Effects of Rumen-Protected L-Tryptophan Supplementation on Productivity, Physiological Indicators, Blood Profiles, and Heat Shock Protein Gene Expression in Lactating Holstein Cows under Heat Stress Conditions.

In this study, we examined the effects of rumen-protected L-tryptophan supplementation on the productivity and physiological metabolic indicators in lactating Holstein cows under heat stress conditions. The study involved eight early lactating Holstein cows (days in milk = 40 ± 9 days; milk yield 30 ± 1.5 kg/day; parity 1.09 ± 0.05, p < 0.05), four cows per experiment, with environmentally controlled chambers. In each experiment, two distinct heat stress conditions were created: a low-temperature and low-humidity (LTLH) condition at 25 °C with 35-50% humidity and a high-temperature and high-humidity (HTHH) condition at 31 °C with 80-95% humidity. During the adaptation phase, the cows were subjected to LTLH and HTHH conditions for 3 days. This was followed by a 4-day heat stress phase and then by a 7-day phase of heat stress, which were complemented by supplementation with rumen-protected L-tryptophan (ACT). The findings revealed that supplementation with ACT increased dry matter intake as well as milk yield and protein and decreased water intake, heart rate, and rectal temperature in the HTHH group (p < 0.05). For plateletcrit (PCT, p = 0.0600), the eosinophil percentage (EOS, p = 0.0880) showed a tendency to be lower, while the monocyte (MONO) and large unstained cells (LUC) amounts were increased in both groups (p < 0.05). Albumin and glucose levels were lower in the HTHH group (p < 0.05). The gene expressions of heat shock proteins 70 and 90 in the peripheral blood mononuclear cells were higher in the ACT group (HTHH, p < 0.05). These results suggest that ACT supplementation improved productivity, physiological indicators, blood characteristics, and gene expression in the peripheral blood mononuclear cells of early lactating Holstein cows under heat-stress conditions. In particular, ACT supplementation objectively relieved stress in these animals, suggesting that L-tryptophan has potential as a viable solution for combating heat-stress-induced effects on the cattle in dairy farming.

Paper-Based Diagnostic System Facilitating Escherichia coli Assessments by Duplex Coloration.

Laboratory support for low-resource regions is a rising global issue. As microbiological contamination is closely associated with other issues like food safety, water supply sustainability, and public health, bacterial assessments in this setting need to be improved. Herein, we demonstrate a paper-based diagnostic device for point-of-need testing, in which fecal-indicating Escherichia coli and highly pathogenic E. coli are detected by duplex coloration. This device was functionalized by mixing different chromogenic substrates that reflect each bacterial enzymatic phenotype. In the final part of the paper, we describe this microbiological diagnostic system tested with bacteria-contaminated food samples. The device sensitivity was shown to have greatly reduced the total analysis time (below to 4 h) when combined with an enrichment amplification procedure. Notably, this paper device successfully detected 10 cfu/mL of target bacteria in a contaminated milk sample. Our diagnostic system shows acceptable accuracy, short analysis time, and a user-friendly interface, thereby eliminating demands for high-end equipment and a highly trained staff. We expect that this diagnostic system will be a sustainable solution in supporting microbiological or clinical laboratories in low-income countries.

One-step sensing of foodborne pathogenic bacteria using a 3D paper-based device.

Managing food contamination from bacteria has been an ongoing issue in the public health and industrial fields. Enzymatic substrates possessing optical properties, e.g. fluorescence or color manifestation, are widely exploited in pathogenic/non-pathogenic bacteria culture methods. Recently, various chromogenic substrates have been utilized in the development of point-of-care diagnostic tools. Herein, four types of chromogenic substrates were exploited to develop paper-based sensors for major foodborne pathogens. We designed a compact sized three-dimensional paper device with a simple user interface. By inserting functional layers in the middle of multilayers, pre-lysis and pH regulation steps were excluded and the analysis time was subsequently reduced, while only one sample droplet was needed for the whole analysis process. After the enzymatic reactions had proceeded, target-specific colors appeared. When it was combined with enrichment, 101 cfu mL-1 of pathogens were successfully detected in 4-8 hours, while those in milk samples were readily sensed in 12 hours. The proposed bacteria sensor exhibited great advantages of low cost, portability and simple operation, while showing a respectable limit-of-detection as low as 101 cfu mL-1 and below. Significantly, we emphasize that it takes fewer steps than existing methods and provides a reduced analysis time owing to the layer functionalization.

First report of Anaplasma phagocytophilum infection in Holstein cattle in the Republic of Korea.

Global warming has increased the incidence and risk of tick-borne diseases in domestic animals and humans in the Republic of Korea (ROK). In this study, we investigated the prevalence of Anaplasma phagocytophilum in Holstein cattle (n = 214) in the ROK using specific PCR assays. A. phagocytophilum infection was detected in only two animals (0.93%, 2/214). Our findings showed that PCR assay using the 16S rRNA gene, but not groEL, was suitable for detection of A. phagocytophilum in cattle. Phylogenetic analysis based on the16S rRNA gene showed that A. phagocytophilum was divided into two clades. Clade 1 included Korean isolates, such as those from dogs, cats, Korean water deer, and ticks, while A. phagocytophilum identified in Holstein cattle formed clade 2. Our results suggest that there is genetic variability among isolates of A. phagocytophilum circulating in the ROK. This is the first study to report A. phagocytophilum infection in Holstein cattle in the ROK. As A. phagocytophilum has zoonotic potential, additional epidemiological studies are needed to investigate the prevalence and genetic characterization of A. phagocytophilum from different regions and hosts.

Biotinylation of the rhizobial cyclic ß-glucans and succinoglycans crucial for symbiosis with legumes.

The cyclic ß-glucans and succinoglycans produced by rhizobia are required for nodulation during symbiosis with legume hosts. However, only gene deletion analyses have been used to investigate their biological importance. For future studies on the physiological activity of those during symbiosis, biochemical methods need to be developed with separate carbohydrate compounds. Here, we isolated and purified rhizobial cellular carbohydrates using various chromatographic methods. Purified cyclic ß-glucans, cyclosophoraoses, were monofunctionalized with biotin using the following three steps: tosylation, azidation, and amination. The mono-6-amino-cyclosophoraoses were linked with biotinamidohexanoic acid N-hydroxysuccinimide ester. Succinoglycans and monomers were tagged with biotinamidocaproyl hydrazide at the reducing sugar via reductive amination. The resulting biotinylated rhizobial carbohydrates were characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and electrospray ionization mass spectrometry. The resulting neoglycoconjugates can be used as solid probes to study putative plant receptors and for non-invasive imaging for in vivo tracing.

Cholesterol reduction from milk using ß-cyclodextrin immobilized on glass.

ß-Cyclodextrin (ß-CD) was converted into ß-CD-undecenyl ether by chemical modification and subsequently covalently attached to a glass surface. The functionalized glass surface was characterized by static water contact angle and x-ray photoelectron spectroscopy. Both techniques confirmed that an excellent monolayer of ß-CD was formed on the glass surface. The ß-CD solid surface was used to reduce cholesterol levels in milk. In 4h, 73.6% of the cholesterol was extracted at 25°C with shaking at 170rpm. This is the highest value ever reported for milk using ß-CD immobilized on a solid surface. The same surface was repeatedly used for 10 cycles and maintained its efficiency with 72±2% cholesterol reduction observed in all the cycles. X-ray photoelectron spectroscopy analysis completed after 5 and 10 cycles of cholesterol reduction showed that the ß-CD on the glass surface was not degraded. The high efficiency and long-term stability of the functional monolayer was attributed to the specific structure of ß-CD, which is composed of a relatively low number of functional groups and long spacer chain lengths that provide great flexibility.

Development of protein-cage-based delivery nanoplatforms by polyvalently displaying ß-cyclodextrins on the surface of ferritins through copper(I)-catalyzed azide/alkyne cycloaddition.

Protein cages are spherical hollow macromolecules that are attractive platforms for the construction of nanoscale cargo delivery vehicles. Human heavy-chain ferritin (HHFn) is modified genetically to control the number and position of functional groups per cage. 24 ß-CDs are conjugated precisely to the modified HHFn in specific locations through thiol-maleimide Michael-type addition followed by copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC). The resulting human ferritins displaying ß-CDs (ß-CD-C90 HHFn) can form inclusion complexes with FITC-AD, which can slowly release the guest molecule reversibly in a buffer solution via non-covalent ß-CD/AD interactions. ß-CD-C90 HHFn can potentially be used as delivery vehicles for insoluble drugs.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric behavior of succinoglycan monomers, dimers, and trimers isolated from Sinorhizobium meliloti 1021.

Low-molecular-weight (LMW) succinoglycans (monomers, dimers, and trimers) were isolated from Sinorhizobium meliloti 1021 and have been firstly investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using 2,4,6-trihydroxyacetophenone (THAP) as an optimal matrix in the negative ion mode. The main fractions of LMW succinoglycans contain molecules assembled of octasaccharide subunits. MALDI-TOF mass spectra of the LMW succinoglycan monomers, the dimers, and the trimers showed the daughter ions resulting from the losses of the terminal galactose residues at the reducing ends, clearly indicating that the galactosyl linkages are more labile than the other glucosyl linkages. Furthermore, the losses of the acetyl groups as substituents rather than the succinyl and pyruvyl ester linkages by prompt fragmentation primarily occurred during MALDI-TOF analysis, suggesting the greater instability of acetyl linkages compared to pyruvyl and succinyl linkages.

Stereoisomeric separation of some flavanones using highly succinate-substituted α-cyclosophoro-octadecaoses as chiral additives in capillary electrophoresis.

α-Cyclosophoro-octadecaoses (α-C18), produced by Rhodobacter sphaeroides, are mostly homogeneous in size with 18 glucose units per ring as the predominant form. α-C18s are linked by ß-(1→4)-linkages and one α-(1→6)-linkage and are also known to be highly substituted by acetyl (0-2 per mol) and/or succinoyl groups (1-7 per mol). We isolated and purified α-C18 and successfully used it in capillary electrophoresis (CE) as a chiral additive for the separation of five flavanones and flavanone-7-O-glycosides, including naringenin, hesperetin, eriodictyol, homoeriodictyol, isosakuranetin, and hesperidin. Throughout the CE experiment with unsubstituted α-C18 (uα-C18) obtained after alkaline treatment of the isolated α-C18, we found that successful chiral separation critically depends on the presence of succinate substituents attached to α-C18 in CE, suggesting that succinoylation of α-C18 is decisive for effective stereoisomeric separation.

Carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis.

A carboxymethylated cyclosophoraose (CM-Cys) was synthesized by the chemical modification of neutral Cys, which was isolated from Rhizobium trifolii TA-1. CM-Cys was successfully applied as a novel chiral selector for the separation of some flavonoids including catechin, 3,5,7,3',4'-pentahydroxyflavanone, hesperidin, hesperetin, isosakuranetin, naringenin, naringin, and eriodictyol. The effects of pH, chiral additive concentration, and temperature on resolution and migration time were also studied.

Chiral separation and discrimination of catechin by sinorhizobial octasaccharides in capillary electrophoresis and (13)C NMR spectroscopy.

Succinoglycan, a sinorhizobial exopolysaccharide produced by Sinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high-molecular-weight and low-molecular-weight (M(r)<10,000) succinoglycans which consist of monomers, dimers, or trimers. Succinoglycan monomers were isolated and further purified in the monomer series (M1, M2, and M3) by the degree of succinylation. We used sinorhizobial octasaccharides (M1, M2, and M3) as chiral additives in capillary electrophoresis (CE) for chiral separation of catechin and also as chiral shift reagents with (13)C NMR spectroscopy for chiral discrimination of catechin. Chiral separation of catechin took place when sinorhizobial octasaccharides (M2 and M3) were added to the background electrolyte (BGE) in CE. NMR signal splittings were also observed in the interactions of sinorhizobial octasaccharides with the enantiomers of catechin. Both chiral separation and discrimination of catechin depend on the presence of succinate substituents of the linear monomeric octasaccharide in CE and NMR spectroscopy, suggesting that succinylation of sinorhizobial octasaccharide is decisive for the effective chiral separation and discrimination of catechin.

Synthesis of selenium nanowires morphologically directed by Sinorhizobial oligosaccharides.

Shinorhizobial cyclosophoraose (cyclic beta-(1-->2)-glucan) or succinoglycan monomer (SGM 2), which has one acetyl, pyruvyl, and succinyl group, functions as a morphology-directing agent for the synthesis of pure trigonal selenium nanowires by using ascorbic acid (vitamin C) as the reducing agent. The synthesis was achieved in water at room temperature. Under these experimental conditions, the diameters of the as-prepared Se nanowires were varied in the range of 34-120 nm by cyclosophoraose and of 33-66 nm by SGM 2, in which the nanowires were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy. Through this study, we propose that Shinorhizobial cyclic and linear oligosaccharides have morphologically directing functions for the synthesis of single-crystalline selenium nanowires by green chemical methods.

Low-energy collision-activated dissociation electrospray ionization tandem mass spectrometric analysis of Sinorhizobial succinoglycan monomers.

Succinoglycan monomers (M1, M2, and M3) are octasaccharides with acetyl, pyruvyl, and/or succinyl groups as substituents derived from Sinorhizobium meliloti 1021. The dissociation patterns of the octasaccharides caused by low-energy collision-activated dissociation (CAD) were investigated using triple quadrupole tandem mass spectrometry (MS) equipped with an electrospray ionization (ESI) source with increasing collision energy (CE) in negative ion mode. None of the succinoglycan monomers were fragmented at a CE of -25eV. When the CE was applied to -50 or -70eV, the loss of the terminal Gal residue and/or the succinyl group of the monomers was observed in the product ion scan mode. Interestingly, the acetyl and the pyruvyl groups in the succinoglycan monomers were not lost even when a CE of -70eV was applied, indicating that the substituents are more stable than the succinyl group in the octasaccharides.

Enantiomeric separation of some flavanones using shinorhizobial linear octasaccharides in CE.

Succinoglycan, a shinorhizobial exopolysaccharide produced by Shinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high-molecular-weight and low-molecular-weight (M(r)<10 000) succinoglycans that consisted of monomer, dimer, or trimer of an octasaccharide unit. We isolated and purified the monomer among low-molecular-weight succinoglycans and used this microbial linear octasaccharide as a novel chiral additive for enantiomeric separation of some flavanones such as homoeriodictyol, hesperetin, naringenin, and isosakuranetin in CE. Throughout the present investigation, we firstly used noncyclic oligosaccharides for the chiral separation of flavanones. We also found that successful enantioseparation of four flavanones depends on the presence of succinate substituents of the linear monomeric octasaccharide in CE, suggesting that succinylation of succinoglycan monomer is decisive for the effective enantiomeric separation.

Cyclosophorohexadecaose and succinoglycan monomers as catalytic carbohydrates for the Strecker reaction.

Some microbial carbohydrates have been used as catalysts for the multicomponent Strecker reaction using trimethylsilyl cyanide (TMSCN). Alpha-Cyclosophorohexadecaose (alpha-C16) derived from Xanthomonas species and succinoglycan monomers derived from Rhizobium species acted as catalytic carbohydrates in the mixture solutions of methanol and water. Malonaldehyde bis(phenylimine) as a substrate was completely converted (yield: 100%) into its product to 100% by both alpha-C16 and the succinoglycan monomer (M2), having acetyl, pyruvyl, and succinyl groups as substituents after 1h. The catalytic abilities of the carbohydrates were dependent on the inherent structures of the substrates used in this study, where substrate 1 having a symmetrical structure rather than the others was favorably reacted with the alpha-C16 and M2. Through this study, we suggest that the microbial carbohydrates used in this study could be expected to be environmentally-benign catalysts for the synthesis of alpha-aminonitriles.

Enantioseparation of some chiral flavanones using microbial cyclic beta-(1-->3),(1-->6)-glucans as novel chiral additives in capillary electrophoresis.

Cyclic beta-(1-->3),(1-->6)-glucans, microbial cyclooligosaccharides produced by Bradyrhizobium japonicum USDA 110, were used as novel chiral additives for the enantiomeric separation of some flavanones such as eriodictyol, homoeriodictyol, hesperetin, naringenin, and isosakuranetin in capillary electrophoresis (CE). Among the flavanones, eriodictyol was separated with the highest resolution (R(s) 5.66) and selectivity factor (alpha 1.18) when 20mM cyclic beta-(1-->3),(1-->6)-glucans were added to the background electrolyte (BGE) at pH 8.3.