Cause and consequence of heterogeneity in human mesenchymal stem cells: Challenges in clinical application.

Human mesenchymal stem cells (hMSCs) are mesoderm-derived adult stem cells with self-proliferation capacity, pluripotent differentiation potency, and excellent histocompatibility. These advantages make hMSCs a promising tool in clinical application. However, the majority of clinical trials using hMSC therapy for diverse human diseases do not achieve expectations, despite the prospective pre-clinical outcomes in animal models. This is partly attributable to the intrinsic heterogeneity of hMSCs. In this review, the cause of heterogeneity in hMSCs is systematically discussed at multiple levels, including isolation methods, cultural conditions, donor-to-donor variation, tissue sources, intra-tissue subpopulations, etc. Additionally, the effect of hMSCs heterogeneity on the contrary role in tumor progression and immunomodulation is also discussed. The attempts to understand the cellular heterogeneity of hMSCs and its consequences are important in supporting and improving therapeutic strategies for hMSCs.

Long-term efficacy, safety, and cumulative retention rate of antitumor necrosis factor-alpha treatment for patients with Behcet's uveitis: A systematic review and meta-analysis.

AIM: This study aims to evaluate the long-term efficacy, safety, and cumulative retention rate of antitumor necrosis factor-alpha (anti-TNF-α) therapy for patients with Behcet's uveitis (BU) using meta-analysis. METHODS: We searched the Web of Science and PubMed databases for eligible studies up to December 1, 2022. The quality of each identified study was assessed using the Joanna Briggs Institute's case series literature quality assessment tool. Statistical analysis was conducted using Stata 16.0 software with a random-effects model. RESULTS: Twelve studies comprising 1156 patients with BU were included in our analysis. We found that 85.0% of patients achieved ocular inflammation remission after receiving anti-TNF-α treatment, with a 95% confidence interval (CI) ranging from 78.7% to 90.5%. Additionally, 77.4% (95% CI: 57.5%-92.5%) experienced an improvement in visual acuity (VA). Moreover, the pooled dose reduction of glucocorticoids (GCs) was 11.08 mg (95% CI: -13.34 mg to -8.83 mg). Throughout the follow-up period, the cumulative retention rate of the medication was 67.3% (95% CI: 53.7%-79.6%). Serious adverse events occurred in 5.8% (95% CI: 3.1%-8.9%) of cases, with the three most common types being severe infusion or injection reactions (2.7%; 95% CI: 0.8%-5.4%), tuberculosis (1.3%; 95% CI: 0.0%-3.9%), and bacterial pneumonia (1.3%; 95% CI: 0.1%-3.4%). Subgroup analysis revealed that ocular inflammation remission rates were 89.3% (95% CI: 81.2%-95.5%) for adalimumab treatment and 83.7% (95% CI: 75.3%-90.8%) for infliximab treatment. The drug retention rate after adalimumab therapy was 70.3% (95% CI: 62.0%-78.0%) compared to 66.4% (95% CI: 48.6%-82.2%) for infliximab treatment. Furthermore, the incidence of severe infusion or injection reactions was 2.2% (95% CI: 0.1%-5.8%) following adalimumab treatment and 3.5% (95% CI: 0.7%-7.7%) following infliximab treatment. CONCLUSIONS: Anti-TNF-α therapy represents an effective treatment for BU patients with favorable safety profile and high drug retention rate and a potential advantage of adalimumab over infliximab in terms of ocular inflammation remission, drug retention, and the incidence of severe infusion or injection reactions.

Site-specific N-glycan changes during semen liquefaction.

Normal liquefaction of semen is one of the key steps to ensure the smooth progress of fertilization, and glycosylation has been reported to be involved in the whole process of fertilization. Till now, it is still unclear whether and how glycosylation changes during the liquefaction process of semen. In this study, by performing a glycoproteomic analysis of human semen with the liquefaction process (liquefaction time of semen: 0 min vs 30 min) using our recently developed StrucGP software combined with the Tandem Mass Tags (TMT) based quantification, we identified 25 intact glycopeptides (IGPs) from 10 glycoproteins in semen that were significantly changed during liquefaction, including 23 up-regulated and two down-regulated. Among the 23 up-regulated glycopeptides, half were modified with sialylated glycans, suggesting that sialylated glycans may play a key role in the semen liquefaction process. The data provide an invaluable resource for further studies on the role of glycosylation during semen liquefaction.

Recognition of Core-Fucosylated Glycopeptides Based on the Y1+Fuc/Y1 Ratio in Low-Energy HCD Spectra.

Accurate identification of core fucosylation on N-glycopeptides remains challenging due to fucose migration during mass spectrometry analysis. Here, we introduce a simple and straightforward method for core-fucosylated glycopeptide recognition based on the relative intensities of Y1+Fuc ions compared with their corresponding Y1 ions (labeled as Y1+Fuc/Y1 or simply Y1F/Y1 ratio > 0.1) in low-energy HCD-based spectra. The method was first developed by systematically evaluating the influence of fucose migration on the Y1F ion from antenna fucoses based on the distribution of the Y1F/Y1 ratios in the MS/MS spectra of antenna-fucosylated glycopeptides from Fut8-/- mouse brain. The feasibility of the method was then confirmed by using two standard glycoproteins, comparison with glycopeptides in Fut8+/+ mouse brain with/without in silico core-fucosylation removal, and Y1F/Y1 ratio alterations under a lower HCD energy. This method will be applicable to the manual interpretation and software-based high-throughput analysis of core-fucosylated glycopeptides.

Glycoproteomic analysis reveals the effects of bisecting GlcNAc in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second major subtype of primary liver cancer and has caused more and more attention with increasing incidence and mortality worldwide. Our previous study found that bisecting N-glycans are commonly increased in ICC, while the effects and potential functions of bisecting GlcNAc in ICC are still largely unclear. In this study, we further confirmed that the structures of bisecting GlcNAc were significantly up-regulated in ICC compared with paracancer tissues by glycoproteomic data and lectin histochemistry. The expression of its glycosyltransferase MGAT3 was also up-regulated in ICC tissues at both mRNA and protein levels, and expression of MGAT3 is negatively correlated with overall survival explored by bioinformatic analyses and published datasets from 255 patients. Next, the silencing of MGAT3 could inhibit the growth and invasion of ICC cells, and overexpressing of MGAT3 only promoted ICC cell invasion. Further glycoproteomic analysis showed that the commonly glycoproteins modified by bisecting GlcNAc after MGAT3-overexpression in two ICC cell lines were mainly involved in cell movement-related biological processes, such as cell adhesion, integrin-related and ECM-receptor interaction. This study sheds light on the potential effects of bisecting GlcNAc in ICC cells and suggests that MGAT3 might be used as a potential target in the therapy of ICC.

IL-17A Promotes the Migration, Invasion and the EMT Process of Lung Cancer Accompanied by NLRP3 Activation.

Background: Lung cancer is a deadly cancer worldwide, and its pathogenesis and treatment methods require continuous research and exploration. As a representative factor of adaptive immunity, the role of interleukin-17A (IL-17A) in lung cancer is still unclear. The purpose of the present study was to investigate the effect of IL-17A on the biological behaviour of lung cancer cells and the relative mechanism. Methods: The human lung adenocarcinoma A549 and H1299 cell lines were used for in vitro study. The effects of IL-17A on cell proliferation, migration and invasion were assessed by CCK-8 assay, wound-healing assay, transwell invasion assay and real-time cell analysis (RTCA). The expression levels of marker proteins in the process of epithelial-mesenchymal transition (EMT) were detected by western blot analysis. Caspase-1 activity and the concentration of IL-1ß after NLRP3 inflammasome activation were measured by a Caspase-1 Activity Assay Kit and an IL-1ß ELISA kit, respectively. Results: Compared to the control group, IL-17A treatment did not affect the proliferation of A549 and H1299 cells in vitro, but it promoted cell migration, invasion and the EMT process. IL-17A treatment increased NLRP3 expression, caspase-1 activity and IL-1ß level. Blockade of NLRP3 alleviated the cell migration, invasion and the EMT process induced by IL-17A. Conclusions: In conclusion, these findings indicated that NLRP3 participates in the migration, invasion and the EMT process of IL-17A-stimulated lung cells in vitro.

Evaluation of absorbent cotton for glycopeptide enrichment.

Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200µL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.

Precision Structural Interpretation of Site-Specific N-Glycans in Seminal Plasma.

N-Linked glycoproteins are rich in seminal plasma, playing various essential roles in supporting sperm function and the fertilization process. However, the detailed information on these glycoproteins, particularly site-specific glycan structures, is still limited. In this study, a precision site-specific N-glycoproteome map of human seminal plasma was established by employing the site-specific glycoproteomic approach and a recently developed glycan structure interpretation software, StrucGP. A total of 9567 unique glycopeptides identified in human seminal plasma were composed of 773 N-linked glycan structures and 1019 N-glycosites from 620 glycoproteins. These glycans were comprised of four types of core structures and 13 branch structures. The majority of identified glycoproteins functioned in response to stimulus and immunity. As we reported in human spermatozoa, heavy fucosylation (fucose residues ≥6 per glycan) was also detected on seminal plasma glycoproteins such as clusterin and galectin-3-binding protein, which were involved in the immune response of biological processes and reactome pathways. Comparison of site-specific glycans between seminal plasma and spermatozoa revealed more complicated glycan structures in seminal plasma than in spermatozoa, even on their shared glycoproteins. These present data will be greatly beneficial for the in-depth structural and functional study of glycosylation in the male reproduction system.

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa.

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.

Optimization of Sperm Management and Fertilization in Zebrafish (Danio rerio (Hamilton)).

The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0-2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40-60%, respectively, with milt stored for 1.5 h in the E400 extender at 0-2 °C.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Level of in vitro storage of the European catfish (Silurus glanis L.) eggs at different temperatures.

European catfish (Silurus glanis) is a commercially important freshwater fish originating from Eastern Europe. The objective of this study was to examine the short-term storage of its eggs especially in relation to maintaining a low level of malformation in newly hatched fry. The eggs from freshly spawned individuals were stored separately in cell incubators at 17 and 22 °C under aerobic conditions. Changes in fertilization, hatching, and malformation were examined in eggs stored at 1, 3, 5, and 7 h post-stripping. The sperm used for fertilization showed very good motility rates (84-90%) and curvilinear velocity (110-125 µm/s), and straight-line velocity did not drop below 77 µm/s. For all females, a temperature of 17 °C was better than 22 °C for egg storage in vitro. Egg fertilization and total hatching decreased rapidly after 7 h storage at 17 °C. The storage time of eggs in vitro to fertilization should therefore not exceed 5 h at 17 °C. In all females, there was no difference in the total number of eggs hatching between 1 and 3 h of egg storage at 17 °C. The storage time of eggs did not correlate with the level of malformations of the fry. However, the level of hatching and malformations was clearly affected by the storage temperature of eggs when it was > 17 °C. Analysis showed that the storage time of eggs, temperature of storage, and individual females had a significant influence on fertilization and total hatching rates. Regression analysis confirmed a low correlation of fertilization and hatching rates with storage time of eggs.

Biological Functions and Large-Scale Profiling of Protein Glycosylation in Human Semen.

Glycosylation is one of the most important post-translational modifications of proteins and plays an essential role in spermatogenesis, maturation, extracellular quality control, capacitation, sperm-egg recognition, and final fertilization. Spermatozoa are synthesized in the testes inactively with a thick glycocalyx and passed through the epididymis for further modification by glycosylation, deglycosylation, and integration to reach maturation. Subsequently, sperm capacitation and further fertilization require redistribution of glycoconjugates and dramatic glycocalyx modification of the spermatozoa surface. Furthermore, glycoproteins and glycans in seminal plasma are functional in maintaining spermatozoa structure and stability. Therefore, aberrant glycosylation may cause alteration of semen function and even infertility. Currently, mass spectrometry-based technologies have allowed large-scale profiling of glycans and glycoproteins in human semen. Quantitative analysis of semen glycosylation has also indicated many involved glycoproteome issues in male infertility and the potential biomarkers for diagnosis of male infertility in clinical. This review summarizes the role of glycosylation during spermatozoa development, the large-scale profiling of glycome and glycoproteome in human semen, as well as the association of aberrant glycosylation with infertility.

Improving motility and fertilization capacity of low-quality sperm of sterlet Acipenser ruthenus during storage.

Improvement of sperm quality with low motility by storage could ensure higher success of fertilization and maintain higher genetic diversity, especially for sturgeons, which as endangered species have limited broodstock and gametes. Sperm was collected from mature male sterlet Acipenser ruthenus and motility was evaluated using the CASA system; samples were categorized as GS 'good sperm' (>80%) or BS 'bad sperm' (<20%). Samples from both groups were incubated with seminal plasma from good- (GSP) and bad-quality sperm (BSP), respectively for 15 min, 6 h, 24 h and 96 h at 4 °C. Motility of BS incubated in GSP increased after different storage times compared to BS incubated in BSP, while the motility and velocity of GS incubated in BSP decreased compared to GS incubated in GSP. Fertilization rates were evaluated with samples stored for 15 min and 6 h post-stripping; fertilization and hatching rate of BS after incubation in GSP increased significantly compared to the BS incubated in BSP. Inorganic ion (Na+, K+, Cl-) concentrations and osmolality of BSP were significantly lower than that of GSP. These results indicated that sterlet sperm quality can be revitalized by incubation with GSP. Further, fertilization capacity of BS after incubation in GSP can reach similar levels to the good quality sperm (∼70%). Low ion concentration and osmolality in BSP may be a partial cause of low sperm quality. The current study is the first report on the capability to revitalize low quality sterlet sperm by storage in GSP.

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii.

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN3 ), glycolysis (2-deoxy-D-glucose, DOG) and ß-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN3 resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O2 min-1 (109 spz)-1 ) was significantly higher than in NAM (8.2 ± 1.5 nmol O2 min-1 (109 spz)-1 ). The OCR significantly declined with addition of NaN3 to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.

Optimization of sterlet (Acipenser ruthenus) egg incubation.

Sterlet Acipenser ruthenus was used to assess egg and embryo development when incubated at 17 °C in Petri dishes placed in a hatchery tank (300 L recirculating dechlorinated water) with incubation occurring in a static tabletop system in an air-conditioned laboratory, or in a 700 L Q-cell incubator. Eggs in each dish were placed in a plastic box with 300 mL dechlorinated water. Separated eggs from three individual females were fertilized using pooled sperm from four males with there being four replicates. There were no differences (P > 0.05) in mean percentages of neurulation and embryos undergoing cleavage for eggs incubated in the hatchery tank and with use of the static tabletop system. Furthermore, there were no differences (P >  0.05) in percentage of embryos undergoing cleavage, neurulation and hatching for each female when eggs were incubated using the two systems. Results indicate a Petri dish placed in a small plastic box with 300 mL of dechlorinated water was adequate for incubation of sterlet eggs. Results of the study also indicate that with the static system: 1) eggs should be fertilized from each female to retain individual identity; 2) eggs should be dispersed in Petri dishes to avoid clumping; 3) water should be changed at 24 h, but not at 48 h (neurulation) post-fertilization; and 4) embryos that do not optimally develop should be removed the day after neurulation (72 h of post-fertilization period) and water should be exchanged every day subsequent to the 48 h time-point post-fertilization.

Transferrin Identification in Sterlet (Acipenser ruthenus) Reproductive System.

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Gene-Wide Analysis of Aquaporin Gene Family in Malus domestica and Heterologous Expression of the Gene MpPIP2;1 Confers Drought and Salinity Tolerance in Arabidposis thaliana.

The aquaporins (AQPs) are a family of integral membrane proteins involved in the transcellular membrane transport of water and other small molecules. A scan of the apple (Malus domestica) genome revealed the presence of 42 genes encoding putative AQPs. Based on a phylogenetic analysis of the deduced peptide sequences of the AQPs generated by Arabidopsis thaliana, poplar (Populus trichocarpa), and rubber (Hevea brasiliensis), the apple AQPs were each assigned membership of the five established AQP subfamilies, namely the PIPs (eleven members), the TIPs (thirteen members), the NIPs (eleven members), the SIPs (five members), and the XIPs (two members). The apple AQPs included asparagine-proline-alanine (NPA) motifs, an aromatic/arginine (ar/R) selectivity filter, and the Froger's positions. The heterologous expression of MpPIP2;1 in A. thaliana was shown to enhance the level of tolerance exhibited against both drought and salinity.

In vitro antioxidant enzyme activity and sperm motility at different temperatures in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss.

Influence of in vitro temperature on sperm antioxidant enzyme activity, thiobarbituric acid-reactive substance (TBARS) content and motility parameters was evaluated in sterlet Acipenser ruthenus and rainbow trout Oncorhynchus mykiss. Sperm activation was conducted at 4, 14 and 24 °C in both species. Duration of motility was significantly longer at 4 °C than at 14 and 24 °C in both species. At 60 s post-activation, the velocity of sterlet spermatozoa was highest at 24 °C. This trend continued to 420 s post-activation. In rainbow trout, at 10 s post-activation, the highest velocity was observed at 14 °C. Significantly higher catalase activity was seen at 4 °C in both species. No significant difference in spermatozoon superoxide dismutase activity among temperatures was observed. In sterlet, TBARS content was significantly higher at 24 °C compared to other temperatures, but, in rainbow trout, it was highest at 4 °C. The results presume species-specific level of antioxidant enzyme activity and TBARS content at studied temperatures.