Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5^LacZ/− transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6^+/−, KRT5^LacZ/− corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6^+/− mice.     

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics.     

Crimean-Congo hemorrhagic fever virus-infected hepatocytes induce ER-stress and apoptosis crosstalk

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tick-borne member of the Nairovirus genus (Bunyaviridae) with a high mortality rate in humans. CCHFV induces a severe disease in infected patients that includes, among other symptoms, massive liver necrosis and failure. The interaction between liver cells and CCHFV is therefore important for understanding the pathogenesis of this disease. Here, we described the in vitro CCHFV-infection and -replication in the hepatocyte cell line, Huh7, and the induced cellular and molecular response modulation. We found that CCHFV was able to infect and replicate to high titres and to induce a cytopathic effect (CPE). We also observed by flow cytometry and real time quantitative RT-PCR evidence of apoptosis, with the participation of the mitochondrial pathway. On the other hand, we showed that the replication of CCHFV in hepatocytes was able to interfere with the death receptor pathway of apoptosis. Furthermore, we found in CCHFV-infected cells the over-expression of PUMA, Noxa and CHOP suggesting the crosstalk between the ER-stress and mitochondrial apoptosis. By ELISA, we observed an increase of IL-8 in response to viral replication; however apoptosis was shown to be independent from IL-8 secretion. When we compared the induced cellular response between CCHFV and DUGV, a mild or non-pathogenic Nairovirus for humans, we found that the most striking difference was the absence of CPE and apoptosis. Despite the XBP1 splicing and PERK gene expression induced by DUGV, no ER-stress and apoptosis crosstalk was observed. Overall, these results suggest that CCHFV is able to induce ER-stress, activate inflammatory mediators and modulate both mitochondrial and death receptor pathways of apoptosis in hepatocyte cells, which may, in part, explain the role of the liver in the pathogenesis of CCHFV.     

A least-squares identification algorithm for estimating squat exercise mechanics using a single inertial measurement unit

This study investigated the possibility of estimating lower-limb joint kinematics during a squat exercise performed in the sagittal plane based on data collected from a single inertial measurement unit located on the lower trunk. The human body was modeled as a three-degrees-of-freedom planar chain and the relevant joint angles (ankle, knee, and hip) are represented by Fourier series. A least-squares approach based on the minimization of the difference between the measured and estimated linear accelerations and the angular velocity of the lower trunk was used to solve the related analytical problem. The approach was validated on ten healthy young volunteers (ten trials each) using a force plate and a stereophotogrammetric system to collect reference data. The root mean square differences between the estimated joint angles and those reconstructed with the stereophotogrammetric system were lower than 4° with correlation coefficients higher than 0.99. The ankle joint resultant vertical force component was estimated with an accuracy of about 3% and a high correlation coefficient of r=0.95, whereas much lower percentage accuracies were found for the horizontal force and couple components. The latter accuracies were similar to those affecting these force and couple components as estimated through inverse dynamics and the stereophotogrammetric data in conjunction with the same mechanical model, which suggests that only minor errors were introduced by the proposed algorithm and measurement tools.     

Primary aldosteronism: the role of confirmatory tests

The high prevalence of primary aldosteronism among hypertensives justifies large scale screening by the aldosterone-renin ratio; however, this test is subject to several variables responsible for false-positive results. Functional tests to confirm autonomous aldosterone secretion are commonly used, with the fludrocortisone suppression test considered the gold standard, and saline infusion or captopril challenge, the most practical. However, each of these tests has sub-optimal sensitivity and specificity and none has been so far prospectively validated by comparing the results with the lateralization by adrenal vein sampling and the results of surgery. Their role in confirming the diagnosis of primary aldosteronism due to unilateral adenoma remains incompletely resolved.     

Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. II: population and infection models

Tick-borne encephalitis is an emerging vector-borne zoonotic disease reported in several European and Asiatic countries with complex transmission routes that involve various vertebrate host species other than a tick vector. Understanding and quantifying the contribution of the different hosts involved in the TBE virus cycle is crucial in estimating the threshold conditions for virus emergence and spread. Some hosts, such as rodents, act both as feeding hosts for ticks and reservoirs of the infection. Other species, such as deer, provide important sources of blood for feeding ticks but they do not support TBE virus transmission, acting instead as dead-end (i.e., incompetent) hosts. Here, we introduce an eco-epidemiological model to explore the dynamics of tick populations and TBE virus infection in relation to the density of two key hosts. In particular, our aim is to validate and interpret in a robust theoretical framework the empirical findings regarding the effect of deer density on tick infestation on rodents and thus TBE virus occurrence from selected European foci. Model results show hump-shaped relationships between deer density and both feeding ticks on rodents and the basic reproduction number for TBE virus. This suggests that deer may act as tick amplifiers, but may also divert tick bites from competent hosts, thus diluting pathogen transmission. However, our model shows that the mechanism responsible for the dilution effect is more complex than the simple reduction of tick burden on competent hosts. Indeed, while the number of feeding ticks on rodents may increase with deer density, the proportion of blood meals on competent compared with incompetent hosts may decrease, triggering a decline in infection. As a consequence, using simply the number of ticks per rodent as a predictor of TBE transmission potential could be misleading if competent hosts share habitats with incompetent hosts.     

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3)

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14–16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3–1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.     

Diving patterns of breeding female rockhopper penguins (<Emphasis Type="Italic">Eudyptes chrysocome)</Emphasis>: Noir Island,Chile

The diving behaviour of female southern rockhopper penguins (Eudyptes chrysocome) was studied at Noir Island (54°30′S–73°00′W), Chile, in the southeast Pacific Ocean. This isolated island is located at the edge of the continental shelf in an area where the Humboldt Current originates, and holds a population of more than 150,000 breeding pairs. On 13 December 2005, four TDRs were successfully attached to females at the end of the brooding period and recorded diving activity at intervals of 2 s over the next 4 weeks. In total, 40 complete foraging trips were recorded. Trip duration was on average 42.4 ± 40.1 h and the proportion of overnight trips (60%) was the highest value found so far for this species. Mean dive depth and dive duration was 20.6 ± 19.4 m and 63.7 ± 36.4 s, respectively. The diving effort was higher than that of brooding females from the Indian Ocean and comparable with that of conspecifics from colonies in the southwest Atlantic in terms of diving rate (38 ± 14.2 dives h⁻¹), but slightly lower as regards the proportion of time spent underwater (61 ± 10.5%). This study confirms that the diving behaviour of rockhopper penguins varies as a function of the physical and biological characteristics of the foraging areas and of the particular stage of the breeding season.