Fucosylation and galactosylation in N-glycans of bovine intestinal alkaline phosphatase and their role in its enzymatic activity.

Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-ß-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-ß-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-ß-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially ß-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.

Distinct properties of putative trophoblast stem cells established from somatic cell nuclear-transferred pig blastocysts.

BACKGROUND: Genetically modified pigs are considered ideal models for studying human diseases and potential sources for xenotransplantation research. However, the somatic cell nuclear transfer (SCNT) technique utilized to generate these cloned pig models has low efficiency, and fetal development is limited due to placental abnormalities. RESULTS: In this study, we unprecedentedly established putative porcine trophoblast stem cells (TSCs) using SCNT and in vitro-fertilized (IVF) blastocysts through the activation of Wing-less/Integrated (Wnt) and epidermal growth factor (EGF) pathways, inhibition of transforming growth factor-ß (TGFß) and Rho-associated protein kinase (ROCK) pathways, and supplementation with ascorbic acid. We also compared the transcripts of putative TSCs originating from SCNT and IVF embryos and their differentiated lineages. A total of 19 porcine TSCs exhibiting typical characteristics were established from SCNT and IVF blastocysts (TSCsNT and TSCsIVF). Compared with the TSCsIVF, TSCsNT showed distinct expression patterns suggesting unique TSCsNT characteristics, including decreased mRNA expression of genes related to apposition, steroid hormone biosynthesis, angiopoiesis, and RNA stability. CONCLUSION: This study provides valuable information and a powerful model for studying the abnormal development and dysfunction of trophoblasts and placentas in cloned pigs.

Thermomonas paludicola sp. nov., isolated from a lotus wetland.

A Gram-stain-negative, aerobic, rod-shaped and motile bacterium, designated IMCC34681T, was isolated from a lotus wetland in the Republic of Korea. Cellular growth occurred at 10-37 °C (optimum, 30 °C), pH 6-9 (optimum, pH 7) and with 0-2 % (w/v) NaCl (optimum, 0.5 % NaCl). The results of 16S rRNA gene sequence analysis indicated that IMCC34681T represented a member of the genus Thermomonas, sharing 95.3-96.9 % similarities with type strains of species of the genus. The whole-genome sequence of IMCC34681T was 2.72 Mbp in size with 66.2 % DNA G+C content. The IMCC34681T genome shared the highest average nucleotide identity (ANI) value, 82.8 %, with that of Thermomonas brevis KACC 16975T among species of the genus Thermomonas, indicating that the strain represents a novel genomic species. The major respiratory quinone of the strain was ubiquinone-8 (Q-8) and the predominant cellular fatty acids were iso-C15 : 0 (25.7 %) and iso-C14 : 0 (20.8 %). The strain harboured diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid as major fatty polar lipids. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characteristics, IMCC34681T was assigned to the genus Thermomonas as the type strain of a novel species, for which the name Thermomonas paludicola sp. nov. is proposed. The type strain is IMCC34681T (=KACC 21793T=NBRC 114635T).

The subjective controllability of exotropia and its effect on surgical outcomes in patients with intermittent exotropia.

BACKGROUND/AIMS: We evaluate the clinical characteristics of intermittent exotropia with controllability and compare surgical outcomes between patients with and without controllability. METHODS: We reviewed the medical records of patients aged 6-18 years with intermittent exotropia who underwent surgery between September 2015 and September 2021. Controllability was defined as the patient's subjective awareness of exotropia or diplopia associated with the presence of exotropia and ability to instinctively correct the ocular exodeviation. Surgical outcomes were compared between patients with and without controllability, with a favorable surgical outcome defined as an ocular deviation between ≤ 10 PD of exotropia and ≤ 4 PD of esotropia at distance and near. RESULTS: Among 521 patients, 130 (25%, 130/521) had controllability. The mean age of onset (7.7 years) and surgery (9.9 years) were higher in patients with controllability than in those without controllability (p < 0.001). The mean control scores of patients with controllability (distance: 1.9, near: 1.5) were lower compared with patients without controllability (distance: 3.0, near: 2.2), reflecting a better level of control. Patients with controllability had a better surgical outcome than those without controllability, as analyzed by log-rank test (p < 0.001). Larger preoperative ocular exodeviation at distance (hazard ratio [HR] = 1.083, confidence interval [CI] = 1.018-1.151, p = 0.012) and near (HR = 1.102, CI = 1.037-1.172, p = 0.002) were significantly related to recurrence in patients with controllability. CONCLUSIONS: Patients with controllability showed better surgical outcomes, later exotropia onset, and better level of control than patients without controllability. Preoperative ocular exodeviation was a significant factor influencing favorable outcomes in patients with controllable exotropia.

Multiscale Engineering of Nanofiber-Aerogel Composite Nanogenerator with Tunable Triboelectric Performance Based on Multifunctional Polysuccinimide.

Multiscale polymer engineering, involving chemical modification to control their triboelectric polarities as well as physicomechanical modification to maximize charge transfer and structural durability, is paramount to developing a high-performance triboelectric nanogenerator (TENG). This report introduces a highly efficient and comprehensive strategy to engineer high-performance TENG based on multifunctional polysuccinimide (PSI). With the ability of PSI to undergo facile nucleophilic addition with amines, sodium sulfate and quaternary ammonium chlorides having opposite charged groups are conjugated to PSI in varying densities. The resulting Sulfo-PSI and TMAC-PSI, respectively, processed into nanofibrous films, demonstrate highly enhanced and variable triboelectric properties based on the charge type and density. To further enhance the mechanical toughness and biocompatibility necessary for wearable applications, these PSI nanofibers are processed into alginate aerogel (AG). The sustained triboelectric performance of this nanofiber-AG TENG as a wearable energy harvester and biosensor is examined and validated in detail.

Exploring the mechanism of trehalose: dual functions of PI3K/Akt and VPS34/mTOR pathways in porcine oocytes and cumulus cells†.

Autophagy, an intracellular recycling system, is essential for the meiotic maturation of porcine oocytes. Trehalose has been reported as a novel mammalian target of rapamycin (mTOR)-independent autophagy inducer in many cells. Furthermore, we previously have demonstrated that trehalose supplementation during in vitro maturation of porcine oocytes improves the developmental competence of parthenogenetic embryos, possibly via autophagic activation, whereas the underlying mechanisms remain unclear. Therefore, the aim of this study was to address this issue. We found that trehalose plays a role as an autophagy activator by autophagic flux assay and determined that it promotes phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) inhibition and vacuolar protein sorting 34 (VPS34)/mTOR activation by immunoblotting, both in cumulus cells (CCs) and oocytes. However, interestingly, the effects and the mechanisms regulated by trehalose were different in them, respectively. In CCs, the autophagy was activated through the improvement of lysosomal function/autophagic clearance viability by upregulation of coordinated lysosomal expression and regulation genes via PI3K/Akt inhibition. Whereas in oocytes, autophagy was activated via induction of VPS34, which directly influences autophagosome formation, and the precise meiotic process was ensured via Akt inhibition and mTOR activation. Taken together, this study furtherly elucidates the novel detailed mechanism of trehalose during porcine oocyte maturation, thus laying the biological foundations for pharmacological application.

Deep learning-based classification of breast cancer cells using transmembrane receptor dynamics.

MOTIVATION: Motions of transmembrane receptors on cancer cell surfaces can reveal biophysical features of the cancer cells, thus providing a method for characterizing cancer cell phenotypes. While conventional analysis of receptor motions in the cell membrane mostly relies on the mean-squared displacement plots, much information is lost when producing these plots from the trajectories. Here we employ deep learning to classify breast cancer cell types based on the trajectories of epidermal growth factor receptor (EGFR). Our model is an artificial neural network trained on the EGFR motions acquired from six breast cancer cell lines of varying invasiveness and receptor status: MCF7 (hormone receptor positive), BT474 (HER2-positive), SKBR3 (HER2-positive), MDA-MB-468 (triple negative, TN), MDA-MB-231 (TN) and BT549 (TN). RESULTS: The model successfully classified the trajectories within individual cell lines with 83% accuracy and predicted receptor status with 85% accuracy. To further validate the method, epithelial-mesenchymal transition (EMT) was induced in benign MCF10A cells, noninvasive MCF7 cancer cells and highly invasive MDA-MB-231 cancer cells, and EGFR trajectories from these cells were tested. As expected, after EMT induction, both MCF10A and MCF7 cells showed higher rates of classification as TN cells, but not the MDA-MB-231 cells. Whereas deep learning-based cancer cell classifications are primarily based on the optical transmission images of cell morphology and the fluorescence images of cell organelles or cytoskeletal structures, here we demonstrated an alternative way to classify cancer cells using a dynamic, biophysical feature that is readily accessible. AVAILABILITY AND IMPLEMENTATION: A python implementation of deep learning-based classification can be found at https://github.com/soonwoohong/Deep-learning-for-EGFR-trajectory-classification. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Uliginosibacterium aquaticum sp. nov., Isolated from a Freshwater Lake.

A Gram-stain-negative, aerobic, chemoheterotrophic, rod-shaped bacterium motile by a polar flagellum, designated IMCC34675T, was isolated from Chungju Lake, an artificial freshwater reservoir in Republic of Korea. The 16S rRNA gene sequence analysis indicated that strain IMCC34675T belongs to the genus Uliginosibacterium, sharing ≤ 97.1% sequence similarities with the type strains of the genus. Whole genome sequencing of strain IMCC34675T revealed a 4.1 Mbp of genome size with 62.4% of the DNA G + C content. The IMCC34675T genome shared 73.3% of average nucleotide identity and 19.9% of digital DNA-DNA hybridization values to the genome of Uliginosibacterium gangwonense DSM 18521T, the type species of the genus. The major fatty acids of strain IMCC34675T were summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c) and C16:0. The respiratory quinone detected in the strains was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminophospholipid, one aminolipid, and five unidentified lipids. Based on the phylogenetic and phenotypic characterization, strain IMCC34675T was considered to represent a novel species within the genus Uliginosibacterium, for which the name Uliginosibacterium aquaticum sp. nov. is proposed with IMCC34675T (= KACC 21758T = NBRC 114418T) as the type strain.

Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells.

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.

Characterization and comparison of genomic profiles between primary cancer cell lines and parent atypical meningioma tumors.

BACKGROUND: Meningiomas are the second most common primary tumors of the central nervous system. However, there is a paucity of data on meningioma biology due to the lack of suitable preclinical in vitro and in vivo models. In this study, we report the establishment and characterization of patient-derived, spontaneously immortalized cancer cell lines derived from World Health Organization (WHO) grade I and atypical WHO grade II meningiomas. METHODS: We evaluated high-resolution 3T MRI neuroimaging findings in meningioma patients which were followed by histological analysis. RT-qPCR and immunostaining analyses were performed to determine the expression levels of meningioma-related factors. Additionally, flow cytometry and sorting assays were conducted to investigate and isolate the CD133 and CD44 positive cells from primary atypical meningioma cells. Further, we compared the gene expression profiles of meningiomas and cell lines derived from them by performing whole-exome sequencing of the blood and tumor samples from the patients, and the primary cancer cell lines established from the meningioma tumor. RESULTS: Our results were consistent with earlier studies that reported mutations in NF2, SMO, and AKT1 genes in atypical meningiomas, and we also observed mutations in MYBL2, a gene that was recently discovered. Significantly, the genomic signature was consistent between the atypical meningioma cancer cell lines and the tumor and blood samples from the patient. CONCLUSION: Our results lead us to conclude that established meningioma cell lines with a genomic signature identical to tumors might be a valuable tool for understanding meningioma tumor biology, and for screening therapeutic agents to treat recurrent meningiomas.

Graft Architecture Guided Simultaneous Control of Degradation and Mechanical Properties of In Situ Forming and Fast Dissolving Polyaspartamide Hydrogels.

Polyaspartamide, derived from polysuccinimide (PSI), has the advantage of conveniently presenting desired functional groups by ring-opening addition of amine-based nucleophiles to the succinimidyl ring moieties of PSI. Using diamines with varying lengths of poly(ethylene glycol) linker, polyaspartamide presenting amine groups with controllable grafting density and length, namely, poly(2-hydroxyethyl aspartamide)-g-amino-poly(ethylene glycol) (PHEA-PEGAm) could be synthesized. This PHEA-PEGAm was then used to develop in situ forming hydrogels by Schiff base formation with aldehyde-containing alginate (Alg-ALD). By modulating the graft architecture (i.e., grafting length and density), the mechanical properties of the resulting Alg-PHEA hydrogels could be controlled in a broad range. Remarkably, the hydrogels were shown to undergo facile degradation and complete dissolution in physiological conditions, regardless of hydrogel mechanics, by the expedited hydrolysis through the action of remaining amine groups, which was also heavily influenced by the graft architecture. Moreover, the rate of degradation could be further controlled by additional ionic cross-linking of alginate. The potential application as an injectable drug delivery system was demonstrated by measuring drug release kinetics and monitoring degradation ex vivo.

Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium.

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-ß (TGF-ß) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 µM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

Efficacy of intravitreal bevacizumab for macular edema following branch retinal vein occlusion stratified by baseline visual acuity.

PURPOSE: To compare the clinical features and bevacizumab efficacy for macular edema (ME) following branch retinal vein occlusion (BRVO) stratified by baseline visual acuity. METHODS: This retrospective study included a total 117 eyes from 117 consecutive patients with ME following BRVO, who received PRN intravitreal bevacizumab injection and were followed for more than 6 months. The eyes were categorized into three groups according to baseline best-corrected visual acuity (BCVA) (group A, BCVA <20/200; group B, BCVA ≥20/200 and ≤20/40; group C, BCVA >20/40). Baseline demographics, clinical features, BCVA, and central retinal thickness (CRT) at 1, 3, 6, and 12 months after injection and the number of injections were compared. RESULTS: Groups A-C included 11, 83, and 23 eyes, respectively. The mean baseline CRT was thickest in group A (810.1, 580.8, and 473.5 µm in groups A-C, respectively; p < 0.001) and the percentage of eyes with macular ischemia increased in the worst BCVA group (45.5, 25.0, and 4.3 % in groups A-C, respectively; p = 0.005). The mean BCVA and CRT improved at 1, 3, 6, and 12 months after treatment compared to baseline values in all groups (all, p < 0.001). The number of injections for 6 months was greater in the worst BCVA group (3.2, 2.3, and 1.9 injections in groups A-C, respectively; p = 0.009). CONCLUSION: In ME following BRVO, baseline visual acuity correlates with macular ischemia and baseline CRT. Intravitreal bevacizumab treatment results in significant anatomical and functional improvement regardless of baseline visual acuity.

COMPARISON OF THE LAMINA CRIBROSA THICKNESS OF PATIENTS WITH UNILATERAL BRANCH RETINAL VEIN OCCLUSION AND HEALTHY SUBJECTS.

PURPOSE: To compare the lamina cribrosa (LC) thickness of healthy subjects and patients with unilateral branch retinal vein occlusion (BRVO), and to determine possible correlations between the LC thickness and the BRVO subtypes. METHODS: This prospective, cross-sectional study included a total of 46 patients with naive, untreated, unilateral BRVO and 31 healthy control subjects. The occlusion site was divided into two BRVO types: arteriovenous crossing BRVO (AV-BRVO) and optic nerve BRVO (ON-BRVO). The optic nerve head was scanned using enhanced-depth imaging with the Spectralis optical coherence tomography system. RESULTS: The mean LC thickness of both eyes in patients with BRVO was thinner than that of eyes (274.0 µm) of the healthy subjects (both, P < 0.001). Although the LC thickness of the BRVO-affected eyes was slightly thinner than that of the fellow eyes (237.0 µm vs. 241.4 µm, respectively), there was no statistically significant difference. In addition, there were no significant differences in the LC thicknesses of both eyes according to the site of occlusion. CONCLUSION: A thinner LC was observed in both eyes of unilateral BRVO patients compared with those of healthy subjects. This finding suggests that thin LC may contribute to the pathogenesis of BRVO as a local mechanical factor in addition to systemic factors.

Parental perspectives and concerns regarding exotropia surgery and comparison with clinicians' predictions.

PURPOSE: To evaluate parental perspectives and concerns regarding exotropia surgery and compare them with clinicians' predictions of parental responses in Korean pediatric patients with intermittent exotropia. METHODS: This survey study included the parents of pediatric patients with intermittent exotropia who underwent surgery and clinicians at five hospitals from June 2022 to February 2023, who participated in the Survey of Parental Attitude and Concerns of Exotropia surgery (SPACE) study 1. Parental attitudes and concern about exotropia surgery were assessed using a questionnaire. Clinicians' estimation of each item corresponding to the parental questionnaire was also assessed and compared with parental responses. RESULTS: A total of 266 parents and 41 clinicians were included. More parents responded that information about surgery was most helpful or most commonly received from clinicians than clinicians estimated (P = 0.001). More parents reported actively communicating with the child about surgery than clinicians estimated (P < 0.001). Parents showed a higher level of concern for general anesthesia and the hospital environment than clinicians thought they would (P = 0.002 and P < 0.001, resp.). In the postoperative follow-up items, parents showed high levels of concern regarding postoperative infection (P < 0.001), conjunctival redness (P = 0.040), persistent overcorrection (P < 0.001), and glasses wearing (P = 0.019). CONCLUSIONS: Parental perspectives and concerns regarding pediatric intermittent exotropia surgery differed from clinicians' estimations thereof. More parents obtain information on exotropia surgery from clinicians and actively talk about surgery with their child than estimated by clinicians. Parents had a higher level of concern regarding general anesthesia, hospital environment, postoperative infection, conjunctival redness, persistent overcorrection, and glasses wearing compared with clinician estimations.

A general analytical solution for fluid flow and heat convection through arbitrary-shaped triangular ducts: A variational analysis.

This paper presents an analytical solution for fluid flow and heat transfer inside arbitrarily-shaped triangular ducts for the first time. The former analytical solutions are limited to the special case of isosceles triangular ducts. The literature has no report about the analytical solution for the general case of arbitrarily-shaped triangular ducts. Due to the significant role of fluid flow through non-circular channels in industry and the large number of triangular shapes, a method for solving the heat transfer problem for all triangular shapes is needed. The heat transfer of a fluid flow through a channel with an arbitrary triangular cross-section for the case of constant heat flux at the walls is solved in this work for the first time, considering viscous dissipation. Here, the functionals of flow and heat transfer equations are derived, and the resulting Euler-Lagrange equations are solved using the Ritz method. The effect of the duct geometry on the velocity profile and friction coefficient is studied in detail. The effect of the Brinkman number on the temperature distribution and Nusselt number is investigated for both cooling and heating cases. The results reveal that the critical Brinkman Number distinguishes between the cooling and heating cases and represents the critical point at which the Nusselt number approaches infinity. The value of the Nusselt number decreases with the increase of the Brinkman number in both the wall cooling and heating modes. It is also found that the equilateral triangle exhibits the minimum friction coefficient and the maximum value of the Poiseuille number.

Structural identification and quantification of unreported sialylated N-glycans in bovine testicular hyaluronidase by LC-ESI-HCD-MS/MS.

Bovine testicular hyaluronidase (BTH), which accelerates the absorption and dispersion of drugs by decomposing hyaluronan in subcutaneous tissues, has been used in medical applications, including local anesthesia, ophthalmology, and dermatosurgery. The requirement of N-glycans for the activity of human hyaluronidase has been reported, and BTH has greater activity than human hyaluronidase. However, the N-glycan characteristics of BTH are unclear. From a commercial BTH source containing additional proteins, purified BTH (pBTH) was obtained using size exclusion chromatography, and the structures and quantities of its N-glycans were analyzed using liquid chromatography (LC)-electrospray ionization-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS). In pBTH, 32 N-glycans were identified, with 12 sialylations (39.0% of total N-glycan content), nine core-fucosylations (31.5%), six terminal galactosylations (14.6%), five high-mannosylations (13.7%), and four bisecting N-acetylglucosamine structures (7.8%). The presence of sialylated glycopeptides in pBTH was confirmed by nano-LC-HCD-MS/MS analysis. The absolute quantity of all N-glycans was calculated as 1.4 pmol (0.6 pmol for sialylation) in pBTH (1.0 pmol). The sialylation level (related to half-life, thermal stability, resistance to proteolysis, and solubility) was 24.4 times higher than that of human hyaluronidase. The hyaluronan degradation activity of de-sialylated pBTH decreased to 41.2 ± 4.2%, showing that sialylated N-glycans were required for pBTH activity as well. This is the first study to identify and quantify 32 N-glycans of pBTH and investigate their structural roles in its activity. The presence of larger amounts of sialylated N-glycans in pBTH than in human hyaluronidase suggests a greater utilization of pBTH.

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1).

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

Experimental study on flow and turbulence characteristics of jet impinging on cylinder using three-dimensional Lagrangian particle tracking velocimetry.

When a round jet impinges on a convex cylindrical surface, complex three-dimensional (3D) flow structures occur, accompanied by the Coanda effect. To characterize the flow and turbulence properties of the general system, ensemble averages of 3D Lagrangian particle tracking velocimetry measurements were taken. The radial bin-averaging method was used in post-processing the tracked particles and corresponding instantaneous velocity vectors to generate appropriate ensemble-averaged statistics. Two impinging angles were selected, and at a fixed Reynolds number, the ensemble-averaged volumetric velocity field and turbulent stress tensor components were measured. The flow and turbulence characteristics of the impinging jet on the cylinder were notably different based on the impinging angle, especially in the downstream region. Surprisingly, the attached wall jet with a half-elliptic shape was abruptly thickened in the wall-normal direction, similar to the axis switching phenomenon observed in elliptic jets in the case of oblique impingement. In the jet-impinging region, the flow spread in all directions with high mean vorticity values. With the development of a 3D curved wall jet, both the Coanda effect and centrifugal force played a significant role in the flow behavior. A notable feature of the self-preserving region was the similarity of mean velocity profiles with scaling by the maximum velocity and the jet half-width for both impinging angle cases. Local isotropy of turbulent normal stresses was observed in this region, supporting the existence of self-preservation in the 3D curved wall jet. The volumetric ensemble-averaged Reynolds stress tensor revealed strong inhomogeneous turbulence in the boundary layer region and the curvature effect on the Reynolds shear stress in the free shear layer.