Human Leukemia Inhibitory Factor Research Articles

Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.

AB0605 Levels of the Circulating Leukaemia Inhibitory Factor (LIF) in Patients with Vasculitis under Immunosuppressive Therapy

BackgroundLeukaemia inhibitory factor (LIF) is a member of the IL-6 family of cytokines. It is one of the key mediators in various inflammatory processes such as acute-phase reaction, tissue damage,...

Human leukemia inhibitory factor produced by the ExpressTec method from rice (Oryza sativa L.) is active in human neural stem cells and mouse induced pluripotent stem cells

Stem cell-based therapy has the potential to treat an array of human diseases. However, to study the therapeutic potential and safety of these cells, a scalable cell culture medium is needed that is free of human or bovine-derived serum proteins. Thus, cost-effective recombinant serum proteins and cytokines are needed to produce such mediums. One such cytokine, leukemia inhibitory factor (LIF), has been shown to be a critical paracrine factor that maintains stem cell pluripotency in murine embryonic stem cells and human naïve stem cells while simultaneously inhibiting differentiation. We recently produced recombinant human LIF (rhLIF) in a rice-based protein expression system known as ExpressTec.12 We described expression of rice-derived rhLIF and demonstrated its biological equivalency to E. coli-derived rhLIF in traditional and embryonic mouse stem cell systems. Here we describe the expression yield of rice-derived rhLIF and the scale up production capacity. We provide further evidence of the efficacy of rice-derived rhLIF in additional stem cell systems including human neural stem cells and mouse induced pluripotent stem (iPS) cells. The expression level, biological activity, and potential for production at commercial scale of rice-derived rhLIF provides a proof-of-principal for ExpressTec-derived proteins to produce regulatory-friendly, high performance, and dependable stem cell media.

Correction: Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

Correction: Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells

Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

91 EFFECT OF LEUKEMIA INHIBITORY FACTOR (LIF) ON MATURATION OF PORCINE OOCYTES IN VITRO MATURATION AND DEVELOPMENT OF PARTHENOGENETIC EMBRYOS

Cytokines have been suggested to have an important role for successful implantation. Among the cytokines, leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interlukin-6 family that has been confirmed for its significance in implantation in human and animal studies. Furthermore, it has been reported that LIF enhanced in vitro maturation (IVM) in cattle and sheep, blastocyst formation and hatching rate of embryos and pregnancy rate in mouse, human, cattle and sheep. However, to our knowledge, there has been no report on the effects of human LIF (hLIF) on pig oocytes IVM and embryos development. Therefore, we designed and performed this study to examine the effect of hLIF treatment on pig IVM and oocyte developmental competence. We investigated the effect of hLIF treatment during pig oocyte in vitro maturation (IVM) and in vitro culture (IVC) on parthenogenetic embryos. Three groups of different hLIF concentrations were used: 0, 0.5, and 1.0 ng mL–1. In experiment 1, hLIF was contained on IVM media, in experiment 2, hLIF was contained on IVC media for 7 days, and experiment 3, hLIF was contained on IVM and IVC media. In experiment 1, hLIF in IVM media significantly increased cleavage rate in 0.5 ng mL–1 group [hLIF 0 ng mL–1; 46.56 ± 3.1 (%), 0.5 ng mL–1; 58.43 ± 3.6 (%), 1.0 ng mL–1; 52.05 ± 2.7 (%)] and total cell number of blastocysts in hLIF 1.0 ng mL–1 group (hLIF 0 ng mL–1; 35.00 ± 2.3, 0.5 ng mL–1; 40.71 ± 3.1, 1.0 ng mL–1; 51.06 ± 3.7) but no significant differences were found in oocyte maturation rate or blastocyst formation rate. In experiment 2, total cell number of blastocysts showed significant difference in hLIF 1.0 ng mL–1 in IVC media (hLIF 0 ng mL–1; 43.81 ± 1.6, hLIF 0.5 ng mL–1; 45.97 ± 2.0, hLIF 1.0 ng mL–1; 52.10 ± 2.9). Finally, total cell number of blastocysts increased in hLIF 0.5 ng mL–1 in IVM and IVC media [hLIF 0 ng mL–1; 46.50 ± 2.3 (%), 0.5 ng mL–1; 55.11 ± 2.9 (%)]. In conclusion, hLIF supplementation to IVM and IVC medium improved porcine embryo development in terms of increasing total cell number of blastocysts. This study was supported by Korean MKE (#10033839), the Research Institute for Veterinary Science.

Recombinant leukemia inhibitory factor suppresses human medullary thyroid carcinoma cell line xenografts in mice

Medullary thyroid carcinoma (MTC) is a neoplasm of the endocrine system, which originates from parafollicular C-cells of the thyroid gland. For MTC therapy, the Food and Drug Administration recently approved vandetanib and cabozantinib, multi-kinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. Previously, we reported that expression of activated Ras or Raf in the human MTC cell lines, TT and MZ-CRC-1, can induce growth arrest and RET downregulation via a leukemia inhibitory factor (LIF)-mediated autocrine/paracrine loop. In this study, we aimed to evaluate bacterially-produced recombinant human LIF for its efficacy to suppress human MTC xenografts in mice. Here, we report that, consistent with its effects in vitro, locally or systemically administered recombinant LIF effectively suppressed growth of TT and MZ-CRC-1 xenografts in mice. Further, as predicted from its effects in TT and MZ-CRC-1 cell cultures in vitro, recombinant LIF activated the JAK/STAT pathway and downregulated RET and E2F1 expression in tumors in mice. These results suggest that LIF is a potent cytostatic agent for MTC cells, which regulates unique mechanisms that are not targeted by currently available therapeutic agents.

High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli

Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2×YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.

Preferential expansion of umbilical cord blood‐derived CD34‐positive cells on human leukemia inhibitory factor transgenic feeder cells cultured on regenerated silk fibroin film

In vitro expansion of transplantable hematopoietic stem cells (HSCs) is a very promising approach for different clinical applications. We have recently developed a new culture system that facilitates in vitro expansion of transplantable cord blood HSCs. In our study, we constructed a recombinant adenovirus Ad-GFP/human leukemia inhibitory factor (hLIF) expressing hLIF. The hLIF gene was delivered into human embryo lung fibroblast cell line WI-38 via infection with Ad-GFP/hLIF. Then, the transgenic cells were cultured on regenerated silk fibroin (SF) films as feeder layer cells for expansion of cord blood CD34(+) cells. Our results showed that the hLIF transgenic WI-38 cells cultured on SF could express hematopoiesis-related cytokines at higher levels compared with control groups. The hLIF-expressing feeder layer cells cultured on SF in combination with cytokines more efficiently expanded CD34(+) cells and CD34(+) CD38(-) cells. The percentages of adhesion molecules on the expanded CD34(+) cells in transgenic feeder layer cells cultured on SF were higher than those of control groups. Interestingly, the migration rate assessed by transwell assay was also significantly higher than those of control groups, which suggests that transgenic feeder layer cells cultured on SF has powerful ability to maintain the homing capacity of expanded CD34(+) cells.

281 USE OF SMALL MOLECULES ENHANCES REPROGRAMMING SUCCESS IN BOVINE DERMAL FIBROBLASTS

Reprogramming of differentiated cells to induced pluripotency holds tremendous potential for livestock reproduction. Here we report use of small molecules to support the generation of stem cell-like colonies from bovine dermal fibroblasts that show characteristics of pluripotent cells. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc through a lentiviral drug-inducible polycistronic vector. Several small molecules were tested to support reprogramming. A combination of 3 small molecules (sodium butyrate, PD0325901, and SB431542; NaB-PD-SB) in iPS medium [Minimum Essential Medium Alpha, 20% fetal bovine serum, 1× insulin-transferrin-selenium (ITS), 2 mM Glutamax, 100 µM NEAA, 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the kinetics of the reprogramming process. Colonies appeared at 12 days of culture compared with 21 days in iPS medium alone (P < 0.01, n = 5). Colonies in NaB-PD-SB iPS medium had consistent morphology, with small cells in compact dome formations with well-defined colony borders. Cells were not passaged but were either harvested for mRNA extraction or differentiated into embryoid bodies. Gene expression of the reprogramming factors in stem cell-like colonies was confirmed by qRT-PCR, and further gene expression analysis revealed activation of Nanog and ALPL mRNA expression (P < 0.01, n = 2). Embryoid bodies were analysed for gene expression by quantitative RT-PCR. Silencing of the transgene was not observed in embryoid bodies despite withdrawal of the inducer doxycycline. Expression of the endoderm marker FOXA2, ectoderm markers NES and TUBB3, and mesoderm marker DES was strongly increased in embryoid bodies compared with non-reprogrammed bovine fibroblasts (P < 0.01, n = 2), confirming expression of marker genes for the 3 germ layers.

Generation of Porcine-Induced Pluripotent Stem Cells by Using OCT4 and KLF4 Porcine Factors

Induced pluripotent stem cells (iPSCs) can be artificially reprogrammed from somatic cells by overexpression of exogenous transcription factors. The pig has increasingly become an important large animal model for preclinical tests and studies of human diseases; thus, the generation of porcine iPSCs will facilitate research into the efficacy and safety of stem cell therapy. A current major problem facing the generation of porcine iPSCs is the failure to silence exogenous transgenes. We hypothesized that this problem can be resolved by reducing the number of transcriptional factors used for porcine iPSCs induction. Here, we report the successful generation of porcine iPSCs using the porcine factors Oct4 and Klf4 in combination with specific small molecules. In comparison with high oxygen conditions (20%), the efficiency of porcine iPSC generation was higher under low oxygen conditions (5%). Porcine iPSCs exhibited a normal karyotype and morphology, like mouse embryonic stem cells (ESCs), and could proliferate in the absence of basic fibroblast growth factor (bFGF) and in the presence of human leukemia inhibitory factor (hLIF) and mouse embryonic fibroblast feeder cells. These iPSCs also expressed ESC-like markers (Oct4, Nanog, Klf4, c-Myc, Bmp4, bFgf). Importantly, the porcine iPSCs showed pluripotency, as evidenced by differentiation into three germ layers in vitro following embryoid body formation, as well as by efficiently forming teratomas containing three germ layers in immunodeficient mice. Thus, pluripotent porcine iPSCs can be generated from somatic stem cells by using only two porcine transcription factors in combination with small molecules. These attempts represent the first step toward generating truly pluripotent porcine iPSCs with fewer exogenous genes and less integration.

Assessment of leukemia inhibitory factor and glycoprotein 130 expression in endometrium and uterine flushing: a possible diagnostic tool for impaired fertility

BackgroundUterine receptivity and implantation are complex processes requiring coordinated expression of molecules by zygote and uterus. Our objective was to evaluate the role of the endometrial expression of leukemia inhibitory factor (LIF) and its glycoprotein 130 (gp130) receptor molecules and their secretion in uterine flushing during the window of implantation in cases of primary unexplained infertilityCase presentationThe study was conducted on 25 infertile women with unexplained infertility for at least two years and 10 normal fertile women as a control group . Endometrial tissue and uterine flushing were obtained. Each tissue specimen was divided into two pieces; one piece was used for histological dating of the endometrium and for immunostaining of progesterone receptors, and the second was used for RNA extraction and PCR assay of LIF and gp130 mRNA expression. Serum estrogen and progesterone were measured for all subjects. LIF mRNA was expressed in the endometrium of all normal fertile women but significantly decreased in infertile women. LIF was not detectable in 88% of infertile women while it was fairly detectable in 12% of them. Gp130 mRNA was hardly detectable in both fertile and infertile women with no difference between them. Infertile women secreted significantly less LIF and gp130 molecules in the uterine flushing compared with normal fertile women.ConclusionsExpression of LIF mRNA in endometrium could be used as a molecular marker of unexplained infertility. Assessment of secreted LIF and gp130 molecules in uterine flushing could be another useful and safe method for predicting successful implantation as well as for diagnosing and eventually treating women with impaired fertility using recombinant human LIF.

Codon Optimization, Cloning and Expression of the Human Leukemia Inhibitory Factor (hLIF) in E. coli

Background: Leukemia inhibitor factor (LIF) is a very important pleiotropic cytokine which belongs to interleukin-6 (IL-6) family. LIF exerts multiple effects on different types of cells and tissues with numerous regulatory effects in vivo and in vitro. It is a lymphoid factor, which performs a number of activities including cholinergic neuron differentia‌tion, control of stem cell pluripotency, bone and fat metabolism, and is important for embryo implantation and promoting megakaryocytes production in vivo. Human LIF is a potential therapeutic candidate for some diseases such as multiplesclerosis (MS). Objectives: Because of aforementioned applications of the LIF protein in biological sys‌tems, the LIF gene has been cloned in various species. In this study a useful novel method was used to clone an optimized LIF sequence. Materials and Methods: In this study, the optimized cDNA form of human leukemia inhibitory factorwas cloned into the pET-28a (+) expression vector under control of T7lacpromoter using BamHI and XbaI restriction enzymes. The recombinant vector was transformed into Escherichia coli strain BL21 (DE3). The cloned hLIF cDNA was expressed as a fusion protein with His-tag. Cloning of hLIF cDNA was confirmed by digestion and DNA sequencing. Appropriate expression of recombinant hLIF was examined by SDS-PAGE af‌ter induction with isopropylthio-β-galactoside (IPTG). Results: The results confirmed the expression of the 19.7 kDa rhLIF protein in the bacte‌rial expression system. Conclusions: We could show that codon optimization might increase the production of recombinant hLIF in the E. coli. This result is useful in similar cases, in which the level of expressed gene is critical.

Comparison of the activity and pluripotency maintaining potential of human leukemia inhibitory factor (LIF) produced in E.coliand CHO cells

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In murine stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. To explore if the glycosylation and/or other post-translational modifications are affecting this activity, we produced and isolated LIF from either eucaryotic cells (Chinese hamster ovary (CHO) cells) or procaryotes (E.coli) and compared their biological activities in this study.

Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.

High-level Soluble Expression, Purification, and Functional Characterization of the Recombinant Human Leukemia Inhibitory Factor: A Potential General Strategy for the Recombinant Expression of Cytokines Consisting of Four &amp;#945;-Helices in a Bundle

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95? purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four α-helices in a bundle for basic research, as well as clinical applications.

A preferable approach to clone hLIF cDNA from the genomic DNA

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.

Incomplete and inconsistent reporting of maternal and fetal outcomes in infertility treatment trials

Pregnancy outcomes and adverse outcomes in infertility trials are reported to varying extents; for example, 35% of clinical trials reported no information on pregnancy loss, only 43% reported adverse events during the preconception treatment period, and only 7% reported any serious adverse events. Incomplete reporting limits the value of these studies in counseling patients on the risk-benefit ratio of treatment to themselves and their infants.