Hyaluronan and Hyaluronidase, which is better for embryo development?

Our aim was to examine size-specific effects of Hyaluronan (HA) on preimplantation embryo development. We investigated the effects of Hyalovet (HA, 500–750 kDa; the size produced by HA synthase-3, which is abundant in the oviduct), or HA treated with Hyaluronidase-2 (Hyal2; also expressed in the oviduct that breaks down HA into 20 kDa fragments). In experiment 1 (in vivo), oviducts of synchronized and superovulated ewes (n = 20) were surgically exposed on Day 2 post-mating, ligated, and infused with either Hyalovet, Hyalovet + Hyal2, Hyal2, or PBS (control). Ewes were killed 5 days later for recovery of embryos and oviductal epithelial cells (OEC). Blastocyst rates were significantly higher in Hyal2 and Hyalovet + Hyal2 oviducts. Hyaluronidase-2 infusion resulted in higher blastocyst cell numbers and hatching rates. This was associated with increased HSP70 expression in OEC. In contrast, Hyalovet resulted in the lowest development to blastocyst stage and lowest hatching rates, and decreased IGF2 and IGFBP2 expression in OEC. IGF1 and IL1α expression were not affected. In experiment 2, to rule out indirect effects of oviductal factors, ovine embryos were produced and cultured with the same treatments in vitro from Day 2 to 8. Hyaluronidase-2, but not Hyalovet, enhanced blastocyst formation and reduced inner cell mass apoptosis. Hyalovet inhibited hatching. In conclusion, the presence of large-size HA (500–750 kDa) in the vicinity of developing embryos appears to disturb the oviductal environment and embryo development in vivo and in vitro. In contrast, we show evidence that breakdown of HA into smaller fragments is required to maximize embryo development and blastocyst quality

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis

BACKGROUND: Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. RESULTS: In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. CONCLUSION: Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies

New miRNA labeling method for bead-based quantification

<p>Abstract</p> <p>Background</p> <p>microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.</p> <p>Results</p> <p>Here we have applied with an innovative approach, the Luminex^®xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.</p> <p>Conclusions</p> <p>We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex^®xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.</p

High IGFBP2 expression correlates with tumor severity in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS

Analysis of a fast-locking scheme for a high performance DPLL at 10GHz with improved disturb robustness

LAUREA MAGISTRALEI Phase-Locked Loop (PLL) digitali stanno diventando sempre più attraenti nel campo della sintesi di frequenza per le comunicazioni wireless grazie ad un’occupazione di area estremamente ridotta e una bassa dissipazione di potenza, insieme ad una semplicità intrinseca di calibrazioni PVT (Process, Voltage, Temperature) e dell’implementazione di algoritmi per il miglioramento delle prestazioni. Particolare attenzione è stata posta sulla semplificazione della progettazione del rivelatore di fase per evitare l’uso di complessi time to digital converter (TDC) ad elevato consumo di potenza. è stato dimostrato con successo che i rivelatori di fase a un solo bit (bang-bang PDs) consentono un funzionamento a basso jitter per PLL ad alte prestazioni. Tuttavia, in caso di perturbazioni della frequenza di uscita, il BBPD non è in grado di fornire alcuna informazione oltre alla semplice differenza binaria di anticipo/ritardo. Perciò la dissipazione del transitorio richiederebbe un tempo non accettabile. Per mitigare queste prestazioni evitando l’utilizzo di TDC a grande range e linearità, molte tecniche di riduzione del locking time presenti in letteratura fanno uso di algoritmi alternativi di rilevamento delle differenze di frequenza e fase. I tempi dichiarati di locking time si riferiscono spesso solamente a salti di frequenza programmati, ad esempio quando il sistema di ricezione e trasmissione deve saltare da un canale di comunicazione a un altro. Non vengono quindi considerati ai fini delle prestazioni di locking le variazioni PVT e glitch di frequenza non programmati, che potrebbero portare il PLL fuori dal suo stato stazionario. Per questo motivo, il lavoro di tesi presentato investiga l’esistenza di un convertitore da tempo a digitale (TDC) statico, nonlineare e a bassa complessità che minimizzi il tempo di locking su qualsiasi perturbazione della frequenza di uscita, senza avere alcun impatto sul jitter in stato stazionario.La concezione di una natura lineare è motivata dall’esclusione di un’ampia gamma di tecniche attive di riduzione del tempo di locking nel caso venga applicato un modello lineare per la progettazione del TDC. Pertanto, verrà proposto uno schema di un TDC non lineare, che seleziona in modo ottimale la caratteristica di uscita necessaria per garantire la migliore efficienza nella dissipazione del transitorio di locking e verrà fatta una comparazione con lo stato dell’arte. La validazione dell’analisi matematica avverrà mediante l’ultilizzo di simulazioni effettuate in ambiente MATLAB.Digital Phase-Locked-Loops are becoming more attractive in the field of frequency synthesis for wireless communications. They enable extremely low area, low power consumption, robustness against technology parameter variations together with ease of implementation of PVT (Process, Voltage, Temperature) calibrations and performance enhancing algorithms. Particular focus in the design of these systems is put towards simplifying the phase detector design to avoid the use of high complexity and power hungry time to digital converters (TDC). Research has successfully demonstrated that one bit phase detectors (bang-bang PDs) enable extremely low jitter operation for high performance PLLs. However, in case of any output frequency perturbation, the BBPD is not capable of providing any information other than a simple lead-lag phase difference output and thus it will take an unacceptably long time to restore the locking condition. To mitigate the out-of-lock performance while avoiding the use of large range and linear TDCs altogether, many re-locking schemes found in literature make use of alternative frequency and phase detection algorithms. Most of the times, the claimed locking times refer to just programmed frequency jumps, e.g. when the transceiver needs to hop from a communication channel to another, while response to temporary PVT variations and unprogrammed glitches which could bring the PLL out-of-lock are seldom discussed in literature. For his reason, this thesis investigates the existence of a low-complexity, static nonlinear TDC that minimizes the lock-time on any frequency perturbation without impacting the steady-state jitter. Such nonlinear concept comes from the fact that a LTI model applied to digital PLLs limits the design to large range, high resolution TDC only, and precludes the use of a variety of active locking-time reduction techniques altogether. The designed TDC will then act on the auxiliary loops of the PLL that are used to boost the locking performance. Therefore, a highly non-linear scheme is proposed, which optimally selects the least amount of deadzones needed in order to guarantee the best out-of-lock performance and a comparison with the state-of-the-art will be performed. The mathematical analysis will be supported by simulations carried out using MATLAB