Expression Of Molecular Markers Research Articles

P-412 3D live-imaging reconstruction of the human embryo implantation ex vivo

Abstract Study question How do human embryos implant in physiological conditions and develop beyond blastocyst stage? Summary answer Human embryos apply forces during invasion of the matrix, the mural trophectoderm undergoes a quick compaction leading to a fast radial expansion of polar side What is known already Human embryos can be cultured beyond blastocyst stage using supplements of animal origin, such as fetal bovine serum. However, two main issues limit our understanding of the implantation of the human embryo. First, human embryos do not express fluorescent proteins, severely limiting the access to advanced live-imaging tools. Second, the animal serums may not represent the physiological peri-implantation conditions of the human embryo, thus limiting the reproducibility of the in vitro experiments. Here we combine label-free multiphoton imaging with media formulations including clinical-grade protein supplements from human plasma unlocking the observation of the human embryo in 3D and physiological conditions Study design, size, duration We have cultured 150 human embryos from D3 until blastocyst stage and also more than 600 mouse embryos from zygote until blastocyst stage and then transferred them into a 3D implantation platform. All embryos were supplemented with clinical-grade human-derived serum, which is rich in globulins and growth factors, and commercial basal media. The implantation was monitored for 3-to-5 days up until day 11. Traction-force microscopy and molecular imaging was employed to quantify implantation markers. Participants/materials, setting, methods Implantation was measured using our novel version of traction-force microscopy, which reveals the force applied by entire embryos during live-imaging experiments. Multiphoton illumination of autofluorescent molecules allows to reconstruct images of live human embryos on 3D at unprecedented resolution. Key molecular markers for the formation of the epiblast rosette (OCT4), extra-embryonic visceral endoderm (GATA4), extra-embryonic ectoderm and trophoblast (CDX2) and primary yolk sac and pre-amniotic cavity were reconstructed in 3D using high resolution confocal imaging. Main results and the role of chance The implantation of human embryos was visualized in 3D movies at high resolution, during time-resolved experiments and compared to implantation in mouse embryos. Human embryos undergo a remarkable compaction upon attachment of the polar trophectoderm and thereafter they implant by engaging the matrix through many different points (>8). The post-implantation compaction leads to a strong invasion of the polar trophectoderm, digging large holes in the matrix and expanding radially. Some trophoblast cells escape the embryo colonizing the matrix. Human embryos expressed the correct pattern of molecular markers by day 11 of culture. Actin staining and OCT4 expression revealed the formation of a central rosette defining the pre-amniotic cavity. GATA6 expression defined the visceral endoderm and the formation of a primordial yolk sac. Mouse embryos conversely, compact upon implantation of the mural trophectoderm and are less invasive, typically anchoring to the matrix through 2-to-3 points. Post-implantation culture of mouse embryos starting at zygote was possible until the formation and large expansion of the amniotic cavity, which constituted 2/3 of the whole embryo size, corresponding with the initial steps of gastrulation, longer than any previous protocol Transfer of mouse blastocysts back to receptive mothers resulted in birth of viable litter. Limitations, reasons for caution This study has been performed with human embryos ex vivo using bioengineering technology to implant embryos outside the mother uterus. The bio-compatibility of our serum supplements was validated by live birth of transferred embryos in animal models, but would require further evaluation in clinical trials. Wider implications of the findings Our work stresses the safety and efficiency of supplementing embryo culture and particularly embryo transfer, with human plasma-derived serums of clinical-grade. The correct expression of main molecular markers indicates that this type of supplementation promotes implantation physiologically, which opens the possibility to employ these serums in the IVF laboratory. Trial registration number Not applicable

Expression of PD-L1 and PD-1 in Pathological Tissue of Patients Newly Diagnosed with Diffuse Large B-Cell Lymphoma

To investigate the expression of PD-L1 and PD-1 in pathological tissue of patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL). Data of DLBCL patients who visited the Department of Hematology, Peking University Third Hospital from May 2014 to March 2017 were collected, and a total of 21 patients with pathological tissue sections which were still available at the initial treatment were selected. The patients were divided into complete remission (CR) group and refractory relapse (RR) group according to clinical outcome. The expression and proportion of PD-1 and PD-L1 in pathological tissue sections were detected by multiplex fluorescence immunohistochemical staining, and the differences in the expression of different molecular markers in different clinical characteristics and different prognosis were compared using non-parametric test. The ratio of PD-L1+ cells to PD-1+ cells (PD-L1+ : PD-1+) was 5.14±3.825 in increased lactate dehydrogenase (LDH) group, which was significantly higher than 0.76±0.563 in non-increased LDH group (P=0.001). The ratio of PD-L1+ : PD-1+ in increased Treg cells group was 1.41±1.454, which was lower than 6.42±4.426 in decreased Treg cells group (P=0.023). The increased expression ratio of PD-L1 to PD-1 in pathological tissue sections of newly diagnosed DLBCL patients is associated with poor prognostic clinical characteristics.

GRK2, a novel regulator of skeletal development

STUDY OBJECTIVEDuring skeletal development, the cartilaginous growth plate (GP) allows for bone elongation, composed of chondrocytes organized into three histological zones: resting (RZ), proliferative (PZ), and hypertrophic (HZ). Resting chondrocytes proliferate, become prehypertrophic, then hypertrophic, to create room for bone deposition. The G‐protein coupled receptor (GPCR) Parathyroid Hormone Receptor‐1 (PTH1R) expressed on the chondrocytes of the prehypertrophic zone, decelerates the transition to hypertrophy. PTH1R signaling is regulated by GPCR kinase 2 (GRK2); GRK2 phosphorylates PTH1R leading to its internalization and signal termination. Recent studies showed that loss of function mutations in GRK2 result in skeletal deformities, and we demonstrated that GRK2 promotes chondrocyte hypertrophy (CH) in adult arthritic cartilage. However, the role of GRK2 in molecular signaling in GP chondrocytes has yet to be explored.HYPOTHESISWe hypothesized that GRK2 desensitizes PTH1R to promote CH in the GP.METHODSWe utilized the Cre‐Lox system to generate transgenic mice with inducible ‐conditional‐knockout of GRK2 (GRK2‐icKO) in chondrocytes. KO was initiated at postnatal day 3 (PN3) by Tamoxifen ip injections and mice were sacrificed at PN10 to examine the effects on early and late stages of skeletal development. We performed H&E staining to analyze histological changes in GP structure. To determine changes in molecular signaling, we used ISH and IF staining to quantify expression of GRK2, PTH1R, BrdU (proliferation marker), and ColX (CH marker). At the chondro‐osseous junction, we quantified apoptosis (TUNEL staining), bone deposition (Col1 and Runx2 IF), and matrix mineralization (Alizarin Red (AR) staining). We used in vitro experiments with primary chondrocyte culture to confirm in vivoresults. GRK2 knockdown (KD) was achieved with siRNA before inducing CH. Gene and protein expression of the same molecular markers were analyzed with qPCR and WB, and matrix mineralization quantified by AR.RESULTSGKR2‐icKO mice had: (1) Shorter bones (2) Retained PTH1R expression in the HZ, with reduced gene expression (Fig. 1A), as confirmed in vitro (Fig. 1D) (3) Longer PZ and more proliferative chondrocytes confirmed by BrdU, (4) shorter HZ and less hypertrophic chondrocytes, confirmed by ColX IF (Fig. 1B). (5) At the chondro‐osseous junction, GRK2‐icKO mice had reduced chondrocyte apoptosis, bone deposition, and matrix mineralization (Fig. 1C). In vitroexperiments demonstrated increased GRK2 expression with CH, where GRK2 KD reduced CH and matrix mineralization (Fig. 1D).CONCLUSIONSWe show a novel role of GRK2 in skeletal development as a major regulator of PTH1R in GP chondrocytes. GRK2‐icKO increases chondrocyte proliferation and reduces hypertrophy and apoptosis, leading to reduced bone deposition, matrix mineralization, and shorter bones. Altogether, this presents GRK2 as a novel therapeutic target for skeletal abnormalities.

Transcriptome profile of white, brown and beige adipose tissue

Adipose tissue dysregulation lies at the core metabolic syndrome. White adipose tissue (WAT) has the capacity to store energy, whereas brown adipose tissue (BAT) can dissipate energy through thermogenesis. Newly discovered beige adipocytes are brown like adipocytes recruited in WAT in response to b3‐adrenergic stimulation (CL316,243) or cold temperatures. Despite the important metabolic role, information regarding differential expression of molecular markers between different adipose tissue, especially beige and other depots is scarce. The objective of this study was to identify the shared and unique transcript signatures that characterize WAT, BAT and beige adipose tissue. We collected inguinal WAT (iWAT), gonadal WAT (gWAT) and BAT from male C57BL6/J mice fed a control diet (17% fat) for 13 weeks. Beige WAT (iWATCL) was isolated from mice treated with b3‐adrenergic agonist CL316, 243 for 7 days (1 mg/kg BW, ip). RNA sequencing was used to generate transcriptional profiles. Using gWAT as control, differentially expressed genes (DEGs) were determined with the DEseq2 and considered when the false discovery rate corrected P‐value was ≤ 0.05 and the fold change ≥ 4. Principal components analysis revealed that the transcriptional profiles differed across BAT, gWAT and iWAT and iWATCL. Across adipose tissues, 208 DEGs were shared. Between BAT and iWATCL, 223 DEGs were shared and were associated with muscle related pathways. Unique DEGs were 1130, 549, and 40 for BAT, iWAT, and iWATCL, respectively. Top upregulated unique genes included Lncbate10, Zic1, and Gm32468 in BAT; Dsc3, Cd22 and H2‐M2 in iWAT; and Cacng5, Mup11 and Mup9 in iWATCL. Among other processes, genes upregulated in BAT were associated with cellular respiration and fatty acid biosynthesis, in iWAT with immune responses and in iWATCL with sodium ion homeostasis and cellular response to lipids. Overall results show transcriptional heterogeneity across adipose tissues depots, especially the unique set of transcripts in CL316, 243 induced beige adipocytes that can serve specific markers.

Botulinum toxin injection in masseter muscle evokes musculoskeletal impairment of the masticatory system

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non‐desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA‐injection in masseter muscle of adult mice.Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks‐old) by single‐injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral‐ and medial‐pterygoid) and mandibles were dissected after 2‐7‐14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC‐Universidad de Chile (# 20381‐ODO‐UCH). T‐test or Wilcoxon‐test were used for statistical comparisons between BoNTA‐ and Control‐side (n=3‐10, p<0,05).The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA‐side, as early as 2‐7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time‐dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid’s volumes significantly reduced in the BoNTA‐injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA‐injected side, starting as early as 7d and worsening 14d after the intervention.In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early‐bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho‐functional monitoring.

Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Early Expression of MMP-9 Predicts Recovery of Tibialis Anterior after Sciatic Nerve Crush Injury.

Background:The purpose of this study was to assess the expression of molecular markers and epineural blood flow after differing degrees of nerve injury to identify potential tools to predict nerve recovery in a rat sciatic nerve model.Methods:A total of 72 rats were divided into nine groups. Each group was subjected to one of three crush injuries, created by applying one of three vascular clamps for 30 seconds. Vascularity was assessed with laser Doppler flowmetry before and after crush, and at nonsurvival surgery. Nonsurvival surgeries were performed 6 hours, 2 weeks, or 6 weeks later with nerve conduction studies and muscle strength testing. Expression of matrix metalloproteinase 9 (MMP-9) and matrix metalloproteinase 2 (MMP-2) in each nerve was quantified using with enzyme linked immunosorbent analysis.Results:Persistent hyperemia was noted in the zone of injury compared with baseline at 2 weeks and 6 weeks in the groups that displayed incomplete recovery. Expression of MMP-9 at 6 hours increased with increasing severity of crush and was inversely related to tibialis anterior muscle force recovery. The ratio of MMP-9:MMP-2 expression correlated well with recovery of compound nerve action potential amplitude at 6 weeks.Conclusions:Resolution of nerve hyperemia may correlate with nerve recovery from trauma, but early measures of nerve blood flow after injury are not prognostic of recovery. Ratio of MMP-9:MMP-2 expression 6 hours after injury correlates with recovery of compound nerve action potential at 6 weeks, while MMP-9 expression alone predicts tibialis anterior recovery. These findings together suggest that increased MMP-9 expression is a potentially useful marker of more severe nerve injury.

Repair-related molecular changes during recovery phase of ischemic stroke in female rats

BackgroundSome degree of spontaneous recovery is usually observed after stroke. Experimental studies have provided information about molecular mechanisms underlying this recovery. However, the majority of pre-clinical stroke studies are performed in male rodents, and females are not well studied. This is a clear discrepancy when considering the clinical situation. Thus, it is important to include females in the evaluation of recovery mechanisms for future therapeutic strategies. This study aimed to evaluate spontaneous recovery and molecular mechanisms involved in the recovery phase two weeks after stroke in female rats.MethodsTransient middle cerebral artery occlusion was induced in female Wistar rats using a filament model. Neurological functions were assessed up to day 14 after stroke. Protein expression of interleukin 10 (IL-10), transforming growth factor (TGF)-β, neuronal specific nuclei protein (NeuN), nestin, tyrosine-protein kinase receptor Tie-2, extracellular signal-regulated kinase (ERK) 1/2, and Akt were evaluated in the peri-infarct and ischemic core compared to contralateral side of the brain at day 14 by western blot. Expression of TGF-β in middle cerebral arteries was evaluated by immunohistochemistry.ResultsSpontaneous recovery after stroke was observed from day 2 to day 14 and was accompanied by a significantly higher expression of nestin, p-Akt, p-ERK1/2 and TGF-β in ischemic regions compared to contralateral side at day 14. In addition, a significantly higher expression of TGF-β was observed in occluded versus non-occluded middle cerebral arteries. The expression of Tie-2 and IL-10 did not differ between the ischemic and contralateral sides.ConclusionSpontaneous recovery after ischemic stroke in female rats was coincided by a difference observed in the expression of molecular markers. The alteration of these markers might be of importance to address future therapeutic strategies.

Head and neck cancer organoids as a promising tool for personalized cancer therapy: A literature review.

Background and AimChemotherapy and targeted therapy are used in treating head and neck cancers (HNCs) either alone or in combination with surgery, especially in advanced tumors but these treatments have resulted in variable outcomes in different patients. This, along with the introduction of new therapies to improve the survival of patients makes it necessary to search for models that can predict the response to treatment among different patients. Organoids, as three‐dimensional culture models, have been studied more widely in non‐HNCs and to a lesser extent in HNCs as tools to predict treatment outcomes. We aimed to conduct a review to validate the use of organoids as a preclinical tool for the treatment of HNCs patients.MethodsA comprehensive literature search was separately performed by both authors in PubMed and google scholar databases, using the following keywords: “organoid,” “head and neck cancer,” “personalized medicine,” “chemotherapy,” and “targeted therapy.” The articles published up to September 2021 were included in this review and selected according to a quality appraisal method.ResultsExamination of HNC‐derived organoids made in various studies showed that these organoids had the ability to recapitulate original tumor features, including histopathological properties, functional characteristics, and expression of molecular markers in almost all of the studies. Differential sensitivity to chemotherapy drugs similar to in vivo was observed in sensitivity testing. Epidermal growth factor receptor (EGFR) expression levels were different between organoids from different patients and EGFR expression level was found to correlate with the response to anti‐EGFR targeted therapy. A similar result was reported for organoids derived from salivary adenoid cystic carcinoma.ConclusionSince HNC‐derived organoids seem to recapitulate characteristics of original tumors and to show differential responses to different chemotherapy and targeted therapy agents, these organoids might have the potential to be used as preclinical prediction tools for the treatment of HNC patients.

Immunological regulation of the active fraction from Polygonatum sibiricum F. Delaroche based on improvement of intestinal microflora and activation of RAW264.7 cells

Ethnopharmacological relevancePolygonatum sibiricum, known as “Huangjing” in Chinese traditional medicine, of which functions include invigorating Qi and nourishing Yin, tonifying spleen and kidney, which are considered to replenish energy, and strengthen immunity. However, both the active components and mechanism of the immune-enhancing effect of Polygonatum sibiricum have not been clarified. Aim of the studyTo evaluate the immunoregulation effects of PSE30 (Polygonatum sibiricum ethanol 30) and PSE75 (Polygonatum sibiricum ethanol 75). The gut microbial and activation of RAW264.7 cells were also evaluated for exploring the mechanism of PSE75. Materials and methodsFemale ICR mice were randomly divided into different groups, which were pretreated with 0.9% saline, Yupingfeng granules, different dosage of PSE30 or PSE75. And the immunosuppressed mice model was constructed using cyclophosphamide. And the total duration of the experiment was 15 d. After that, the serum Immunoglobulins G (IgG) and Immunoglobulins M (IgM) antibody, regular blood testing, assessment of natural killer cell activity, and histological observation of spleen in immunosuppressed mice were measured to evaluate the immunoregulation effects of PSE30 and PSE75. Besides, effects of PSE75 on gut microbial were evaluated using 16s rRNA sequence. And the mRNA expression and cytokine secretion of RAW264.7 cell were evaluated to analyze the immunoregulation mechanism of PSE75. ResultsThe content of serum IgG, IgM was significantly elevated by PSE75 (P＜0.05, P＜0.001). The NK cells killing activity in splenocytes against K562 cells induced by PSE30, and PSE75 was pronounced higher than that of the model group (P < 0.05). Both mRNA expression of Th1 molecular markers including interleukin (IL)-2, interferon (IFN)-γ, and signal transducers and activators of transcription (STAT) 4, and Th2 molecular markers including IL-4 in splenocytes were markedly enhanced by PSE30, and PSE75 (P < 0.05, P < 0.01, or P < 0.001). Besides, the result of 16s rRNA sequence indicated that PSE75 could recover the gut microbial community disturbed by cyclophosphamide. PSE75 could markedly promote the secretion of IL-6, IL-10, and IL-12 p40 from RAW264.7 cell (P＜0.01, or P＜0.001). ConclusionsPSE75 was proved to be a more promising immunomodulation agent, of which may enhance the immunity of immunosuppressed mice by improving gut microbial and activating macrophages. And PSE75 could be developed as a good immune booster in the future.

Culture of patient-derived multicellular clusters in suspended hydrogel capsules for pre-clinical personalized drug screening.

A personalized medication regimen provides precise treatment for an individual and can be guided by pre-clinical drug screening. The economical and high-efficiency simulation of the liver tumor microenvironment (TME) in a drug-screening model has high value yet challenging to accomplish. Herein, we propose a simulation of the liver TME with suspended alginate-gelatin hydrogel capsules encapsulating patient-derived liver tumor multicellular clusters, and the culture of patient-derived tumor organoids(PDTOs) for personalized pre-clinical drug screening. The hydrogel capsule offers a 3D matrix environment with mechanical and biological properties similar to those of the liver in vivo. As a result, 18 of the 28 patient-derived multicellular clusters were successfully cultured as PDTOs. These PDTOs, along with hepatocyte growth factor (HGF) of non-cellular components, preserve stromal cells, including cancer-associated fibroblasts (CAFs) and vascular endothelial cells (VECs). They also maintain stable expression of molecular markers and tumor heterogeneity similar to those of the original liver tumors. Drugs, including cabazitaxel, oxaliplatin, and sorafenib, were tested in PDTOs. The sensitivity of PDTOs to these drugs differs between individuals. The sensitivity of one PDTO to oxaliplatin was validated using magnetic resonance imaging (MRI) and biochemical tests after oxaliplatin clinical treatment of the corresponding patient. Therefore, this approach is promising for economical, accurate, and high-throughput drug screening for personalized treatment.

Characteristics of Whale Müller Glia in Primary and Immortalized Cultures.

Müller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Müller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Müller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Müller glia to neurons. Here, the Müller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Müller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Müller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Müller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Müller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Müller cells, with an increase in dedifferentiation markers like α-SMA and β-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal’s age and size on these cells, and on their senescence.

Hexadecanoic acid-enriched extract of Halymenia durvillei induces apoptotic and autophagic death of human triple-negative breast cancer cells by upregulating ER stress

Objective: To investigate the effect of the hexane solvent fraction of Halymenia durvillei (HDHE) on triple-negative breast cancer. Methods: The phytochemical profile of HDHE was investigated by GC-MS. The cytotoxicity of HDHE against MDA-MB-231 cells was determined. The apoptotic and autophagic effects of HDHE were analyzed. The expression of molecular markers controlling apoptosis, autophagy, DNA damage, and endoplasmic reticulum (ER) stress was determined. Results: HDHE contains a mixture of fatty acids, mainly hexadecanoic acid. HDHE at a cytotoxic concentration induced apoptotic death of MDA-MB-231 cells through mitochondrial membrane dysfunction, and induction of apoptosis markers, and increased the expression of proteins related to DNA damage response. HDHE also induced the expression of LC-3, a marker of autophagic cell death at a cytotoxic concentration. Moreover, HDHE modulated the expression of ER stress genes. Conclusions: The hexadecanoic acid-enriched extract of Halymenia durvillei promotes apoptosis and autophagy of human triple-negative breast cancer cells. This extract may be further explored as an anticancer agent for triple-negative breast cancer.

Paclitaxel Induces Epidermal Molecular Changes and Produces Subclinical Alterations in the Skin of Gynecological Cancer Patients.

Simple SummarySkin toxicity is one of paclitaxel’s adverse effects. However, its real impact on the skin could be underestimated as these alterations can also appear asymptomatic. We have observed that paclitaxel modifies gene and protein expression of skin markers in a 3D epidermis model, and impairs physical, physiological, and biomechanical properties of the skin in gynecologic cancer patients. These subclinical alterations might be avoided by using prophylactic measures during treatment to prevent possible future adverse reactions.Background: Paclitaxel is a microtubule-stabilizing chemotherapeutic agent. Despite its widespread use, it damages healthy tissues such as skin. The goal of this study was to prove that the real impact of paclitaxel-induced skin toxicity could be underestimated because the adverse events might appear asymptomatic. Methods: Gynecological cancer patients were recruited. Skin parameters measurements were taken after three and six paclitaxel cycles. Measurements were conducted using specific probes which measure hydration, transepidermal water loss (TEWL), sebum, elasticity and firmness, erythema, roughness, smoothness, skin thickness, and desquamation levels. Further, a 3D epidermis model was incubated with paclitaxel to analyze gene and protein expression of aquaporin 3, collagen type 1, elastin, and fibronectin. Results: Paclitaxel induced alterations in the skin parameters with no visible clinical manifestations. Gynecological cancer patients under paclitaxel treatment had a decrease in hydration, TEWL, sebum, elasticity, and thickness of the skin, while erythema, roughness, and desquamation were increased. The molecular markers, related to hydration and the support of the skin layers, and analyzed in the 3D epidermis model, were decreased. Conclusions: Results suggest that paclitaxel modifies gene and protein expression of skin-related molecular markers, and impairs different physical, physiological, and biomechanical properties of the skin of cancer patients at a subclinical level.

SOX8 promotes cetuximab resistance via HGF/MET bypass pathway activation in colorectal cancer.

Cetuximab is an essential drug for the treatment of wild-type K-RAS colorectal cancer (CRC). It improves the overall survival of patients. However, acquired resistance prevents its clinical efficacy. Tumor heterogeneity may be a nonnegligible reason for cetuximab resistance. We attempted to explore the corresponding molecular mechanism. Cetuximab-resistant CRC cell RKO and cetuximab-sensitive CRC cell Caco-2 were applied in this study. Cells were centrifuged to determine the concentration in the culture supernatant (CS). MTT, EdU, and colony formation assays were utilized to evaluate cell survival and proliferation. Chromatin immunoprecipitation (ChIP) and promoter-luciferase reporter assays were employed to confirm the direct binding of transcription factors. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) assays were used to detect the expression of molecular markers in the pathway. Hepatocyte growth factor (HGF) was up-regulated in RKO cell culture supernatant and induced cetuximab resistance in Caco-2 cells. SRY-Box Transcription Factor 8 (SOX8) bound to the promoter sequence of HGF. HGF activated the HGF/MET bypass pathway and induced cetuximab resistance in Caco-2 cells. The SOX8/HGF/MET axis played a crucial role in the communication between cetuximab-resistant cells and cetuximab-sensitive cells, inducing treatment resistance.

Thymopentin-Mediated Inhibition of Cancer Stem Cell Stemness Enhances the Cytotoxic Effect of Oxaliplatin on Colon Cancer Cells.

Thymopentin (TP5) is an immunomodulatory pentapeptide that has been widely used in malignancy patients with immunodeficiency due to radiotherapy and chemotherapy. Here, we propose that TP5 directly inhibits the stemness of colon cancer cells HCT116 and therefore enhances the cytotoxicity of oxaliplatin (OXA) in HCT116 cells. In the absence of serum, TP5 was able to induce cancer stemness reduction in cultured HCT116 cells and significantly reduced stemness-related signals, such as the expression of surface molecular markers (CD133, CD44 and CD24) and stemness-related genes (ALDH1, SOX2, Oct-4 and Nanog), and resulted in altered Wnt/β-catenin signaling. Acetylcholine receptors (AchRs) are implicated in this process. OXA is a common chemotherapeutic agent with therapeutic effects in various cancers. Although TP5 had no direct effect on the proliferation of HCT116, this pentapeptide significantly increased the sensitivity of HCT116 to OXA, where the effect of TP5 on the stemness of colon cancer cells through stimulation of AchRs may contribute to this process. Our results provide a promising strategy for increasing the sensitivity of colon cancer cells to chemotherapeutic agents by incorporating immunomodulatory peptides.

Immune-Related lncRNA Pairs Clinical Prognosis Model Construction for Hepatocellular Carcinoma.

BackgroundLong non-coding RNA (lncRNA) plays an essential regulatory role in the occurrence and development of hepatocellular carcinoma (HCC). This paper aims to establish an immune-related lncRNA (irlncRNA) pairs model independent of expression level for risk assessment and prognosis prediction of HCC.MethodsTranscriptome data and corresponding clinical data were downloaded from TCGA. HCC patients were randomly divided into training group and test group. Univariate Cox regression analysis, LASSO regression analysis, and stepwise multiple Cox regression analysis were used to establish a prognostic model. The prediction ability of the model was verified by ROC curves. Next, the patients were divided into low-risk and high-risk groups. We compared the differences between the two groups in survival rate, clinicopathological characteristics, tumor immune cell infiltration status, chemotherapeutic drug sensitivity and immunosuppressive molecules.ResultsA prognosis prediction model was established based on 7 irlncRNA pairs, namely irlncRNA pairs (IRLP). ROC curves of the training group and test group showed that the IRLP model had high sensitivity and specificity for survival prediction. Kaplan–Meier analysis showed that the survival rate of the high-risk group was significantly lower than that of the low-risk group. Immune cell infiltration analysis showed that the high-risk group was significantly correlated with various immune cell infiltration. Finally, there were statistically significant differences in chemosensitivity and molecular marker expression between the two groups.ConclusionThe prognosis prediction model established by irlncRNA pairs has a certain guiding significance for the prognosis prediction of HCC. It may provide valuable clinical applications in antitumor immunotherapy.

Oral Contraceptive Use and Breast Cancer Risk According to Molecular Subtypes Status: A Systematic Review and Meta-Analysis of Case-Control Studies.

Simple SummaryBreast cancer (BrCa) is a heterogeneous disease with multiple intrinsic tumor subtypes evidenced by the joint expression of molecular tumor markers. Data from epidemiologic studies provide evidence supporting differential effects of oral contraceptives on risk of developing the distinct subtypes of breast cancer; while some studies suggest increased risk, others show its lack. Toward this objective, we conducted meta-analysis of case-control trials devoted to this topic. The results of our study suggest that the oral contraceptive use has different effects on the risk of developing the various molecular breast cancer subtypes.We conducted a systematic review and meta-analysis to investigate the effect of oral contraceptives (OCs) on risk of breast cancer (BrCa) by status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). We searched the MEDLINE (PubMed), Embase and the Cochrane Library database and bibliographies of pertinent articles published up to 2020. Therein, we identified nineteen eligible case-control studies which provided data by breast cancer subtypes: ER-positive (ER+), ER-negative (ER−), HER2-positive (HER2+) and Triplet-negative (TN). Summary risk estimates (pooled OR [pOR]) and 95% confidence intervals (CIs) were calculated using fixed/random effects models. The summary meta-analysis showed that over-use of OCs led to significant increased risk of TNBrCa (OR = 1.37, 95% CI; 1.13 to 1.67, p = 0.002), as well as of ER−BrCa (OR = 1.20, 95% CI: 1.03 to 1.40, p = 0.019). There was also a significant reduction in the risk of ER+BrCa (OR = O.92, 95% CI: 0.86 to 0.99, p = 0.026,) and a slight reduction in the risk of HER2+BrCa (OR = 0.95, 95% CI; 0.79 to 1.14, p = 0.561) after taking OCs. Meta-analysis indicated that OC use has different impacts on risk of breast cancer subtypes defined by receptor status. The identified differences between individual subtypes of breast cancer may reflect different mechanisms of carcinogenesis.

Early development of the pulmonary vascular system: An anatomical and histochemical reinvestigation of the pulmonary venous return development in mice

Pulmonary venous return development establishes the fetal circulation and is critical for the formation of pulmonary circulation independent of systemic circulation at birth. Anomalous returns lead to inappropriate drainage of blood flow, sometimes resulting in neonatal cyanosis and cardiac failure. While many classical studies have discussed the anatomical features of the pulmonary venous system development, the cellular dynamics of the endothelia based on the molecular marker expression remain unknown. In the present study, we examined the expression of several endothelial markers during early pulmonary vascular system development of murine embryos. We show that Endomucin and CD31 are expressed early in endothelial cells of the splanchnic plexus, which is the precursor of the pulmonary vascular system. Three-dimensional analyses of the expression patterns revealed the spatiotemporal modification of the venous returns to systemic venous systems or sinoatrial canal during the formation of the pulmonary plexus. We herein report the results of spatiotemporal analyses of the early pulmonary venous system development with histochemistry as well as a delineation of the anatomical features of the tentative drainage pathways.

Curcumin and resveratrol inhibit chemoresistance in cisplatin-resistant epithelial ovarian cancer cells via targeting P13K pathway.

The activation of the PI3K/AKT/mTOR pathway has been proved to be associated with survival as well as proliferation of various tumour cells in multiple cancer types, including epithelial ovarian cancer (EOC). Moreover, the activation of the PI3K/AKT/mTOR pathway is the key mechanism responsible for higher invasiveness and migratory capacities of ovarian cancer cells. Furthermore, PI3K is crucial for activation of the PI3K/AKT/mTOR pathway; therefore, its inhibition might be an effective strategy against cancer. The combination approach is now an established strategy against cancer. So, the present study evaluated molecular mechanics behind the synergistic effects of curcumin and resveratrol along with cisplatin treatment on inhibition of the PI3K pathway in ovarian cancer cells. The present study confirmed significant inhibition of the PI3K/AKT/mTOR pathway as observed by Matrigel invasion assay, Western blot expression of important molecular markers and apoptotic markers. The present study concludes that the combination of curcumin and resveratrol significantly sensitized the EOC cells to cisplatin treatment, thereby inhibiting chemoresistance in ovarian cancer cells by significant inhibition of the PI3K/AKT/mTOR pathway.