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Malaria is responsible for almost half a million deaths annually. The role of Vγ9Vδ2 γδ T cells in malaria is still unclear. Studies have reported an association between this cell subset and malaria symptoms and severity. Profiles of Vγ9Vδ2 γδ T cells in bigger cohorts with different levels of clinical severity have not been described. Proportion, numbers, and activation status of Vγ9Vδ2 γδ T cells were measured by flow cytometry in 59 healthy controls (HCs), 58 children with uncomplicated malaria (UM) and 67 with cerebral malaria (CM,) during acute malaria and in convalescence 28 days later. Vγ9Vδ2 γδ T cell were lower in children presenting with UM and CM than in HCs. Cell counts did not vary with malaria severity (CM median counts 40 x 103 cells/μL, IQR [23–103]; UM median counts 30 x 103 cells/μL [10–90], P = 0.224). Vγ9Vδ2 γδ T cell counts increased during convalescence for UM (70 [40–60] x 103 cells/μL and CM (90 [60–140] x 103 cells/μL), to levels similar to those in HCs (70 [50–140] x 103 cells/μL), p = 0.70 and p = 0.40 respectively. Expression of the activation markers CD69 and HLA-DR on Vγ9Vδ2 γδ T cells was higher in malaria cases than in controls (HCs vs UM or CM, p < 0.0001) but was similar between UM and CM. HLA-DR expression remained elevated at 28 days, suggesting sustained activation of Vγ9Vδ2 γδ T cells during recovery. Vγ9Vδ2 γδ T cell proportions and cells counts were suppressed in acute disease and normalized in convalescence, a phenomenon previously hypothesized to be due to transient migration of the cells to secondary lymphoid tissue. The presence of highly activated Vγ9Vδ2 γδ T cells suggests that this T cell subset plays a specific role in response to malaria infection. [ABSTRACT FROM AUTHOR]

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-β signaling and that pharmacological blockage of TGF-β signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-β signaling. If the TGF-β pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-β signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors. [ABSTRACT FROM AUTHOR]

Background: The presence of infections is one of the main factors that leads to delays in healing or non-closure of cutaneous wounds. Although the goal of antibiotic use is to treat or prevent infection, there is currently no agreement on the effectiveness of these products. Aim: The aim of this study was to evaluate the efficacy of antibiotic use during the healing process of skin wounds in animal models not intentionally infected, as well as to analyze the advances and limitations of the studies carried out in this field. Main methods: This systematic review was performed according to the PRISMA guidelines, using a structured search on the MedLine (PubMed) and Scopus platforms to retrieve studies published until August 29, 2018, 13:35p.m. The studies included were limited to those that used excision or incision wound models and that were not intentionally infected. The data for the animal models, antibiotic used, and the main results of the studies were extracted, and compared where possible. Bias analysis and methodological quality assessments were examined through the SYRCLE's Risk of Bias tool. Key findings: Twenty-seven studies were selected. Overall, the effects of the antibiotic on the wound decreased inflammatory cell infiltration and promoted an increased number of fibroblasts, extracellular matrix constituents, re-epithelialization and tissue strength. A great deal of important information about the methodology was not presented, such as: the statistical analysis used, the animal model (sex and age), antibiotic dosage, blinding and randomization of the animals chosen. Significance: Based on the results found, we believe that antibiotic therapy can be considered a viable alternative for the treatment of cutaneous wounds. However, current evidence obtained from the methodological quality analysis points towards a high risk of bias. This is due to the incomplete characterization of the experimental design and treatment protocol, which compromises the reproducibility of the studies. [ABSTRACT FROM AUTHOR]

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin. [ABSTRACT FROM AUTHOR]

Endothelial nitric oxide synthase (eNOS)-related vessel relaxation is a highly coordinated process that regulates blood flow and pressure and is dependent on caveolae. Here, we investigated the role of caveolar plasma membrane stabilization by the dynamin-related ATPase EHD2 on eNOS-nitric oxide (NO)-dependent vessel relaxation. Loss of EHD2 in small arteries led to increased numbers of caveolae that were detached from the plasma membrane. Concomitantly, impaired relaxation of mesenteric arteries and reduced running wheel activity were observed in EHD2 knockout mice. EHD2 deletion or knockdown led to decreased production of nitric oxide (NO) although eNOS expression levels were not changed. Super-resolution imaging revealed that eNOS was redistributed from the plasma membrane to internalized detached caveolae in EHD2-lacking tissue or cells. Following an ATP stimulus, reduced cytosolic Ca2+ peaks were recorded in human umbilical vein endothelial cells (HUVECs) lacking EHD2. Our data suggest that EHD2-controlled caveolar dynamics orchestrates the activity and regulation of eNOS/NO and Ca2+ channel localization at the plasma membrane. [ABSTRACT FROM AUTHOR]

Thrips transmit one of the most devastating plant viruses worldwide–tomato spotted wilt tospovirus (TSWV). Tomato spotted wilt tospovirus is a type species in the genus Orthotospovirus and family Tospoviridae. Although there are more than 7,000 thrips species, only nine thrips species are known to transmit TSWV. In this study, we investigated the molecular factors that could affect thrips ability to transmit TSWV. We assembled transcriptomes of a vector, Frankliniella fusca [Hinds], and a non-vector, Frankliniella tritici [Fitch], and performed qualitative comparisons of contigs associated with virus reception, virus infection, and innate immunity. Annotations of F. fusca and F. tritici contigs revealed slight differences across biological process and molecular functional groups. Comparison of virus cell surface receptors revealed that homologs of integrin were present in both species. However, homologs of another receptor, heperan sulfate, were present in F. fusca alone. Contigs associated with virus replication were identified in both species, but a contig involved in inhibition of virus replication (radical s-adenosylmethionine) was only present in the non-vector, F. tritici. Additionally, some differences in immune signaling pathways were identified between vector and non-vector thrips. Detailed investigations are necessary to functionally characterize these differences between vector and non-vector thrips and assess their relevance in orthotospovirus transmission. [ABSTRACT FROM AUTHOR]

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment. [ABSTRACT FROM AUTHOR]

Background: Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. Methods: We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. Results: CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Conclusions: Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin. [ABSTRACT FROM AUTHOR]

In the pig, intrauterine competition (IUC) greatly affects postnatal traits, such as birth weight, but also locomotor capacities. In a previous study, our group discovered a lower motor performance in piglets with a low birth weight and low vitality (L piglets), compared to piglets with a normal birth weight and normal vitality (N piglets). In order to explain the force deficit causing this reduced motor performance, in a subsequent study, we investigated whether this deficit in L piglets was caused by a lower force generating capacity (FGC) of the extensors of the hind limb and/or a lower number of type II (fast-twitch) fibers in m. vastus lateralis. L piglets had a lower absolute FGC, but surprisingly, a higher relative FGC (to birth weight) in the hind limb, compared to N piglets. In addition, we found no differences in fiber composition of m. vastus lateralis. In the present study, we assessed whether this higher relative FGC is a common feature for front and hind limb locomotor muscles of L piglets. To that end, the physiological cross-sectional area of the main extensor muscles of the front limb was calculated from their volume and fiber length, in order to calculate both the absolute and the relative FGC. By immunohistochemical staining of m. triceps brachii caput longum, the percentage of type II (fast-contracting) fibers could be determined. Similar to the results of the hind limb, we found a smaller absolute FGC, but a larger relative FGC in the front limb of L piglets, compared to N piglets. In addition, m. triceps brachii caput longum did not have a different muscle fiber composition in L and N piglets. As such, we can conclude that IUC affects the locomotor muscles in the front and hind limb in a similar way and that the observed force deficit in L piglets cannot be explained by a different force generating capacity or a lower percentage of type II muscle fibers. [ABSTRACT FROM AUTHOR]

Transforming growth factor‑β1 (TGF-β1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-β1 requires release of the growth factor from pro-complexes and is regulated through TGF-β binding proteins. Two types of TGF-β binding partners, latent TGF-β-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-β1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-β1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-β1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-β1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-β1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-β1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-β regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer. [ABSTRACT FROM AUTHOR]

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and hyperplasia, as well as cartilage and bone destruction. Several proteins are associated with the pathogenesis of the disease. Galectin-9 belongs to the family of lectins that are involved in various biological processes and have anti-inflammatory activity. Objective: To investigate associations between the SNPs of the GAL-9 gene (LGALS9) and serum levels in rheumatoid arthritis patients. We extracted DNA from 356 subjects, 156 RA patients and 200 healthy controls from northeastern Brazil. Three polymorphisms (rs4795835, rs3763959, and rs4239242) in the LGALS9 gene were selected and genotyped using TaqMan SNP genotyping assay. Serum concentrations of galectin-9 were analyzed by ELISA. Results: The rs4239242 TT genotype showed a positive association with RA (p = 0.0032, odds ratio = 0.28), and heterozygous TC were prevalent in the control group compared to RA patients (p = 0.0001, odds ratio = 7.99). Galectin-9 serum levels were significantly increased in RA patients compared to the control group (p<0.0001). Patients in remission had high levels of galectin compared to the moderate activity group (p<0.0001). Regarding the Clinical Disease Activity Index (CDAI), patients in remission or low activity presented high levels of galectin when compared to patients in severity (p<0.0001). Patients performing moderate activity had a significant value compared to patients who were in high disease severity (p = 0.0064). Interestingly, the AG genotype (rs3763959) has been associated with a higher presence of bone erosion in RA patients (p = 0.0436). The SNP rs4239242 TT genotype showed a positive association with RA in comparison to the control group. The AG genotype (rs3763959) has been associated with a higher presence of bone erosion in RA patients. [ABSTRACT FROM AUTHOR]

Artemisinin resistance is threatening global efforts for malaria control and elimination. Primaquine (PQ) and methylene blue (MB) are gametocytocidal drugs that can be combined with artemisinin-based combination therapy (ACT) to reduce malaria transmission, including resistant strains. Children (6–59 months) with uncomplicated falciparum malaria in Burkina Faso were treated with artesunate-amodiaquine (AS-AQ) and randomized to MB (15 mg/kg/day for 3 days) or PQ (0.25 mg/kg at day 2) with the aim to show non-inferiority of the MB regimen with regard to haematological recovery at day 7 (primary endpoint). MB-AS-AQ could not be shown to be non-inferior to PQ-AS-AQ (mean Hb difference between treatment groups on day 7 was -0.352, 95% CI -0.832–0.128, p = 0.0767), however, haemoglobin recovery following treatment was alike in the two study arms (day 7: mean 0.2±1.4 g/dl vs. 0.5±0.9 g/dl, p = 0.446). Occurrence of adverse events was similar in both groups, except for vomiting, which was more frequent in the MB than in the PQ arm (20/50 vs 7/50, p = 0.003). Adequate clinical and parasitological response was above 95% in both groups, but significantly more asexual parasites were cleared in the MB arm compared to the PQ arm already on day 1 (48/50, 96%, vs 40/50, 80%, p = 0.014). Moreover, P. falciparum gametocyte prevalence and density were lower in the MB arm than in the PQ arm, which reached statistical significance on day 2 (prevalence: 2/50, 4%, vs 15/49, 31%, p<0.001; density: 9.6 vs 41.1/μl, p = 0.024). However, it should be considered that PQ was given only on day 2. MB-ACT appears to be an interesting alternative to PQ-ACT for the treatment of falciparum malaria. While there is a need to further improve MB formulations, MB-ACT may already be considered useful to reduce falciparum malaria transmission intensity, to increase treatment efficacy, and to reduce the risk for resistance development and spread. Trial registration: ClinicalTrials.gov . [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases 4 and 6 (CDK4/6) play critical roles in the G1 to S checkpoint of the cell cycle and have been shown to be overactive in several human cancers. Small-molecule inhibitors of CDK4/6 have demonstrated significant efficacy against many solid tumors. Since CDK4/6 inhibition is thought to induce cell cycle arrest at the G1/S checkpoint, much interest has been focused on combining CDK4/6 inhibitors with cytotoxic agents active against the S or M phase of the cell cycle to enhance therapeutic efficacy. However, it remains unclear how best to combine these two classes of drugs to avoid their potentially antagonistic effects. Here, we test various combinations of highly selective and potent CDK4/6 inhibitors with commonly used cytotoxic drugs in several cancer cell lines derived from lung, breast and brain cancers, for their cell-killing effects as compared to monotherapy. All combinations, either concurrent or sequential, failed to enhance cell-killing effects. Importantly, in certain schedules, especially pre-treatment with a CDK4/6 inhibitor, combining these drugs resulted in reduced cytotoxicity of cytotoxic agents. These findings urge cautions when combining these two classes of agents in clinical settings. [ABSTRACT FROM AUTHOR]

Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients. [ABSTRACT FROM AUTHOR]

H3N2 canine influenza viruses are prevalent in Asian and North American countries. During circulation of the viruses in dogs, these viruses are occasionally transmitted to cats. If this canine virus causes an epidemic in cats too, sporadic infections may occur in humans because of the close contact between these companion animals and humans, possibly triggering an emergence of mutant viruses with a pandemic potential. In this study, we aimed to gain an insight into the mutations responsible for inter-species transmission of H3N2 virus from dogs to cats. We found that feline CRFK cell-adapted viruses acquired several mutations in multiple genome segments. Among them, HA1-K299R, HA2-T107I, NA-L35R, and M2-W41C mutations individually increased virus growth in CRFK cells. With a combination of these mutations, virus growth further increased not only in CRFK cells but also in other feline fcwf-4 cells. Both HA1-K299R and HA2-T107I mutations increased thermal resistance of the viruses. In addition, HA2-T107I increased the pH requirement for membrane fusion. These findings suggest that the mutations, especially the two HA mutations, identified in this study, might be responsible for adaptation of H3N2 canine influenza viruses in cats. [ABSTRACT FROM AUTHOR]

Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2. [ABSTRACT FROM AUTHOR]

Background: PRR14 (Proline rich protein 14) was firstly identified for its ability to specify and localize heterochromatin during cell cycle progression. Aberrant expression of PRR14 is associated with the tumorigenesis and progression of lung cancer. However, its involvement in colon cancer remains unknown. Herein, we report the role of PRR14 in colon cancer. Methods: Colon cancer tissue microarray was used to analyze and compare the expression of PRR14 among some clinicopathological characteristics of colon cancer. HCT116 and RKO cells were transfected with siRNA to downregulate PRR14 expression. The roles of PRR14 in proliferation, migration and invasion of the cell lines were determined using cell counting kit-8, colony formation assay, wound healing assay and transwell assays respectively. The expression of PRR14 was measured using immunofluorescence, qRT- PCR and western blot. Epithelial-mesenchymal transition (EMT) markers were determined by western blot. Results: PRR14 was highly expressed in colon cancer tissues, and the expression level was correlated with tumor size, distant metastasis and Tumor Node Metastasis stages. Functional study revealed that downregulation of PRR14 inhibited colon cancer cells growth, migration and invasion. Furthermore, knockdown of PRR14 inhibited epithelial-mesenchymal transition (EMT) process, cell cycle-associated proteins expression and p-AKT level. Conclusion: PRR14 may promote the progression and metastasis of colon cancer, and may be a novel prognostic and therapeutic marker for the disease. [ABSTRACT FROM AUTHOR]

Introduction: Patients suffering from Sickle Cell Disease (SCD) are at increased risk for complications due to influenza virus. Annual influenza vaccination is strongly recommended but few clinical studies have assessed its immunogenicity in individuals with SCD. The aim of this study was to explore the biological efficacy of annual influenza vaccination in SCD patients by characterizing both their humoral and cell-mediated immunity against influenza antigen. We also aimed to investigate these immunological responses among SCD individuals according to their treatment (hydroxyurea (HU), chronic blood transfusions (CT), both HU and CT or none of them). Methods: Seventy-two SCD patients (49 receiving HU, 9 on CT, 7 with both and 7 without treatment) and 30 healthy controls were included in the study. All subjects received the tetravalent influenza α-RIX-Tetra® vaccine from the 2016–2017 or 2017–2018 season. Results: Protective anti-influenza HAI titers were obtained for the majority of SCD patients one month after vaccination but seroconversion rates in patient groups were strongly decreased compared to controls. Immune cell counts, particularly cellular memory including memory T and memory B cells, were greatly reduced in SCD individuals. Functional activation assays confirmed a poorer CD8+ T cell memory. We also document an imbalance of cytokines after influenza vaccination in SCD individuals with an INFγ/IL-10 ratio (Th1-type/Treg-type response) significantly lower in the SCD cohort. Conclusion: SCD patients undergoing CT showed altered immune regulation as compared to other treatment subgroups. Altogether, the cytokine imbalance, the high regulatory T cell levels and the low memory lymphocyte subset levels observed in the SCD cohort, namely for those on CT, suggest a poor ability of SCD patients to fight against influenza infection. Nevertheless, our serological data support current clinical practice for annual influenza vaccination, though immunogenicity to other vaccines involving immunological memory might be hampered in SCD patients and should be further investigated. [ABSTRACT FROM AUTHOR]

The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P = 0.030) for male embryos than female embryos. Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac. Results indicate that the blastocyst trophoctoderm can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks. [ABSTRACT FROM AUTHOR]

Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality. [ABSTRACT FROM AUTHOR]

Streptococcus suis is an important porcine bacterial pathogen and a zoonotic agent responsible for sudden death, septic shock and meningitis, of which serotype 2 is the most widespread, with serotype 14 also causing infections in humans in South-East Asia. Knowledge of its pathogenesis and virulence are almost exclusively based on these two serotypes. Though serotype 9 is responsible for the greatest number of porcine cases in Spain, the Netherlands and Germany, very little information is currently available regarding this serotype. Of the different virulence factors, the capsular polysaccharide (CPS) is required for S. suis virulence as it promotes resistance to phagocytosis and killing and masks surface components responsible for host cell activation. However, these roles have been described for serotypes 2 and 14, whose CPSs are structurally and compositionally similar, both containing sialic acid. Consequently, we evaluated herein the interactions of serotype 9 with host cells and the role of its CPS, which greatly differs from those of serotypes 2 and 14. Results demonstrated that serotype 9 adhesion to but not invasion of respiratory epithelial cells was greater than that of serotypes 2 and 14. Furthermore serotype 9 was more internalized by macrophages but equally resistant to whole blood killing. Though recognition of serotypes 2, 9 and 14 by DCs required MyD88-dependent signaling, in vitro pro-inflammatory mediator production induced by serotype 9 was much lower. In vivo, however, serotype 9 causes an exacerbated inflammatory response, which combined with persistent bacterial presence, is probably responsible for host death during the systemic infection. Though presence of the serotype 9 CPS masks surface components less efficiently than those of serotypes 2 and 14, the serotype 9 CPS remains critical for virulence as it is required for survival in blood and development of clinical disease, and this regardless of its unique composition and structure. [ABSTRACT FROM AUTHOR]

Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank's balanced salt solution (HBSS) and Tyrode's buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors. [ABSTRACT FROM AUTHOR]

Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene® RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals' expression differences, RNAgard® outperformed PAXgene® RNA, and both showed better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study. [ABSTRACT FROM AUTHOR]

Hypoxia and angiogenesis are critical components in the progression of solid cancer, including gastric cancers (GCs). miR-382 has been identified as a hypoxia-induced miR (hypoxamiR), but the clinical significance in GCs has not been identified yet. To explore the clinical and prognostic importance of miR-382 in GCs, the surgical specimens of 398 patients with GCs in KNU hospital in Korea, the total of 183 patients was randomly selected using simple sampling methods and big data with 446 GCs and 45 normal tissues from the data portal () were analysed. Expression of miR-382 as well as miR-210, as a positive control hypoxamiR by qRT-PCR in histologically malignant region of GCs showed significantly positive correlation (R = 0.516, p<0.001). High miR-210 and miR-382 expression was significantly correlated with unfavorable prognosis including advanced GCs (AGC), higher T category, N category, pathologic TNM stage, lymphovascular invasion, venous invasion, and perinueral invasion, respectively (all p<0.05). In univariate analysis, high miR-210 expression was significantly associated with worse overall survival (OS) (p = 0.036) but not high miR-382. In paired 60 gastric normal and cancer tissues, miR-382 expression in cancer tissues was significantly higher than normal counterpart (p = 0.003), but not miR-210 expression. However, by increasing the patient number from the big data analysis, miR-210 as well as miR-382 expression in tumor tissues was significantly higher than the normal tissues. Our results suggest that miR-382, as novel hypoxamiR, can be a prognostic marker for advanced GCs and might be correlated with metastatic potential. miR-382 might play important roles in the aggressiveness, progression and prognosis of GCs. In addition, miR-382 give a predictive marker for progression of GCs compared to the normal or preneoplastic lesion. [ABSTRACT FROM AUTHOR]

The N-type Ca2+ channel (Cav2.2) is distributed in sympathetic nerves that innervate the tubules, the vessels, and the juxtaglomerular granular cells of the kidney. However, the role of N-type Ca2+ channels in renal disease remains unknown. To address this issue, Cav2.2 knockout mice were utilized. Immunoreactive Cav2.2 was undetectable in normal kidneys of C57BL/6N mice, but it became positive in the interstitial S100-positive nerve fibers after unilateral ureteral obstruction (UUO). There were no significant differences in mean blood pressure, heart rate, and renal function between wild-type littermates and Cav2.2-knockout mice at baseline, as well as after UUO. Cav2.2 deficiency significantly reduced the EVG-positive fibrotic area, alpha-SMA expression, the production of type I collagen, and the hypoxic area in the obstructed kidneys. The expression of tyrosine hydroxylase, a marker for sympathetic neurons, was significantly increased in the obstructed kidneys of wild-type mice, but not in Cav2.2-knockout mice. These data suggest that increased Cav2.2 is implicated in renal nerve activation leading to the progression of renal fibrosis. Blockade of Cav2.2 might be a novel therapeutic approach for preventing renal fibrosis. [ABSTRACT FROM AUTHOR]

Network coupled cells, such as astrocytes, regulate their cellular homeostasis via Ca2+ signals spread between the cells through gap junctions. Intracellular Ca2+ release is controlled by different signaling pathways that can be stimulated by ATP, glutamate and serotonin (5-HT). Based on our findings, all these pathways are influenced by inflammatory agents and must be restored to fully recover the Ca2+ signaling network. An ultralow concentration of the local anesthetic agent bupivacaine reduced 5-HT-evoked intracellular Ca2+ release, and an ultralow concentration of the phosphodiesterase-5 inhibitor sildenafil in combination with vitamin D3 reduced ATP-evoked intracellular Ca2+ release. Combinations of these three substances downregulated 5-HT-, glutamate- and ATP-evoked intracellular Ca2+ release to a more normal Ca2+ signaling state. Furthermore, inflammatory Toll-like receptor 4 expression decreased with a combination of these three substances. Substance P receptor neurokinin (NK)-1 expression was reduced by ultralow concentrations of bupivacaine. Here, bupivacaine and sildenafil (at extremely low concentrations) combined with vitamin D3 have potential anti-inflammatory properties. According to the present study, drug combinations at the right concentrations, especially extremely low concentrations of bupivacaine and sildenafil, affect different cellular biochemical mechanisms and represent a potential solution for downregulating inflammatory parameters, thereby restoring cells or networks to normal physiological homeostasis. [ABSTRACT FROM AUTHOR]

Mouse models of mesial temporal lobe epilepsy recapitulate aspects of human epilepsy, which is characterized by neuronal network remodeling in the hippocampal dentate gyrus. Observational studies suggest that this remodeling is associated with altered Wnt pathway signaling, although this has not been experimentally examined. We used the well-characterized mouse intrahippocampal kainate model of temporal lobe epilepsy to examine associations between hippocampal neurogenesis and altered Wnt signaling after seizure induction. Tissue was analyzed using immunohistochemistry and confocal microscopy, and gene expression analysis was performed by RT-qPCR on RNA extracted from anatomically micro-dissected dentate gyri. Seizures increased neurogenesis and dendritic arborization of newborn hippocampal dentate granule cells in peri-ictal regions, and decreased neurogenesis in the ictal zone, 2-weeks after kainate injection. Interestingly, administration of the novel canonical Wnt pathway inhibitor XAV939 daily for 2-weeks after kainate injection further increased dendritic arborization in peri-ictal regions after seizure, without an effect on baseline neurogenesis in control animals. Transcriptome analysis of dentate gyri demonstrated significant canonical Wnt gene dysregulation in kainate-injected mice across all regions for Wnt3, 5a and 9a. Intriguingly, certain Wnt genes demonstrated differential patterns of dysregulation between the ictal and peri-ictal zones, most notably Wnt5B, 7B and DKK-1. Together, these results demonstrate regional variation in Wnt pathway dysregulation early after seizure induction, and surprisingly, suggest that some Wnt-mediated effects might actually temper aberrant neurogenesis after seizures. The Wnt pathway may therefore provide suitable targets for novel therapies that prevent network remodeling and the development of epileptic foci in high-risk patients. [ABSTRACT FROM AUTHOR]

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex. [ABSTRACT FROM AUTHOR]

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria. [ABSTRACT FROM AUTHOR]

Gastric emptying (GE) can be either delayed or accelerated in diabetes mellitus (DM). However, most research has focused on delayed GE mediated by a chronic hyperglycemic condition in DM. As such, the function of GE in the early stages of DM is not well understood. Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal tract. In the present study, we investigated changes in GE and ICC networks in the early stages of DM using a streptozotocin-induced type 1 diabetic mouse model. The changes in GE were measured by the 13C-octanoic acid breath test. ICC networks were immunohistochemically detected by an antibody for c-Kit, a specific marker for ICC. Our results showed that GE in type 1 DM was significantly accelerated in the early stages of DM (2–4 weeks after onset). In addition, acute normalization of blood glucose levels by a single administration of insulin did not recover normal GE. ICC networks of the gastric antrum were significantly increased in DM and were not affected by the acute normalization of blood glucose. In conclusion, our results suggest that GE is accelerated in the early stages of DM, and it is associated with increased ICC networks. This mechanism may help to clarify a link between the onset of DM and GE disorders. [ABSTRACT FROM AUTHOR]

Although there is good evidence to indicate a major role of intrinsic impairment of the contractile apparatus in muscle weakness seen in several pathophysiological conditions, the factors responsible for control of myofibrillar function are not fully understood. To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force between denervated (DEN) and dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES) training. DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (-74% vs. -10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (-24% vs. -17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in the DEN than in the DEX group (-53% vs. -16%). Moreover, there was a parallel decline in myosin heavy chain (MyHC) and actin content per muscle volume in DEN muscles, but not in DEX muscles, which was associated with upregulation of NADPH oxidase (NOX) 2, neuronal nitric oxide synthase (nNOS), and endothelial NOS expression, translocation of nNOS from the membrane to the cytosol, and augmentation of mRNA levels of muscle RING finger protein 1 (MuRF-1) and atrogin-1. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle. [ABSTRACT FROM AUTHOR]

DNA methylation through the activity of cytosine-5-methyltransferases (C5-MTases) and DNA demethylases plays important roles in genome protection as well as in regulating gene expression during plant development and plant response to environmental stresses. In this study, we report on a genome-wide identification of six C5-MTases (SmelMET1, SmelCMT2, SmelCMT3a, SmelCMT3b, SmelDRM2, SmelDRM3) and five demethylases (SmelDemethylase_1, SmelDemethylase_2, SmelDemethylase_3, SmelDemethylase_4, SmelDemethylase_5) in eggplant. Gene structural characteristics, chromosomal localization and phylogenetic analyses are also described. The transcript profiling of both C5-MTases and demethylases was assessed at three stages of fruit development in three eggplant commercial F1 hybrids: i.e. ‘Clara’, ‘Nite Lady’ and ‘Bella Roma’, representative of the eggplant berry phenotypic variation. The trend of activation of C5-MTases and demethylase genes varied in function of the stage of fruit development and was genotype dependent. The transcription pattern of C5MTAses and demethylases was also assessed in leaves of the F1 hybrid ‘Nite Lady’ subjected to salt and drought stresses. A marked up-regulation and down-regulation of some C5-MTases and demethylases was detected, while others did not vary in their expression profile. Our results suggest a role for both C5-MTases and demethylases during fruit development, as well as in response to abiotic stresses in eggplant, and provide a starting framework for supporting future epigenetic studies in the species. [ABSTRACT FROM AUTHOR]

Hapten contact hypersensitivity (CHS) elicits a well-documented inflammation response that can be used to illustrate training of immune cells through hapten-specific CHS memory. The education of hapten-specific memory T cells has been well-established, recent research in mice has expanded the “adaptive” characteristic of a memory response from solely a function of the adaptive immune system, to innate cells as well. To test whether similar responses are seen in a non-rodent model, we used hapten-specific CHS to measure the ear inflammation response of outbred pigs to dinitrofluorobenzene (DNFB), oxazolone (OXA), or vehicle controls. We adapted mouse innate memory literature protocols to the domestic pig model. Animals were challenged up to 32 days post initial sensitization exposure to the hapten, and specific ear swelling responses to this challenge were significant for 7, 21, and 32 days post-sensitization. We established hapten-specific CHS memory exists in a non-rodent model. We also developed a successful protocol for demonstrating these CHS responses in a porcine system. [ABSTRACT FROM AUTHOR]

Recently, we observed a 15% decrease in the incidence of type 1 diabetes (T1D) in Australian 0-4-year-old children following the introduction of RV vaccination [[2], [3]], suggesting that RV vaccination could contribute to the primary prevention of this autoimmune disease. Australian surveillance data [[11]] show that the prevalence of RV G3 strains increased slightly along with an increase in strain diversity in the post-RV vaccine era, but G3 remains a minor component of disease-causing RV strains. RV was prevalent in nurseries, and the change to rooming-in would have altered the timing of exposure to RV, delaying it until later in the first year of life when, based on NOD mouse studies [[17]-[19]], RV might promote rather than retard development of diabetes. [Extracted from the article]

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing. [ABSTRACT FROM AUTHOR]

Leaves display a remarkable range of forms, from flat sheets with simple outlines to cup-shaped traps. Although much progress has been made in understanding the mechanisms of planar leaf development, it is unclear whether similar or distinctive mechanisms underlie shape transformations during development of more complex curved forms. Here, we use 3D imaging and cellular and clonal analysis, combined with computational modelling, to analyse the development of cup-shaped traps of the carnivorous plant Utricularia gibba. We show that the transformation from a near-spherical form at early developmental stages to an oblate spheroid with a straightened ventral midline in the mature form can be accounted for by spatial variations in rates and orientations of growth. Different hypotheses regarding spatiotemporal control predict distinct patterns of cell shape and size, which were tested experimentally by quantifying cellular and clonal anisotropy. We propose that orientations of growth are specified by a proximodistal polarity field, similar to that hypothesised to account for Arabidopsis leaf development, except that in Utricularia, the field propagates through a highly curved tissue sheet. Independent evidence for the polarity field is provided by the orientation of glandular hairs on the inner surface of the trap. Taken together, our results show that morphogenesis of complex 3D leaf shapes can be accounted for by similar mechanisms to those for planar leaves, suggesting that simple modulations of a common growth framework underlie the shaping of a diverse range of morphologies. [ABSTRACT FROM AUTHOR]

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments. [ABSTRACT FROM AUTHOR]

The striatum plays a fundamental role in motor learning and reward-related behaviors that are synergistically shaped by populations of D1 dopamine receptor (D1R)- and D2 dopamine receptor (D2R)-expressing medium spiny neurons (MSNs). How various neurotransmitter inputs converging on common intracellular pathways are parsed out to regulate distinct behavioral outcomes in a neuron-specific manner is poorly understood. Here, we reveal that distinct contributions of D1R-MSNs and D2R-MSNs towards reward and motor behaviors are delineated by the multifaceted signaling protein neurofibromin 1 (NF1). Using genetic mouse models, we show that NF1 in D1R-MSN modulates opioid reward, whereas loss of NF1 in D2R-MSNs delays motor learning by impeding the formation and consolidation of repetitive motor sequences. We found that motor learning deficits upon NF1 loss were associated with the disruption in dopamine signaling to cAMP in D2R-MSN. Restoration of cAMP levels pharmacologically or chemogenetically rescued the motor learning deficits seen upon NF1 loss in D2R-MSN. Our findings illustrate that multiplex signaling capabilities of MSNs are deployed at the level of intracellular pathways to achieve cell-specific control over behavioral outcomes. [ABSTRACT FROM AUTHOR]

Multipotent trophoblasts undergo dynamic morphological movement and cellular differentiation after conceptus implantation to generate placenta. However, the mechanism controlling trophoblast development and differentiation during peri-implantation development in human remains elusive. In this study, we modeled human conceptus peri-implantation development from blastocyst to early postimplantation stages by using an in vitro coculture system and profiled the transcriptome of 476 individual trophoblast cells from these conceptuses. We revealed the genetic networks regulating peri-implantation trophoblast development. While determining when trophoblast differentiation happens, our bioinformatic analysis identified T-box transcription factor 3 (TBX3) as a key regulator for the differentiation of cytotrophoblast (CT) into syncytiotrophoblast (ST). The function of TBX3 in trophoblast differentiation is then validated by a loss-of-function experiment. In conclusion, our results provided a valuable resource to study the regulation of trophoblasts development and differentiation during human peri-implantation development. [ABSTRACT FROM AUTHOR]

Microbial endosymbiosis is widespread in animals, with major ecological and evolutionary implications. Successful symbiosis relies on efficient vertical transmission through host generations. However, when symbionts negatively affect host fitness, hosts are expected to evolve suppression of symbiont effects or transmission. Here, we show that sex chromosomes control vertical transmission of feminizing Wolbachia endosymbionts in the isopod Armadillidium nasatum. Theory predicts that the invasion of an XY/XX species by cytoplasmic sex ratio distorters is unlikely because it leads to fixation of the unusual (and often lethal or infertile) YY genotype. We demonstrate that A. nasatum X and Y sex chromosomes are genetically highly similar and that YY individuals are viable and fertile, thereby enabling Wolbachia spread in this XY-XX species. Nevertheless, we show that Wolbachia cannot drive fixation of YY individuals, because infected YY females do not transmit Wolbachia to their offspring, unlike XX and XY females. The genetic basis fits the model of a Y-linked recessive allele (associated with an X-linked dominant allele), in which the homozygous state suppresses Wolbachia transmission. Moreover, production of all-male progenies by infected YY females restores a balanced sex ratio at the host population level. This suggests that blocking of Wolbachia transmission by YY females may have evolved to suppress feminization, thereby offering a whole new perspective on the evolutionary interplay between microbial symbionts and host sex chromosomes. [ABSTRACT FROM AUTHOR]

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis. [ABSTRACT FROM AUTHOR]

Recent evidence implicates mononuclear diploid cardiomyocytes as a proliferative and regenerative subpopulation of the postnatal heart. The number of these cardiomyocytes is a complex trait showing substantial natural variation among inbred mouse strains based on the combined influences of multiple polymorphic genes. One gene confirmed to influence this parameter is the cardiomyocyte-specific kinase Tnni3k. Here, we have studied Tnni3k alleles across a number of species. Using a newly-generated kinase-dead allele in mice, we show that Tnni3k function is dependent on its kinase activity. In an in vitro kinase assay, we show that several common human TNNI3K kinase domain variants substantially compromise kinase activity, suggesting that TNNI3K may influence human heart regenerative capacity and potentially also other aspects of human heart disease. We show that two kinase domain frameshift mutations in mice cause loss-of-function consequences by nonsense-mediated decay. We further show that the Tnni3k gene in two species of mole-rat has independently devolved into a pseudogene, presumably associated with the transition of these species to a low metabolism and hypoxic subterranean life. This may be explained by the observation that Tnni3k function in mice converges with oxidative stress to regulate mononuclear diploid cardiomyocyte frequency. Unlike other studied rodents, naked mole-rats have a surprisingly high (30%) mononuclear cardiomyocyte level but most of their mononuclear cardiomyocytes are polyploid; their mononuclear diploid cardiomyocyte level (7%) is within the known range (2–10%) of inbred mouse strains. Naked mole-rats provide further insight on a recent proposal that cardiomyocyte polyploidy is associated with evolutionary acquisition of endothermy. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are the most effective professional antigen-presenting cell. They ferry antigen from the extremities to T cells and are essential for the initiation of an adaptive immune response. Despite interest in how DCs respond to chemical stimuli, there have been few attempts to model DC migration. In this paper, we simulate the motility of DCs by modeling the generation of forces by filopodia and a force balance on the cell. The direction of fliopodial extension is coupled to differential occupancy of cognate chemokine receptors across the cell. Our model simulates chemokinesis and chemotaxis in a variety of chemical and mechanical environments. Simulated DCs undergoing chemokinesis were measured to have a speed of 5.1 ± 0.07 μm·min-1 and a persistence time of 3.2 ± 0.46 min, consistent with experiment. Cells undergoing chemotaxis exhibited a stronger chemotactic response when exposed to lower average chemokine concentrations, also consistent with experiment. We predicted that when placed in two opposing gradients, cells will cluster in a line, which we call the “line of equistimulation;” this clustering has also been observed. We calculated the effect of varying gradient steepness on the line of equistimulation, with steeper gradients resulting in tighter clustering. Moreover, gradients are found to be most potent when cells are in a gradient of chemokine whose mean concentration is close to the binding of the Kd to the receptor, and least potent when the mean concentration is 0.1Kd. Comparing our simulations to experiment, we can give a quantitative measure of the strength of certain chemokines relative to others. Assigning the signal of CCL19 binding CCR7 a baseline strength of 1, we found CCL21 binding CCR7 had a strength of 0.28, and CXCL12 binding CXCR4 had a strength of 0.30. These differences emerge despite both chemokines having virtually the same Kd, suggesting a mechanism of signal amplification in DCs requiring further study. [ABSTRACT FROM AUTHOR]

Message passing between components of a distributed physical system is non-instantaneous and contributes to determine the time scales of the emerging collective dynamics. In biological neuron networks this is due in part to local synaptic filtering of exchanged spikes, and in part to the distribution of the axonal transmission delays. How differently these two kinds of communication protocols affect the network dynamics is still an open issue due to the difficulties in dealing with the non-Markovian nature of synaptic transmission. Here, we develop a mean-field dimensional reduction yielding to an effective Markovian dynamics of the population density of the neuronal membrane potential, valid under the hypothesis of small fluctuations of the synaptic current. Within this limit, the resulting theory allows us to prove the formal equivalence between the two transmission mechanisms, holding for any synaptic time scale, integrate-and-fire neuron model, spike emission regimes and for different network states even when the neuron number is finite. The equivalence holds even for larger fluctuations of the synaptic input, if white noise currents are incorporated to model other possible biological features such as ionic channel stochasticity. [ABSTRACT FROM AUTHOR]

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae–all potential predators of fungi–grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits. [ABSTRACT FROM AUTHOR]

We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025–14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function. [ABSTRACT FROM AUTHOR]

Regime shifts involving critical transitions are a type of rapid ecological change that are difficult to predict, but may be preceded by decreases in resilience. Time series statistics like lag-1 autocorrelation may be useful for anticipating resilience declines; however, more study is needed to determine whether the dynamics of autocorrelation depend on the resolution of the time series being analyzed, i.e., whether they are time-scale dependent. Here, we examined timeseries simulated from a lake eutrophication model and gathered from field measurements. The field study involved collecting high frequency chlorophyll fluorescence data from an unmanipulated reference lake and a second lake undergoing experimental fertilization to induce a critical transition in the form of an algal bloom. As part of the experiment, the fertilization was halted in response to detected early warnings of the algal bloom identified by increased autocorrelation. We tested these datasets for time-scale dependence in the dynamics of lag-1 autocorrelation and found that in both the simulation and field experiment, the dynamics of autocorrelation were similar across time scales. In the simulated time series, autocorrelation increased exponentially approaching algal bloom development, and in the field experiment, the difference in autocorrelation between the manipulated and reference lakes increased sharply. These results suggest that, as an early warning indicator, autocorrelation may be robust to the time scale of the analysis. Given that a time scale can be shortened by increasing sampling frequency, or lengthened by aggregating data during analysis, these results have important implications for management as they demonstrate the potential for detecting early warning signals over a wide range of monitoring frequencies and without requiring analysts to make situation-specific decisions regarding aggregation. Such an outcome provides promise that data collection procedures, especially by automated sensors, may be used to monitor and manage ecosystem resilience without the need for strict attention to time scale. [ABSTRACT FROM AUTHOR]

To understand the effect of attention on neuronal dynamics, we propose a multi-module network, with each module consisting of fully interconnected groups of excitatory and inhibitory neurons. This network shows transitive dynamics among quasi-attractors as its typical dynamics. When the release of acetylcholine onto the network is simulated by attention, the transitive dynamics change into stable dynamics in which the system converges to an attractor. We found that this network can reproduce three experimentally observed properties of attention-dependent response modulation, namely an increase in the firing rate, a decrease in the Fano factor of the firing rate, and a decrease in the correlation coefficients between the firing rates of pairs of neurons. Moreover, we also showed theoretically that the release of acetylcholine increases the sensitivity to bottom-up inputs by changing the response function. [ABSTRACT FROM AUTHOR]

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen. [ABSTRACT FROM AUTHOR]