Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland,the gland of the third eyelid and the knee joint

Background aimsMesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs.MethodsAllogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation.ResultsClinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations.ConclusionsThis is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.     

Effects of Maternal Obstructive Sleep Apnoea on Fetal Growth: A Prospective Cohort Study

Objective

The objective of this study is to determine whether obstructive sleep apnea (OSA) is associated with reduced fetal growth, and whether nocturnal oxygen desaturation precipitates acute fetal heart rate changes.

Study Design

We performed a prospective observational study, screening 371 women in the second trimester for OSA symptoms. 41 subsequently underwent overnight sleep studies to diagnose OSA. Third trimester fetal growth was assessed using ultrasound. Fetal heart rate monitoring accompanied the sleep study. Cord blood was taken at delivery, to measure key regulators of fetal growth.

Results

Of 371 women screened, 108 (29%) were high risk for OSA. 26 high risk and 15 low risk women completed the longitudinal study; 14 had confirmed OSA (cases), and 27 were controls. The median (interquartile range) respiratory disturbance index (number of apnoeas, hypopnoeas or respiratory related arousals/hour of sleep) was 7.9 (6.1–13.8) for cases and 2.2 (1.3–3.5) for controls (p<0.001). Impaired fetal growth was observed in 43% (6/14) of cases, vs 11% (3/27) of controls (RR 2.67; 1.25–5.7; p = 0.04). Using logistic regression, only OSA (OR 6; 1.2–29.7, p = 0.03) and body mass index (OR 2.52; 1.09–5.80, p = 0.03) were significantly associated with impaired fetal growth. After adjusting for body mass index on multivariate analysis, the association between OSA and impaired fetal growth was not appreciably altered (OR 5.3; 0.93–30.34, p = 0.06), although just failed to achieve statistical significance. Prolonged fetal heart rate decelerations accompanied nocturnal oxygen desaturation in one fetus, subsequently found to be severely growth restricted. Fetal growth regulators showed changes in the expected direction- with IGF-1 lower, and IGFBP-1 and IGFBP-2 higher- in the cord blood of infants of cases vs controls, although were not significantly different.

Conclusion

OSA may be associated with reduced fetal growth in late pregnancy. Further evaluation is warranted to establish whether OSA may be an important contributor to adverse perinatal outcome, including stillbirth.     

Genetic and Non-Genetic Determinants of Raltegravir Penetration into Cerebrospinal Fluid: A Single Arm Pharmacokinetic Study

Background

Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C→T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C→T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.

Methods

Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.

Results

The 4-hour CSF concentration correlated more strongly with 2-hour (r²=0.76, P=1.12x10^-11) than 4-hour (r²=0.47, P=6.89x10^-6) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC_0-4h r²=0.86, P=5.15x10^-16). There was no significant association between ABCB1 3435C→T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.

Conclusions

Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C→T genotype but strongly correlate with plasma exposure.

Trial Registration

ClinicalTrials.gov NCT00729924 http://clinicaltrials.gov/show/NCT00729924     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.     

Task-Based Core-Periphery Organization of Human Brain Dynamics

As a person learns a new skill, distinct synapses, brain regions, and circuits are engaged and change over time. In this paper, we develop methods to examine patterns of correlated activity across a large set of brain regions. Our goal is to identify properties that enable robust learning of a motor skill. We measure brain activity during motor sequencing and characterize network properties based on coherent activity between brain regions. Using recently developed algorithms to detect time-evolving communities, we find that the complex reconfiguration patterns of the brain''s putative functional modules that control learning can be described parsimoniously by the combined presence of a relatively stiff temporal core that is composed primarily of sensorimotor and visual regions whose connectivity changes little in time and a flexible temporal periphery that is composed primarily of multimodal association regions whose connectivity changes frequently. The separation between temporal core and periphery changes over the course of training and, importantly, is a good predictor of individual differences in learning success. The core of dynamically stiff regions exhibits dense connectivity, which is consistent with notions of core-periphery organization established previously in social networks. Our results demonstrate that core-periphery organization provides an insightful way to understand how putative functional modules are linked. This, in turn, enables the prediction of fundamental human capacities, including the production of complex goal-directed behavior.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary

Prolific sheep have proven to be a valuable model to identify genes and mutations implicated in female fertility. In the Lacaune sheep breed, large variation in litter size is genetically determined by the segregation of a fecundity major gene influencing ovulation rate, named FecL and its prolific allele FecL^L. Our previous work localized FecL on sheep chromosome 11 within a locus of 1.1 Mb encompassing 20 genes. With the aim to identify the FecL gene, we developed a high throughput sequencing strategy of long-range PCR fragments spanning the locus of FecL^L carrier and non-carrier ewes. Resulting informative markers defined a new 194.6 kb minimal interval. The reduced FecL locus contained only two genes, insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2), and we identified two SNP in complete linkage disequilibrium with FecL^L. B4GALNT2 appeared as the best positional and expressional candidate for FecL, since it showed an ectopic expression in the ovarian follicles of FecL^L/FecL^L ewes at mRNA and protein levels. In FecL^L carrier ewes only, B4GALNT2 transferase activity was localized in granulosa cells and specifically glycosylated proteins were detected in granulosa cell extracts and follicular fluids. The identification of these glycoproteins by mass spectrometry revealed at least 10 proteins, including inhibin alpha and betaA subunits, as potential targets of B4GALNT2 activity. Specific ovarian protein glycosylation by B4GALNT2 is proposed as a new mechanism of ovulation rate regulation in sheep, and could contribute to open new fields of investigation to understand female infertility pathogenesis.     

A Copy Number Variant at the KITLG Locus Likely Confers Risk for Canine Squamous Cell Carcinoma of the Digit

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (P_raw = 1.60×10⁻⁷; P_genome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10⁻⁸). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.