The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism.

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

Semen quality and sperm DNA fragmentation in cancer patients undergoing sperm cryopreservation.

PURPOSE: We compared semen quality and sperm DNA fragmentation in cancer patients who underwent sperm banking and controls who underwent sperm cryopreservation for assisted reproductive technology (ART). MATERIALS AND METHODS: A total of 132 men, 65 cancer patients and 67 controls, were prospectively enrolled and performed sperm cryopreservation for fertility preservation from May 2019 to February 2021. Sperm quality was determined by measuring semen volume, sperm concentration, sperm motility, and sperm DNA fragmentation index (DFI). Sperm quality and sperm DFI were compared in cancer patients and controls. RESULTS: The major cancers of the 65 cancer patients were leukemia (26.2%), testicular cancer (23.1%), and lymphoma (20.0%). Sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls. Sperm DFI was significantly higher in cancer patients than in controls (24.32%±15.69% vs. 19.11%±11.63%; p=0.033). After excluding 8 cancer patients who received chemotherapy before sperm banking, sperm concentration, sperm total motility, and sperm progressive motility were significantly lower in cancer patients than in controls, but there was no significant difference in sperm DFI for cancer patients and controls (23.14%±12.79% vs. 19.11%±11.63%; p=0.069). CONCLUSIONS: Sperm quality was lower in cancer patients than in controls. There was no difference in the sperm DFI of cancer patients prior to chemotherapy and men presenting for sperm cryopreservation for ART. We recommend that all men who are planning cancer therapy should be offered sperm banking prior to gonadotoxic chemotherapy as a standard of fertility preservation.

An integrated view of lipid metabolism in ferroptosis revisited via lipidomic analysis.

Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. This process contributes to cellular and tissue damage in various human diseases, such as cardiovascular diseases, neurodegeneration, liver disease, and cancer. Although polyunsaturated fatty acids (PUFAs) in membrane phospholipids are preferentially oxidized, saturated/monounsaturated fatty acids (SFAs/MUFAs) also influence lipid peroxidation and ferroptosis. In this review, we first explain how cells differentially synthesize SFA/MUFAs and PUFAs and how they control fatty acid pools via fatty acid uptake and ß-oxidation, impacting ferroptosis. Furthermore, we discuss how fatty acids are stored in different lipids, such as diacyl or ether phospholipids with different head groups; triglycerides; and cholesterols. Moreover, we explain how these fatty acids are released from these molecules. In summary, we provide an integrated view of the diverse and dynamic metabolic processes in the context of ferroptosis by revisiting lipidomic studies. Thus, this review contributes to the development of therapeutic strategies for ferroptosis-related diseases.

Effect of male age on reproductive function: A comparison of young and middle-aged men.

PURPOSE: There have been concerns regarding potential effects of older paternal age on male reproductive function. However, currently available data on this topic are insufficient and controversy exists. We analyzed semen characteristics and reproductive hormones in young men and middle-aged men to investigate the effect of age on male reproductive function. MATERIALS AND METHODS: This study examined healthy males of reproductive age who visited a single infertility center from January 2016 to July 2021. The young group consisted of men who were less than 35 years-old, and the middle-age group consisted of men who were more than 45 years-old. RESULTS: The two groups had no significant differences in sperm concentration ([89.9±59.4]×106/mL vs. [104.4±82.1]×106/mL, p=0.108) or sperm morphology (normal forms: 3.6%±1.5% vs. 3.4%±1.6%, p=0.131). However, the middle-age group had a smaller semen volume (3.2±1.5 mL vs. 2.5±1.4 mL, p<0.001), lower sperm motility (42.3%±9.8% vs. 31.2%±12.4%, p<0.001), lower progressive sperm motility (39.2%±10.3% vs. 28.4%±12.6%, p<0.001), and a higher serum follicle-stimulating hormone level. CONCLUSIONS: Our results suggest that advanced male age might have a negative effect on fertility potential, as in women. This finding has important clinical implications because more couples are choosing to have children when they are older. Further studies on this issue, especially those that examine reproductive outcome, are warranted.

Depletion of Janus kinase-2 promotes neuronal differentiation of mouse embryonic stem cells.

Janus kinase 2 (JAK2), a non-receptor tyrosine kinase, is a critical component of cytokine and growth factor signaling pathways regulating hematopoietic cell proliferation. JAK2 mutations are associated with multiple myeloproliferative neoplasms. Although physiological and pathological functions of JAK2 in hematopoietic tissues are well-known, such functions of JAK2 in the nervous system are not well studied yet. The present study demonstrated that JAK2 could negatively regulate neuronal differentiation of mouse embryonic stem cells (ESCs). Depletion of JAK2 stimulated neuronal differentiation of mouse ESCs and activated glycogen synthase kinase 3ꞵ, Fyn, and cyclin-dependent kinase 5. Knockdown of JAK2 resulted in accumulation of GTPbound Rac1, a Rho GTPase implicated in the regulation of cytoskeletal dynamics. These findings suggest that JAK2 might negatively regulate neuronal differentiation by suppressing the GSK-3ß/Fyn/CDK5 signaling pathway responsible for morphological maturation. [BMB Reports 2021; 54(12): 626-631].

Mitochondrial Transplantation as a Novel Therapeutic Strategy for Mitochondrial Diseases.

Mitochondria are the major source of intercellular bioenergy in the form of ATP. They are necessary for cell survival and play many essential roles such as maintaining calcium homeostasis, body temperature, regulation of metabolism and apoptosis. Mitochondrial dysfunction has been observed in variety of diseases such as cardiovascular disease, aging, type 2 diabetes, cancer and degenerative brain disease. In other words, the interpretation and regulation of mitochondrial signals has the potential to be applied as a treatment for various diseases caused by mitochondrial disorders. In recent years, mitochondrial transplantation has increasingly been a topic of interest as an innovative strategy for the treatment of mitochondrial diseases by augmentation and replacement of mitochondria. In this review, we focus on diseases that are associated with mitochondrial dysfunction and highlight studies related to the rescue of tissue-specific mitochondrial disorders. We firmly believe that mitochondrial transplantation is an optimistic therapeutic approach in finding a potentially valuable treatment for a variety of mitochondrial diseases.

Selective elimination of human pluripotent stem cells by Anti-Dsg2 antibody-doxorubicin conjugates.

The self-renewal properties of human pluripotent stem cells (hPSCs) contribute to their efficacy in tissue regeneration applications yet increase the likelihood of teratoma formation, thereby limiting their clinical utility. To address this issue, we developed a tool to specifically target and neutralize undifferentiated hPSCs, thereby minimizing tumorigenicity risk without negatively affecting regenerated and somatic tissues. Specifically, we conjugated a monoclonal antibody (K6-1) previously generated in our laboratory against desmoglein 2 (Dsg2), which is highly differentially expressed in undifferentiated hPSCs versus somatic tissues, to the chemotherapeutic agent doxorubicin (DOX). The K6-1-DOX conjugates were selectively targeted and incorporated into Dsg2-positive hPSCs, leading to pH-dependent endosomal release and nuclear localization of DOX with subsequent cytotoxicity via an apoptotic caspase cascade. Conversely, Dsg2-negative fibroblasts showed minimal conjugate uptake or cytotoxicity, suggesting that K6-1-DOX treatment would yield few side effects owing to off-target effects. Selective removal of undifferentiated stem cells was also supported by in vivo studies using a mouse xenograft model, wherein hIgG-DOX- but not K6-1-DOX-pretreated-hPSC injection led to teratoma development. Together, these results validated the ability of the Dsg2-targeted antibody-anticancer drug conjugate to facilitate the safety of stem cell therapies.

The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene.

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

Nurr1 performs its anti-inflammatory function by regulating RasGRP1 expression in neuro-inflammation.

Nurr1, a transcription factor belonging to the orphan nuclear receptor, has an essential role in the generation and maintenance of dopaminergic neurons and is important in the pathogenesis of Parkinson' disease (PD). In addition, Nurr1 has a non-neuronal function, and it is especially well known that Nurr1 has an anti-inflammatory function in the Parkinson's disease model. However, the molecular mechanisms of Nurr1 have not been elucidated. In this study, we describe a novel mechanism of Nurr1 function. To provide new insights into the molecular mechanisms of Nurr1 in the inflammatory response, we performed Chromatin immunoprecipitation sequencing (ChIP-Seq) on LPS-induced inflammation in BV2 cells and finally identified the RasGRP1 gene as a novel target of Nurr1. Here, we show that Nurr1 directly binds to the RasGRP1 intron to regulate its expression. Moreover, we also identified that RasGRP1 regulates the Ras-Raf-MEK-ERK signaling cascade in LPS-induced inflammation signaling. Finally, we conclude that RasGRP1 is a novel regulator of Nurr1's mediated inflammation signaling.

Rapid differentiation of astrocytes from human embryonic stem cells.

Astrocytes are abundant cells in the brain and have vital roles in various brain functions that include biochemical support of endothelial cells, supplying nutrients to the nervous tissue, maintaining the extracellular ion balance, etc. In developing nervous tissue, the differentiation of astrocytes occurs later compared to neurons. It takes more time and more techniques to obtain mature and pure astrocytes in vitro. In this study, a protocol was developed to culture mature and pure astrocytes from human embryonic stem cells (hESCs). To obtain a high quantity and quality of differentiated astrocytes, first, we efficiently generated neural progenitor cells (NPCs) derived from hESCs through the process of embryoid body (EB) formation by adding SB431542 and LDN193189 and neurosphere step. In the astrocyte differentiation stage, the efficiency of astrocyte differentiation was increased using progenitor medium containing EGF and heparin and astrocyte defined medium containing ciliary neurotrophic factor (CNTF). The cell properties were checked with immunocytochemistry and western blot using antibodies for astrocyte-specific marker proteins. From the FACS analysis, we found that the percentage of astrocytes among the cells differentiated from NPCs was over 80%. To validate the functional properties of the astrocytes, we checked IL-6 release from the astrocytes and support of synaptic formation in a co-culture with neurons. Taken altogether, with our protocol, we obtained mature astrocytes within 4 weeks from NPCs and 6 weeks from hESCs.

Misuse of testosterone replacement therapy in men in infertile couples and its influence on infertility treatment.

OBJECTIVE: We investigated the clinical characteristics of men with testosterone replacement therapy (TRT)-induced hypogonadism and its effect on assisted reproductive technology (ART) in infertile couples. METHODS: This study examined the records of 20 consecutive male patients diagnosed with azoospermia or severe oligozoospermia (<5×106/mL) who visited a single infertility center from January 2008 to July 2018. All patients were treated at a primary clinic for erectile dysfunction or androgen deficiency symptoms combined with low serum testosterone. All men received a phosphodiesterase 5 inhibitor and TRT with testosterone undecanoate (Nebido®) or testosterone enanthate (Jenasteron®). Patients older than 50 years or with a chronic medical disease such as diabetes were excluded. RESULTS: The mean age of patients was 37 years and the mean duration of infertility was 16.3±11.6 months. At the initial presentation, eight patients had azoospermia, nine had cryptozoospermia, and three had severe oligozoospermia. Serum follicle-stimulating hormone levels were below 1.0 mIU/mL in most patients. Three ongoing ART programs with female factor infertility were cancelled due to male spermatogenic dysfunction; two of these men had normal semen parameters in the previous cycle. After withholding TRT, serum hormone levels and sperm concentrations returned to normal range after a median duration of 8 months. CONCLUSION: TRT with high-dose testosterone can cause spermatogenic dysfunction due to suppression of the hypothalamic-pituitary-testicular axis, with adverse effects on infertility treatment programs. TRT is therefore contraindicated for infertile couples attempting to conceive, and the patient's desire for fertility must be considered before initiation of TRT in a hypogonadal man.

Delayed recovery of a patient with obstructive azoospermia and a history of acute epididymitis.

Obstructive azoospermia caused by acute epididymitis is usually permanent, and microsurgical vasoepididymostomy is the only reconstructive treatment option. There have been no reports of delayed recovery of sperm count after over 1 year in a patient with obstructive azoospermia related to history of acute epididymitis. We present a young male patient who had azoospermia and a history of acute epididymitis who experienced delayed recovery, with complete restoration of sperm production and the ability to conceive naturally.

Cellulosimicrobium arenosum sp. nov., Isolated from Marine Sediment Sand.

A Gram-stain-positive, non-spore-forming, yellow pigmented, non-motile, aerobic, short rod-shaped bacterial strain, designated CAU 1455T, was isolated from marine sediment sand. Strain CAU 1455T grew optimally at 30 °C and at pH 7.5 in the presence of 1% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1455T was affiliated to the genus Cellulosimicrobium and was most closely related to Cellulosimicrobium terreum DS-61T (similarity 97.9%). The strain possessed MK-9 (H4) as the predominant menaquinone and anteiso-C15:0 as the major cellular fatty acids. Peptidoglycan type was A4a (L-Lys-D-Glu2). The DNA G+C content was 74.3 mol% and the level of DNA-DNA relatedness between CAU 1455T and C. terreum DS-61T was 27.8%. Based on phenotypic, chemotaxonomic, and genetic data, strain CAU 1455T represents a novel species of the genus Cellulosimicrobium, for which the name Cellulosimicrobium arenosum sp. nov. is proposed. The type strain is CAU 1455T (= KCTC 49039T = NBRC 113062T).

Arthrobacter paludis sp. nov., isolated from a marsh.

A novel Gram-stain-positive, strictly aerobic, non-endospore-forming bacterium, designated CAU 9143T, was isolated from a hydric soil sample collected from Seogmo Island in the Republic of Korea. Strain CAU 9143T grew optimally at 30 °C, at pH 7.0 and in the presence of 1 % (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences revealed that strain CAU 9143T belonged to the genus Arthrobacter and was closely related to Arthrobacter ginkgonis SYP-A7299T (97.1 % similarity). Strain CAU 9143T contained menaquinone MK-9 (H2) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two glycolipids and two unidentified phospholipids as the major polar lipids. The whole-cell sugars were glucose and galactose. The peptidoglycan type was A4a (l-Lys-D-Glu2) and the major cellular fatty acid was anteiso-C15 : 0. The DNA G+C content was 64.4 mol% and the level of DNA-DNA relatedness between CAU 9143T and the most closely related strain, A. ginkgonis SYP-A7299T, was 22.3 %. Based on phenotypic, chemotaxonomic and genetic data, strain CAU 9143T represents a novel species of the genus Arthrobacter, for which the name Arthrobacterpaludis sp. nov. is proposed. The type strain is CAU 9143T (=KCTC 13958T,=CECT 8917T).

Nonlabens halophilus sp. nov., isolated from reclaimed land.

A Gram-stain-negative, orange-pigmented, non-spore-forming, non-motile, aerobic, rod-shaped bacterial strain, designated CAU 1131T, was isolated from reclaimed land. Strain CAU 1131T grew optimally at 30 °C and at pH 6.5 in the presence of 4 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CAU 1131T was grouped into the genus Nonlabens, and was most closely related to Nonlabens. marinus S1-08T (95.9 % 16S rRNA gene sequence similarity). The strain possessed+ MK-6 as the predominant menaquinone and iso-C15 : 0, iso-C15:0 3-OH, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) as the major cellular fatty acids. The polar lipid profile was determined to comprise phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid, an unidentified glycolipid and two unidentified lipids. The DNA G+C content was 38.7 mol%. On the basis of data from phenotypic, chemotaxonomic and phylogenetic inference, strain CAU 1131T represents a novel species of the genus Nonlabens, for which the name Nonlabens halophilus sp. nov. is proposed. The type strain is CAU 1131T (=KCTC 52177T=NBRC 111996T).

Tropicimonasarenosa sp. nov., isolated from marine sand.

A Gram-staining-negative, aerobic, non-motile, rod-shaped bacterial strain, designated CAU 1062T, was isolated from marine sand in Jeju island, Republic of Korea and its taxonomic position was investigated using a polyphasic approach. CAU 1062T grew optimally at 30 °C and pH 8 in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that CAU 1062T formed a distinct lineage within the genus Tropicimonasand was the most closely related to Tropicimonas sediminicola M97T (similarity 96.11 %). The strain had ubiquinone-10 (Q-10) as the predominant respiratory quinone and C18 : 1ω7c as the major cellular fatty acid. The polar lipid pattern of CAU 1062T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an aminolipid, six phospholipids and five lipids. The DNA G+C content was 65.7 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, CAU 1062T represents a novel species of the genus Tropicimonas, for which the name Tropicimonas arenosa sp. nov. is proposed. The type strain is CAU 1062T (=KCTC 52178T=NBRC 111995T).

Profiling analysis of protein tyrosine phosphatases during neuronal differentiation.

During neuronal differentiation, it is generally accepted that many kinases and phosphatases fulfill different roles. In this study, phospho-tyrosine phosphatases were focused on and their expression profiling was evaluated during neuronal differentiation of mouse J1 embryonic stem cells. Among 83 phospho-tyrosine phosphatases, expressions of 21 PTPs were increased but mRNA expressions of 10 PTPs decreased depending on the differentiation. We checked the protein expression patterns for the cases where PTPs mRNA expressions changed. Some of them showed consistent results with the mRNA expressions. In particular, it was found that dual-specific phosphatase23 (DUSP23) affected neuronal differentiation. The knock-down of DUSP23 decreased neuronal differentiation in terms of neuronal outgrowth and the expression of neuronal marker proteins and mRNAs. Taken together, the obtained results show that many PTPs play specific roles during neuronal differentiation and manipulating their activities by activators or inhibitors could adjust neuronal differentiation.

DUSP4 regulates neuronal differentiation and calcium homeostasis by modulating ERK1/2 phosphorylation.

Protein tyrosine phosphatases have been recognized as critical components of multiple signaling regulators of fundamental cellular processes, including differentiation, cell death, and migration. In this study, we show that dual specificity phosphatase 4 (DUSP4) is crucial for neuronal differentiation and functions in the neurogenesis of embryonic stem cells (ESCs). The endogenous mRNA and protein expression levels of DUSP4 gradually increased during mouse development from ESCs to postnatal stages. Neurite outgrowth and the expression of neuron-specific markers were markedly reduced by genetic ablation of DUSP4 in differentiated neurons, and these effects were rescued by the reintroduction of DUSP4. In addition, DUSP4 knockdown dramatically enhanced extracellular signal-regulated kinase (ERK) activation during neuronal differentiation. Furthermore, the DUSP4-ERK pathway functioned to balance calcium signaling, not only by regulating Ca(2+)/calmodulin-dependent kinase I phosphorylation, but also by facilitating Cav1.2 expression and plasma membrane localization. These data are the first to suggest a molecular link between the MAPK-ERK cascade and calcium signaling, which provides insight into the mechanism by which DUSP4 modulates neuronal differentiation.