCP (Gizachew, D., Guo, W., Chohan, K. K., Sutcliffe, M. J., and Oswald, R. E. (2000) Biochemistry 39, 3963-3971). Previous studies (Loh, A. P., Guo, W., Nicholson, L. K., and Oswald, R. E. (1999) Biochemistry 38, 12547-12557) suggest that the regions of Cdc42Hs that bind effectors and regulators have distinct dynamic properties from the remainder of the protein. Here, we describe the backbone dynamics of PBD46 bound to Cdc42Hs.GMPPCP. T(1), T(2), T(1)(rho), and steady-state nuclear Overhauser effects were measured at 500 and 600 MHz. An extension of the Lipari-Szabo model-free analysis was used to determine the order parameters (S(2)) and local correlation times (tau(e)) of the N-H bond vectors within PBD46. Both Cdc42Hs and PBD46 exhibit increased mobility in the free versus the bound state, suggesting that protein flexibility may be required for high-affinity PBD46 binding and, presumably, the activation of PAK. Different backbone dynamics were observed in different regions of the peptide. The beta-strand region of bound PBD46, which makes contacts with beta2 of Cdc42Hs, exhibits low mobility on the pico- to nanosecond timescale. However, the part of PBD46 that interacts with Switch I of Cdc42Hs exhibits greater mobility. Thus, PBD46 and Cdc42Hs form a tight complex that exhibits concerted dynamics.

Cdc42Hs is a member of the Ras superfamily of GTPases and initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously used a 46 amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs [Guo et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the three-dimensional solution structure of the Cdc42Hs. GMPPCP-PBD46 complex. Heteronuclear NMR methods were used to assign resonances in the complex, and approximately 2400 distance and dihedral restraints were used to calculate a set of 20 structures using a combination of distance geometry, simulated annealing, and chemical shift and Ramachandran refinement. The overall structure of Cdc42Hs in the complex differs from the uncomplexed structure in two major aspects: (1) the first alpha helix is reoriented to accommodate the binding of the peptide and (2) the regions corresponding to switch I and switch II are less disordered. As suggested by our previous work (Guo et al., 1998) and similar to the complex between Cdc42Hs and fACK [Mott et al. (1999) Nature 399, 384-388], PBD46 forms an intermolecular beta-sheet with beta2 of Cdc42Hs and contacts both switch I and switch II. The extensive binding surface between PBD46 and Cdc42Hs can account for both the high affinity of the complex and the inhibition by PBD46 of GTP hydrolysis.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

A peptide containing residues 36-59 of the human CD4 receptor includes most of the residues thought to be involved in binding the HIV surface glycoprotein, gp120. This peptide was synthesized and inhibited the binding of gp120 to soluble CD4. NMR relaxation experiments indicated that the peptide was in fast exchange between the free and gp120-bound states. Transferred NOESY NMR showed a number of long-range NOEs, from the gp120-bound state, between residues 38, 40, 45, 48, and 49 of the peptide. NMR evidence also suggested that the Phe43 in the peptide, which corresponds to a critical residue in CD4 for the binding of gp120, makes intimate contact with gp120. The Tr-NOESY cross-peak intensities provided proton-proton distance constraints on the conformation of the gp120-bound peptide. The distance constraints were used in simulated annealing, and a set of 20 very similar structures was obtained for the central region of the gp120-bound peptide. Residues 42-49 of the peptide formed a loop with the side chain of Phe43 pointing away from the rest of the peptide. This Phe43 ring points away from the protein surface in two structures of the amino-terminal domain of CD4 found by X-ray crystallography. Differences in the conformation of CD4 in the two crystal forms suggest that the 36-59 region might be flexible. The NMR data on the 36-59 CD4 peptide predicts a gp120-bound conformation different from either of the CD4 crystal forms in the absence of gp120.

During activation of the neutrophil NADPH oxidase, cytosolic p47(phox) is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91(phox). To investigate this binding site further, we studied the three-dimensional structure of a gp91(phox) C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47(phox) using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47(phox)-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91(phox) residues 555-564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5-Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47(phox) correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91(phox) binds to 47(phox) in an extended conformation between gp91(phox) residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.

Solid-state 13C nuclear magnetic resonance (NMR) spectra of a number of inclusion compounds of 2,6-dimethyl-bicyclo[3.3.1]nonane-exo-2-exo-6-diol (host) with small organic small molecules (guests) have been studied. With 3,4-dichloro-1,2,5-thiadiazole and tetrachloroethylene as guests, line splittings of the host resonances were observed due to the location of the guest in the host lattice. The cross-polarization (CP) dynamics of these inclusion compounds have been studied and shown to be indicative of weakly coupled systems. As expected, the proton spin lattice relaxation times in the rotating frame (T1pH) of the host are increased by the presence of rapidly moving guest because the efficiency of homonuclear dipolar relaxation in the rotating frame is reduced. However, strong transient oscillations were also observed for the guest molecules during the Hartmann-Hahn transfer of magnetisation from the more abundant 1H spins to the 13C spins during spin lattice rotating frame relaxation. These oscillations were found to be greatest for carbons with largest chemical shift anisotropies.