l-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efficacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO significantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase, but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors.

Subthreshold A-type K(+) currents (ISA s) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic ISA s. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore-forming Kv4 subunits and two types of auxiliary subunits: K(+) channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these ISA -expressing pain-related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in ISA -expressing nociceptors and pain-modulating spinal interneurons.

Two studies (ROADMAP and ORIENT) evaluating the renoprotective effects of olmesartan medoxomil (OM) in patients with type 2 diabetes suggested OM is associated with increased cardiovascular mortality. We conducted a thorough QTc study to evaluate the effects of OM on cardiac repolarization. A randomized, double-blind, phase 1 study was conducted per E14 Guidance to assess the effects of single doses of OM therapeutic dose (40 mg), OM supratherapeutic dose (160 mg), placebo, or moxifloxacin (MOXI; 400 mg) on QTc in 56 healthy subjects. The primary endpoint was the baseline-adjusted, placebo-corrected QTc interval using Fridericia's formula (ΔΔQTcF) for OM and MOXI. Assay sensitivity was concluded if lower limit of 1-sided 95%CI > 5 milliseconds of ΔΔQTcF for MOXI. No threshold pharmacologic effect for OM was concluded if upper limit of 1-sided 95%CI <10 milliseconds for ΔΔQTcF at any timepoint. Pharmacokinetics, ECGs, and safety were assessed. Assay sensitivity was demonstrated. The largest upper limit of the 1-sided 95%CI for ΔΔQTcF was <5 milliseconds for OM. No clinically significant changes were observed in ECGs. Pharmacokinetics and safety profile were consistent with previous data. Therapeutic and supratherapeutic OM doses had no clinically significant effect on cardiac repolarization and were well tolerated.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

The evolution of island populations in natural systems is driven by local adaptation and genetic drift. However, evolutionary pathways may be altered by humans in several ways. The wild boar (WB) (Sus scrofa) is an iconic game species occurring in several islands, where it has been strongly managed since prehistoric times. We examined genomic diversity at 49 803 single-nucleotide polymorphisms in 99 Sardinian WBs and compared them with 196 wild specimens from mainland Europe and 105 domestic pigs (DP; 11 breeds). High levels of genetic variation were observed in Sardinia (80.9% of the total number of polymorphisms), which can be only in part associated to recent genetic introgression. Both Principal Component Analysis and Bayesian clustering approach revealed that the Sardinian WB population is highly differentiated from the other European populations (FST=0.126-0.138), and from DP (FST=0.169). Such evidences were mostly unaffected by an uneven sample size, although clustering results in reference populations changed when the number of individuals was standardized. Runs of homozygosity (ROHs) pattern and distribution in Sardinian WB are consistent with a past expansion following a bottleneck (small ROHs) and recent population substructuring (highly homozygous individuals). The observed effect of a non-random selection of Sardinian individuals on diversity, FST and ROH estimates, stressed the importance of sampling design in the study of structured or introgressed populations. Our results support the heterogeneity and distinctiveness of the Sardinian population and prompt further investigations on its origins and conservation status.

Plants display numerous strategies to cope with phosphate (Pi)-deficiency. Despite multiple genetic studies, the molecular mechanisms of low-Pi-signalling remain unknown. To validate the interest of chemical genetics to investigate this pathway we discovered and analysed the effects of PHOSTIN (PSN), a drug mimicking Pi-starvation in Arabidopsis. We assessed the effects of PSN and structural analogues on the induction of Pi-deficiency responses in mutants and wild-type and followed their accumulation in plants organs by high pressure liquid chromotography (HPLC) or mass-spectrophotometry. We show that PSN is cleaved in the growth medium, releasing its active motif (PSN11), which accumulates in plants roots. Despite the overaccumulation of Pi in the roots of treated plants, PSN11 elicits both local and systemic Pi-starvation effects. Nevertheless, albeit that the transcriptional activation of low-Pi genes by PSN11 is lost in the phr1;phl1 double mutant, neither PHO1 nor PHO2 are required for PSN11 effects. The range of local and systemic responses to Pi-starvation elicited, and their dependence on the PHR1/PHL1 function suggests that PSN11 affects an important and early step of Pi-starvation signalling. Its independence from PHO1 and PHO2 suggest the existence of unknown pathway(s), showing the usefulness of PSN and chemical genetics to bring new elements to this field.

Recent clinical trials revealed a surprisingly rapid clearance of red blood cells (RBCs) infected with malaria parasites by the spiroindolone KAE609. Here, we show that ring-stage parasite-infected RBCs exposed to KAE609 become spherical and rigid, probably through osmotic dysregulation consequent to the disruption of the parasite's sodium efflux pump (adenosine triphosphate 4). We also show that this peculiar drug effect is likely to cause accelerated splenic clearance of the rheologically impaired Plasmodium vivax- and Plasmodium falciparum-infected RBCs.

Glucagon-like peptide-1 (GLP-1) acts as a satiety signal and enhances insulin release. This study examined how GLP-1 production from intestinal L-cells is modified by dietary changes.

Mesenchymal stem cell-based therapy has emerged as a promising approach for the treatment of peripheral arterial disease. The purpose of this study was to examine the potential effects of human placenta-derived mesenchymal stem cells (PMSCs) on mouse hindlimb ischemia. PMSCs were isolated from human placenta tissue and characterized by flow cytometry. An in vivo surgical ligation-induced murine limb ischemia model was generated with fluorescent dye (CM-DiI) labelled PMSCs delivered via intramuscular injection. Our data show that PMSCs treatment significantly enhanced microvessel density, improved blood perfusion and diminished pathologies in ischemic mouse hindlimbs as compared to those in the control group. Further immunostaining studies suggested that injected PMSCs can incorporate into the vasculature and differentiate into endothelial and smooth muscle cells to enhance angiogenesis in ischemic hind limbs. This may in part explain the beneficial effects of PMSCs treatment. Taken together, we found that PMSCs treatment might be an effective treatment modality for treatment of ischemia-induced injury to mouse hind limbs by enhancement of angiogenesis.

Galectins (Gals) have emerged as potent immunoregulatory molecules that control chronic inflammation through distinct mechanisms. Gal-8, a tandem-repeat type Gal with unique preference for α2,3-sialylated glycans, is ubiquitously expressed, but little is known about its role in T-cell differentiation. Here we report that Gal-8 promotes the polyclonal differentiation of primary mouse regulatory T (Treg) cells in vitro. We further show that Gal-8 also facilitates antigen-specific differentiation of Treg cells, and that Treg cells polarized in the presence of Gal-8 express cytotoxic T-lymphocyte antigen-4 and interleukin (IL)-10 at a higher frequency than control Treg cells, and efficiently inhibit proliferation of activated T-cells in vitro. Investigation of the mechanism by which Gal-8 promotes Treg conversion revealed that Gal-8 activates transforming growth factor-β signaling and promotes sustained IL-2R signaling. Taken together, these data suggest that Gal-8 promotes the differentiation of highly suppressive Treg cells, which has implications for the treatment of inflammatory and autoimmune diseases.

To evaluate evidence of the capacity for causal inference in studies of associations between parental and offspring alcohol consumption in the general population.

Trajectories toward risk or resilience in psychiatric disorders are influenced by acquired and inherited factors. More recently, evidence from rodent studies suggest that acquired risk factors can be transmitted through non-genomic, epigenetic mechanisms to subsequent generations, potentially contributing to a cycle of disease and disease risk. Here, we review examples of transmission of environmental factors across generations and illustrate the difference between behavioral transmission and epigenetic inheritance. We highlight essential definitions of intergenerational and transgenerational transmission of disease risk with corresponding examples. We then explore how these phenomena may influence our understanding of psychiatric disorders leading toward new prevention and therapeutic approaches.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Chronic obstructive pulmonary disease (COPD) is related to an abnormal chronic inflammatory response of the lung to mainly cigarette smoke (CS) and the disease risk is increased in aged individuals. The source of this chronic inflammation is due to the repeated and progressive activation of immune cells. We hypothesize that in a chronic CS-induced mouse model, the predisposition to COPD pathogenesis in aged mice is characterized by an elevated immune response compared to young animals. We measured several characteristics of COPD in young and old mice (2 and 12 months of age) exposed to CS for 3 months. CS-exposed aged mice exhibited increased lung compliance (0.061 ± 0.008 vs. 0.055 ± 0.006 ml/cm H2O, p < 0.01), emphysema development (35.36 ± 0.71 vs. 25.31 ± 0.005 μm; p < 0.01) and airway remodeling (2.15 ± 0.37 vs. 1.09 ± 0.64 μm3/μm2; p < 0.01) compared to control animals, which was not seen in CS-exposed young mice. Quantification of lung tissue inflammation revealed a significantly greater volume of inducible bronchus-associated lymphoid tissue structures in aged mice after CS exposure (5.94 ± 2.89 vs. 2.37 ± 1.69 μm3/μm2; p < 0.01). Our results indicate that age-induced lung inflammation is further elevated after CS exposure in old mice, potentially via an age-induced change in immune cell susceptibility to CS thereby accelerating the pathophysiological hallmarks of COPD.

Two new bifunctional tris(hydroxypyridinone) (THP) chelators designed specifically for rapid labeling with (68)Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, RGD. Each conjugate can be radiolabeled and formulated by treatment with generator-produced (68)Ga(3+) in over 95% radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60-80 MBq nmol(-1). Competitive binding assays and in vivo biodistribution in mice bearing U87MG tumors demonstrate that the new (68)Ga(3+)-labeled THP peptide conjugates retain affinity for the αvβ3 integrin receptor, clear within 1-2 h from circulation, and undergo receptor-mediated tumor uptake in vivo. We conclude that bifunctional THP chelators can be used for simple, efficient labeling of (68)Ga biomolecules under mild conditions suitable for peptides and proteins.

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l-1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.

Brain-derived neurotrophic factor (BDNF), a neurotrophin that regulates neuronal function and development, is implicated in several neurodegenerative conditions. Preliminary data suggest that a reduction of BDNF concentrations may lead to postchemotherapy cognitive impairment. We hypothesized that a single nucleotide polymorphism (rs6265) of the BDNF gene may predispose patients to cognitive impairment. This study aimed to evaluate the effect of BDNF gene polymorphism on chemotherapy-associated cognitive impairment.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

The aim of this study is to determine the prognostic value of tumor markers, as squamous cell carcinoma antigen (SCCAg) and cytokeratin-19 fragment (CYFRA 21.1) and interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), soluble tumor necrosis factor receptor I (sTNF RI), and sTNF RII in patients with squamous cell carcinoma of the cervix. The subjects of analysis were 138 patients with stage I-IVA according to the International Federation of Gynecology and Obstetrics (FIGO) classification. The collected research material comes from one oncology center. During the 10 years of follow-up, 56 relapses and 53 deaths were observed, and recurrent disease in early stage was confirmed in 45 % of patients. The pretreatment serum levels of SCCAg and CYFRA 21.1, and cytokines IL-6, VEGF, sTNF RI, and sTNF RII were determined in all patients. The probability of disease-free survival (DFS) and overall survival (OS) was evaluated using the log-rank test and the Cox regression model. Based on the ROC curve analysis for patients with recurrence, the largest area under the curve was demonstrated for SCCAg and IL-6 and for patients who died, for SCCAg and VEGF. Cox analysis demonstrated that independent prognostic factor for DFS was only SCCAg and for OS cytokine IL-6 and SCCAg, but in patients with early stage the prognostic value for DFS was VEGF, whereas IL-6 and CYFRA 21.1 for OS. Serum level of VEGF, CYFRA 21.1 and IL-6 before treatment in patients with early stage cervical cancer appears to be an important prognostic factor.

A composite likelihood ratio test implemented in the program sweepfinder is a commonly used method for scanning a genome for recent selective sweeps. sweepfinder uses information on the spatial pattern (along the chromosome) of the site frequency spectrum around the selected locus. To avoid confounding effects of background selection and variation in the mutation process along the genome, the method is typically applied only to sites that are variable within species. However, the power to detect and localize selective sweeps can be greatly improved if invariable sites are also included in the analysis. In the spirit of a Hudson-Kreitman-Aguadé test, we suggest adding fixed differences relative to an out-group to account for variation in mutation rate, thereby facilitating more robust and powerful analyses. We also develop a method for including background selection, modelled as a local reduction in the effective population size. Using simulations, we show that these advances lead to a gain in power while maintaining robustness to mutation rate variation. Furthermore, the new method also provides more precise localization of the causative mutation than methods using the spatial pattern of segregating sites alone.