Sialic Acid Content Research Articles

Colorimetric-assisted photoelectrochemical sensing for dual-model detection of sialic acid via oxidation-power mediator integration

Development of sensitive and accurate methods for sialic acid (SA) determination is of great significance in early cancer diagnosis. Here, a colorimetric-assisted Photoelectrochemical (PEC) sensor was constructed for SA detection based on the two pairs of cis-diol groups in SA molecule. With the specific recognition of SA via the two pairs of cis-diol groups, the prepared gold-modified Bi2S3 (Au [email protected]2S3) and metal organic framework ([email protected]) were introduced to the electrode and formed a sandwich structure. Based on the properties of inert electroconductivity and nanozyme of the PCN-224, dual-readout of photocurrent and visualization was achieved with the presence of 3,3′,5,5′-tetramethylbenzidine (TMB). Moreover, to intensify the visualization signal, Ce3+ was employed as the mediator to boost the catalysis capability by transferring energy from PCN-224 surface to the whole system. With the unique SA recognition and the mediation of Ce3+, both the photocurrent and the visualization signals sensitively responded with the SA concentration linearly. The present method showed greatly high sensitivity, selectivity and accuracy for SA detection with a limit of detection of 1.44 μmol/L and a wide linear range of 5-1000 μmol/L. This method provided a new promising platform for SA detection and a potential strategy for design of novel biosensors with dual-model readout.

Morphofunctional parallels of the stomach in patients with chronic atrophic gastritis

Background. Despite some progress in addressing gastric cancer, a marked reduction in morbidity in all developed countries, the search for changes that precede carcinogenesis remains relevant and far from complete. The purpose of the study: to assess the content of aggressive and protective factors of gastric juice and oral fluid in comparison with morphological changes in patients with chronic atrophic gastritis. Materials and methods. The study included 56 patients: group I included 12 patients with atrophic changes of the gastric mucosa of varying severity, groupII— 24 patients with atrophic changes diagnosed with intestinal metaplasia only in the antrum of the stomach, in group III group— 20 patients with intestinal metaplasia in the body and antrum of the stomach. The control group consisted of 16 healthy individuals. Assessment of the degree of atrophy and inflammation in the gastric mucosa was performed with the subsequent determination of integral indicators— stage and degree of atrophy according to the OLGA system and metaplasia— according to the OLGIM system. According to the morphometric study of sections using a light microscope XSP-139TP (“Ulab“, Ukraine) calculated indicators: nuclear-cytoplasmic ratio, the ellipticity of the nuclei. The content of glycoproteins, sialic acids, fucose, hexosamines was determined in gastric juice and oral fluid. Results. Morphometric study showed a significant decrease in the nuclear-cytoplasmic ratio to (0.12±0.04)% in patients of group III. The coefficient of ellipticity of the nuclei of native cells decreased with the development of intestinal metaplasia: in group I it was equal to (0.76±0.04)%, for group II it was (0.65±0.11)%, and for group III— 0.41±0.12)%. In patients with intestinal metaplasia in 82% of cases with a 3-fold increase in the content of sialic acids in gastric juice was diagnosed with an increase in hexosamines by 35%, while patients with only atrophic changes in the stomach showed a decrease in hexosamines 2 times compared to controls (p<0.05). The spread of intestinal metaplasia in the stomach is associated with an increase in the amount of glycoproteins and hexosamines in the oral fluid of patients, while sialic acids in this biological fluid are increased in all study groups. There are direct relationships between the presence of intestinal metaplasia in the body of the stomach and the content of glycoproteins in both gastric juice (r=0.446, p=0.008) and in oral fluid (r=0.378, p=0.021). The relationship between the content of sialic acids in gastric juice with the degree and stage of gastritis by OLGA (r=0.431, p<0.01; r=0.482, p<0.01), the level of hexosamines in oral fluid with the coefficient of ellipticity of the nuclei (r=0.447, p=0,037). Conclusions. A comprehensive study of morpho-functional changes in the stomach and the ratio of aggressive and projective factors of oral fluid will determine the risk groups of patients with precancerous conditions.

Characteristics and qualities of edible bird's nest beverage as affected by thermal pasteurization and sterilization.

Edible bird's nest beverage (B-nest-Bev) was produced from edible bird's nest (B-nest) flakes using different thermal processes. Pasteurization of B-nest-Bev at a low temperature for a longer time (LTLT) or at a high temperature for a shorter time (HTST) resulted in lower CIE L * , CIE a * , CIE b * -values, and drained weight (p < 0.05) than sterilization (118 or 121°C). Sterilized and pasteurized B-nest-Bev had similar soluble solid contents and pH (p < 0.05). Nevertheless, acidified beverages pasteurized via either LTLT or HTST process had a marked decrease in sialic acid content. In addition, drastic protein degradation occurred in pasteurized acidified beverages, regardless of the conditions used, ascertained by the disappearance of major protein bands. However, polymerization of proteins took place in sterilized samples, irrespective of the temperature used. After digestion in a gastrointestinal tract model system (GIMs), all samples had increased (p < 0.05) antioxidant activities including DPPH and ABTS radical scavenging activities, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC). B-nest-Bev subjected to HTST pasteurization or sterilization at 121°C had the best appearance and acceptability among all the treatments used. Therefore, thermal processes directly affected the properties and acceptability of B-nest-Bev.

Loss of Myosin Vb leads to dysregulation of colonic goblet cell structure and function

BackgroundIntestinal goblet cells secrete a protective mucus layer preventing bacteria and harmful ingested substances from contacting the columnar epithelial cells that line the intestine. Single cell RNAseq data has revealed that goblet cells have high expression of Myosin 5b (Myo5b), a molecular motor that regulates vesicle trafficking in epithelial cells. Myo5b is most widely studied in the small intestine, but it’s role in the colonic epithelium and particularly goblet cells is unknown. A recent study found that Myo5b expression was reduced in inflammatory bowel disease (IBD), a condition that is characterized by dysfunctional mucus; potentially providing a link between Myo5b and goblet cell function. We hypothesized that loss of Myo5b would perturb goblet cell mucus production.MethodsAdult control and intestinal specific tamoxifen inducible VillinCreERT2;Myo5bflox/flox(Myo5b KO) mice were injected intraperitoneally with 2 mg of tamoxifen to deplete intestinal Myo5b. After four days colonic tissue was collected and analyzed.ResultsWe found that loss of Myo5b in vivo results in reduced number of goblet cells in the colon by H&amp;E staining. Muc2 immunostaining and alcian blue staining similarly demonstrated a reduction of mucin within goblet cells. Mucin proteins are heavily o‐glycosylated which makes up 80% of the molecular weight. Normally, mucin structures are terminated by sialic acid which provides a negative charge that protects the mucus protein from bacterial degradation. Staining for sialic acid with the lectin SNA revealed a decrease in sialic acid levels in the mucus of Myo5b KO mice compared to controls. Once sialic acid is removed from mucins, the underlying glycan structures are exposed. Using the lectin WGA to identify the internal glycan oligosaccharide n‐acetyl glucosamine, we observed increased exposure of n‐acetyl glucosamine in Myo5b KO mice compared to control mice; which was consistent with our sialic acid findings. Intestinal goblet cells also secrete wound healing factors including the peptide Trefoil factor 3 (TFF3). Similar to the pattern we observed for Muc2 and sialic acid content, we found decreased levels of TFF3 in the mucosa of Myo5b KO mice.ConclusionsOur data shows that loss of functional Myo5b in the colonic epithelium results in decreased goblet cell numbers and alterations in mucin composition. These data suggest that Myo5b may play an important role in goblet cell mucus production and that mutations affecting Myo5b function may contribute to IBD.

The Effect of Sialic Acid on MiR-320a and Let-7e Expression in Human Glial Cell Line.

Sialic acid is pivotal in various critical physiological events at molecular and cellular levels and pathological processes. Changes in sialic acid concentration are observed in many pathological processes; for example, some available data exist on the evaluated level of sialic acid and neurodegenerative prevalence. Presumably, sialic acid can play a significant role in regulating a diverse range of uncovered neurodegeneration factors and downstream targets. matrix metalloproteinases 9 (MMP9) is one factor that changes the exposure of different concentrations of sialic acid solution. Hence, we aimed to examine the possible effect of sialic acid solution exposure on the glial cell line in the expression patterns of miR-320a and let-7e as two upstream factors. Human glial cell line was prepared from the Pasteur Institute of Iran and cultured in a dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). The IC50 value of sialic acid was obtained by colorimetric assay for assessing cell metabolic activity 3-(4,5-Dimethylthiazol-2-yl (MTT), and the glial cell line was treated with sialic acid in 300, 500, 1000 μg/mL for 24 h to investigate the effect of the sialic acid ligand on the expression pattern of the miR-320a and let-7e. Total RNA was isolated from approximately 10×106 glial cells and was used from each sample for complementary dna (cDNA) synthesis. For quantitative analysis of miR-320a and let-7e, we used real-time polymerase chain reaction (PCR), and for statistical analysis, the SPSS v. 21 software was applied. Analyzing the real-time data revealed that the expression of miR-320a and let-7e was significantly increased (P<0.0001) in 300, 500, and 1000 μg/mL treated glial cells by sialic acid compared to the control group. A possible linkage of sialic acid on miR-320a and let-7e regulation was observed in the glial cell line as proinflammatory factors in the inflammation pathway. Differing in sialic acid concentration is seen in various pathological states.MicroRNAs play a role in numerous biological processes and human disorders.miR-320a and let-7e expression levels displayed a significant increase in different sialic acid concentrations. Inflammation in the nervous system occurs because of numerous factors. Sialic acid is an inflammatory factor that promotes cellular inflammation, particularly in the glial cells. That is why it could serve as a useful model for simulating several neurodegenerative diseases, including Parkinson's and Multiple sclerosis. Changes in sialic acid concentration are observed in many pathological states, which could be a useful marker for identifying the inflammatory process. The present study was carried out to examine the impact of different concentrations of sialic acid on two non-coding RNAs in glial cells. Our research shows that these two microRNAs greatly increased when responding to sialic acid. We suggest that these two microRNAs are contributed to the neuroinflammatory pathways related to sialic acid.

Suppression of fertility by &lt;em&gt;Capparis spinosa&lt;/em&gt; root extract through antioxidant alteration in reproductive system of male rats

Aim: To evaluate the suppression of fertility by oral mode of Capparis spinosa root extract through alteration in antioxidant defence of the reproductive system of male albino rats. Methods: The methanol extract of Capparis spinosa root (100mg/kg/day) was fed for 60 days to male, Wistar strain, albino rats. The fertility and testicular functions were assessed by mating tests, antioxidant defence indices, and spermatogenesis, histometry, and biochemical estimations for fertility suppression along with serum biochemistry and hematology for assessment of toxicology. Results: Capparis spinosa root extract administration for a period of 60 days did not cause body weight loss significantly, while the weight of testes, Cauda epididymis, seminal vesicle, and ventral prostate were significantly reduced. The lipid peroxidation and antioxidants levels i.e. superoxide dismutase, catalase, glutathione reduced, glutathione peroxidase, vitamin C, and Vitamin E in testes, Cauda epididymis, seminal vesicle, and ventral prostate were altered significantly (P<0.001) after the 60 days treatment of Capparis spinosa extract when compared to the controls. The root extract feeding caused a marked reduction in the number of primary spermatocytes, secondary spermatocytes, and spermatids however spermatogonia population showed non-significant change. Sertoli cell population was also affected. The number of mature Leydig cells was decreased and degenerating cells increased proportionately. The seminiferous tubule and Leydig cell nuclear area were reduced significantly (P<0.001) when compared to the controls. The protein and sialic acid content of the testes, Cauda epididymis, seminal vesicle, and ventral prostate were significantly (P<0.001) decreased. The glycogen in testes and fructose in seminal vesicle was also significantly reduced, whereas the testicular cholesterol was elevated as compared with the controls. Reduced sperm count and motility resulted in a total suppression of fertility. The RBC and WBC count, hemoglobin, and hematocrit in whole blood whereas marker enzymes and glucose in serum were found to be within the normal range after the 60 days of Capparis spinosa extract treatment. Conclusion: Capparis spinosa root extract may selectively act on developing germ cells mediated via Sertoli cells through increased oxidative stress or impairment of antioxidant defence system in reproductive organs of male rats, leading to suppression of fertility and suggests the feasibility of developing non-toxic, herbal male contraceptive drug.

Fractionation of edible bird’s nest glycoprotein hydrolysates: characterisation and antioxidative activities of the fractions

Edible bird’s nest (EBN) hydrolysates have been proven to exhibit enhanced bioactivities. However, being a macromolecule, fractions with different molecular weights would have different properties and bioactivities. Hence, this research was aimed to determine the chemical properties and antioxidant activities of freeze-dried (EBNFD) and spray-dried EBN (EBNSD) hydrolysates fractionated using gel permeation chromatography (GPC). Overall, two well-separated fractions were identified (EBNfFD1, EBNfFD2, EBNfSD1 and EBNfSD2). EBNFD demonstrated significantly higher (P ≤ 0.05) peptide (3.6%), total carbohydrate (27.7%) and sialic acid (18.2%) contents than that of EBNSD. Similar trend was observed in low molecular weight fractionates (EBNfFD2 and EBNfSD2). Meanwhile, the first fractionates (EBNfFD1 and EBNfSD1) exhibited significantly higher (P ≤ 0.05) hydroxyl radical (•OH) scavenging activity. Fourier transform infrared (FTIR) spectroscopy demonstrated that all EBN fractionates have similar spectrum, except in the region of N—H (amide II) and C—H alkyl group. In conclusion, EBN fractionates with different molecular weights showed different chemical properties and antioxidant activities.

Effect of Trigonella foenum-graecum L. seed extract on the reproductive system of male mice and possible mechanism of its action on spermatogenesis.

Fenugreek seed exhibits antidiabetic, antineoplastic, hepatoprotective, antidepressant and immunomodulatory properties. Fenugreek also causes antifertility effects in rodents. However, the impact of fenugreek seed on male reproduction and the possible mode of its action are not properly evaluated. Herein, we examined the effect of aqueous seed extract of fenugreek (FSE) and the possible mechanism of its action on male reproductive health in mice. Parkes mice were orally administered FSE (600mg/kg body weight/day) or distilled water for 28 and 56days, respectively. Various sperm parameters, histopathology, serum testosterone level and fertility indices were assessed. Furthermore, steroidogenic enzymes activities, oxidative status and germ cell dynamics in the testis were evaluated. Toxicological endpoints were also assessed. Treatment with FSE caused degenerative changes in the testis histoarchitecture. The treatment also affected various sperm parameters and concentrations of sialic acid and fructose in the epididymis and seminal vesicle, respectively. Fenugreek treatment also had negative impact on oxidative status and germ cell dynamics in the testis; fertility indices were also affected in female mice impregnated by the extract-treated male mice, though libido of the treated male mice remained unaffected. Results show that treatment with FSE caused adverse effects on the male reproductive health and pregnancy outcome in Parkes mice.

IDF21-0217 Sialidase and sialyltransferase activities are modulated in some organs of type 1 and type 2 diabetic rats

Background: Diabetes mellitus leads to an aberrant metabolism of sialoglycoconjugates. One of the main features of the pathophysiology of diabetes is elevated serum level of free sialic acids. The source of serum free sialic acid during diabetes is debatable, although the contribution of endothelial damage has been recognised. Aim: The aim of this study is to investigate the association between the level of sialic acids and activities of sialidase and sialyltranferase in the serum and some diabetes-related organs in animal model of type 1 (T1D) and type 2 (T2D) diabetes. Method: Both types of diabetes induced in albino rats using high and low dose streptozotocin respectively. The level of sialic acid and activities of sialidase and sialyltransferase were measured in the serum, liver, pancreas, skeletal muscle and kidney of the diabetic rats and matching controls at early (3rd week) and late (9th week) phases of the diseases. Results: Serum sialic acid level and sialidase activity were significantly elevated at late stage of both T1D and T2D. The activity of sialyltransferase was not significantly changed in the diabetic animals compared to the non-diabetic controls. The total sialic acid content of the pancreas and kidney significantly increased from the early phase of both T1D and T2D to the late phase. On the other hand, the sialic acid content of the liver significantly decreased in the early phase of T2D and the late phase of T1D.The sialic acid content of the skeletal muscle significantly increased in the late phase of both diabetes types. In the early phase of both T1D and T2D, sialidase activity significantly increased in most of the organs whereas sialyltransfease activity was unchanged. A multiple regression analysis revealed the contribution of the liver to the serum level of sialic acid and the contribution of the pancreas and kidney to the serum activity of sialidase. Discussion: It was concluded that the released of sialic acid from the liver and sialidase from the pancreas and kidney may contribute to the increased serum level of sialic acid during diabetes.

Distribution and Influencing Factors of the Sialic Acid Content in the Breast Milk of Preterm Mothers at Different Stages.

Background and AimsThis study aimed to detect breast milk sialic acid (SA) content and the changing pattern, to understand the various stages of breastfeeding SA secretion, and the influence factors of the human milk SA content.MethodsWe recruited mothers and their infants as our subjects. At days 7, 14, 30, 120, and 365 after delivery, the contents of SA in breast milk were collected and detected through Fluorescence Detector-High Performance Liquid Chromatography. The participants completed the baseline questionnaire at ≤day 7 and were followed up at days 30, 120, and 365.ResultsA total of 95 mothers with 122 infants were included in the analysis, including 22 mothers with 22 term infants, 25 mothers with 35 late preterm infants, 31 mothers with 39 very preterm infants, and 17 mothers with 26 extremely preterm infants. Similar to previous findings, the results of the study showed that, compared with breast milk of term mothers at the same period, breast milk of preterm mothers contained more SA at each time node, and the content of SA in breast milk increased with decreasing gestational weeks. Moreover, maternal age, pre-pregnancy BMI, and delivery mode had significant effects on total SA in breast milk, especially for the preterm infant breast milk. Significant negative associations occurred between SA contents and infant growth status, especially in preterm infants.ConclusionsWe have confirmed the previous observations showing that with the prolongation of lactation time, the content of SA in breast milk gradually decreased, and the content of SA in the breast milk of preterm mothers was higher than that of term mothers. In addition, SA content was associated with maternal age, pre-pregnancy BMI, and delivery mode.

Sialic Acid as a Suitable Marker of Clinical Disease Activity in Patients with Crohn's Disease.

Elevated serum levels of sialic acid (SA) have been verified in patients with various inflammatory conditions. The association between the Crohn's disease (CD) activity and serum SA has been insufficiently studied. Serum SA concentrations were determined using an enzymatic colorimetric assay method, and the correlation of SA with the Harvey-Bradshaw Index (HBI) and other inflammation activity markers was evaluated using the Spearman correlation. The predictive value of SA in estimating CD disease activity was assessed using the receiver operating characteristic. The SA levels were positively correlated with HBI and C-reactive protein (CRP) levels. The correlation of SA with the HBI was superior to that of CRP with the HBI. The area under the curve for SA was higher than that for CRP, with an optimal cutoff value of 53.14 mg/dL for active CD. Serum SA correlates with the HBI score better and has better predictive value in monitoring CD disease activity than CRP or other inflammatory markers.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.

Comparison effects of platelet-rich plasma on healing of infected and non-infected excision wounds by the modulation of the expression of inflammatory mediators: experimental research.

Microbial invasion in soft tissue is believed to cause infectious wounds and increase healthcare costs, anxiety, and distress. The current study was conducted to evaluate the effects of topical use of platelet-rich plasma (PRP) on infected wound-healing process in rats. Following the induction of a circular wound, the animals were divided into three groups, including (1) standard control: infected wounds treated with mupirocin (SDCL), (2) non-infected wounds treated with PRP (PRP), and (3) infected group in which the rats were infected with Staphylococcus epidermidis and Pseudomonas aeruginosa and treated with PRP (INF + PRP). To evaluate the effects of PRP on the wound-healing rate, total bacterial count, histopathological assessment, the serum concentrations of sialic acid, C-reactive protein, haptoglobin, and fibrinogen were assessed. Additionally, IL-1β, IL-6, TNF-α, IL-3, NF-κB, iNOS, PDGF, and EGF mRNA level expressions were assessed employing the qRT-PCR method. The results indicated that topical application of PRP could significantly decrease total bacterial count, the level of C-reactive protein, and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) compared to the SDCL group. The administration of PRP also promoted re-epithelization rate by increasing the expression of EGF mRNA level. We could recommend the use of PRP for the treatment of infected wounds owing to its efficiency in decreasing colonization of tissue bacteria, tissue inflammation, and stimulating wound heal-up.

Correlation between Sialylation Status and Cell Susceptibility to Amyloid Toxicity.

The interaction between the cell membrane and misfolded protein species plays a crucial role in the development of neurodegeneration. This study was designed to clarify the relationship between plasma membrane composition in terms of the differently linked sialic acid (Sia) content and cell susceptibility to toxic and misfolded Aβ-42 peptides. The sialylation status in different cell lines was investigated by lectin histochemistry and confocal immunofluorescence and then correlated with the different propensities to bind amyloid fibrils and with the relative cell susceptibility to amyloid damage. This study reveals that expressions of Sias α2,3 and α2,6 linked to galactose/N-acetyl-galactosamine, and PolySia are positively correlated with Aβ-42-induced cell toxicity. PolySia shows an early strong interaction with amyloid fibrils, favoring their binding to GM1 ganglioside containing α2,3 galactose-linked Sia and a loss of cell viability. Our findings demonstrate that cell lines with a prevailing plastic neuron-like phenotype and high monoSia and PolySia contents are highly susceptible to amyloid Aβ-42 toxicity. This toxicity may involve a change in neuron metabolism and promote a compensative/protective increase in PolySia, which, in turn, could favor amyloid binding to GM1, thus exacerbating cell dysmetabolism and further amyloid aggregation.

Valine feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells.

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity. KEY POINTS: • Valine feeding can mitigate the negative impacts of ammonia on CHO cell growth. • Valine addition assists the ammonia removal mechanism by enriching the TCA cycle. • Ammonia is removed from the media through alanine excretion in valine-fed culture.

Glycoengineering of Darbepoetin-α in CHO-DG44 Cells through Overexpression of α-2,3-sialyl-transferase and CMP-sialic Acid Transporter

Sialic acid plays a very important role in determining the circulation life span of glycoprotein in various organisms. Therefore, having a high content of sialic acid is needed by glycoprotein therapeutic agents to be able to function as desired. For example, Darbepoetin (DPO), the 5 N-linked erythropoietin showed higher bioavailability and efficacy compared to 3 N-linked erythropoietin. However, in the DPO production process, the molecular weight can vary and is highly dependent on the content of sialic acid and its production host. To improve the DPO sialic acid contents in our CHO-DG44 expressing DPO, we have engineered the cells through overexpression of α-2,3-sialyl-transferase (ST) and CMP-sialic acid transporter (CST). The DPO contained in the supernatant of the engineered cells was analyzed by Western blot and characterized by using PNGase-F or neuraminidase enzyme digestions. The results showed that, two clones, overexpressing ST or CST, were obtained. The clones showed higher molecular weight of DPO as compared to DPO expressed by the parental cells, yet retained the same protein backbone. The overexpression of these two genes does not affect cell growth. This suggests that may be these cells beneficial for therapeutic glycoproteins.

BIOCHEMICAL EVALUATION OF SALIVARY SIALIC ACID IN WOMEN UNDER DIFFERENT REPRODUCTIVE PHYSIOLOGICAL CONDITIONS

In human female reproductive physiology predicting ovulation is the basis on which the fertile period is determined. The present study was designed to investigate the level of salivary Sialic acid to find the possibility to develop a biomarker for detection of ovulation by non-invasive method. 42 females of different age group of different reproductive physiological conditions (i.e. prepubertal, parous, non-parous, menopausal, and diabetogenic) were selected for this study. It was observed significant (p&lt;0.001) increase in ovulatory, menopause and diabetic condition and highly significant decrease in non-parous ovulatory and post ovulatory condition in women. The study showed that salivary Sialic acid concentration was elevated during ovulation.

Comparison of dental caries experience and salivary parameters among children with Down syndrome and healthy controls in Chennai, Tamil Nadu.

Although there have been numerous studies on dental caries in children with Down syndrome, the reports are conflicting. Studies on salivary chemical composition of children with Down syndrome are limited. The study aims to evaluate and compare the dental caries experience, salivary flow rate, pH, buffering capacity, and concentration of sodium, potassium, calcium, phosphorus, total proteins, and sialic acid in children with Down syndrome and healthy controls. This was a cross-sectional study. Forty subjects with Down syndrome aged 5-18 years fulfilling the eligibility criteria from six special schools were selected by snowball sampling. Sixty healthy controls from six neighborhood schools fulfilling the eligibility criteria were selected by simple random sampling by matching the age, gender, and socioeconomic status. Sociodemographic data, oral hygiene practices, diet history and dental caries experience were recorded. About 6 mL of stimulated whole saliva was collected. Salivary flow rate, salivary pH, buffering capacity, and the concentration of sodium, potassium, calcium, phosphorus, total proteins, and sialic acid were determined. There was no significant difference in the mean proportional caries rate between the study and control group (P = 0.90). Salivary pH (P = 0.00) and salivary sodium concentration (P = 0.02) were significantly low in the study group than the control group. Salivary buffering capacity was significantly higher in the study group than the control group (P = 0.001). Dental caries experience of children with Down syndrome was similar to the healthy controls. School health programs could be implemented in special schools to improve oral and general health of special children.

Enhanced γ-aminobutyric acid and sialic acid in fermented deer antler velvet and immune promoting effects.

Deer antler velvet is widely used in traditional medicine for its anti-aging, antioxidant, and immunity-enhancing effects. However, few studies have reported on the discovery of probiotic strains for deer antler fermentation to increase functional ingredient absorption. This study evaluated the ability of probiotic lactic acid bacteria to enhance the concentrations of bioactive molecules (e.g., sialic acid and gamma-aminobutyric acid [GABA]) in extracts of deer antler velvet. Seventeen strains of Lactobacillus spp. that were isolated from kimchi and infant feces, including L. sakei, L. rhamnosus, L. brevis, and L. plantarum, and those that improved the life span of Caenorhabditis elegans were selected for evaluation. Of the 17 strains, 2 (L. rhamnosus LFR20-004 and L. sakei LFR20-007) were selected based on data showing that these strains increased both the sialic acid and GABA contents of deer antler extract after fermentation for 2 d and significantly improved the life span of C. elegans. Co-fermentation with both strains further increased the concentrations of sialic acid, GABA, and metabolites such as short-chain fatty acids and amino acids. We evaluated the biological effects of the fermented antler velvet (FAV) on the antibacterial immune response in C. elegans by assessing worm survival after pathogen infection. The survival of the C. elegans conditioned with FAV for 24 h was significantly higher compared with that of the control worm group fed only normal feed (non-pathogenic E. coli OP50) exposed to E. coli O157:H7, Salmonella typhi, and Listeria monocytogenes. To evaluate the protective effects of FAV on immune response, cyclophosphamide (Cy), an immune-suppressing agent was treated to in vitro and in vivo. We found that FAV significantly restored viability of mice splenocytes and immune promoting-related cytokines (interleukin [IL]-6, IL-10, inducible nitric oxide synthase [iNOS], interferon [IFN]-γ, and tumor necrosis factor [TNF]-α) were activated compared to non-fermented deer antlers. This finding indicated the protective effect of FAV against Cy-induced cell death and immunosuppressed mice. Taken together, our study suggests that immune-promoting antler velvet can be produced through fermentation using L. rhamnosus LFR20-004 and L. sakei LFR20-007.