Important Target For Study Research Articles

Application of real-time PCR for the identification of the endangered species Galemys pyrenaicus through faecal samples

BackgroundCurrently, many micromammals are important targets for study. The endangered Galemys pyrenaicus is an outstanding example. Globally, their populations have suffered a substantial decline in last 20 years. In the surveyed area, the capture of desman is legally forbidden due to the high conservation concerns. Reason by non-invasive sampling through faeces is proposed for its monitoring. Furthermore, the confusion between faeces from desman and Mediterranean water shrews must be considered. Thus, the aim of this study was focused on developing RT-PCR assays to determine the presence of Galemys pyrenaicus and N. a. anomalus from non-invasive samples.Methods and resultsThe study was conducted in the mountains of the System Central of Extremadura (Spain). A total of 186 samples were collected from 2018 to 2021 by experts where historically reported and/or our previous studies confirmed their presence. RT-PCR assays using hydrolysis probes were designed to detect genetic material from both desman and Mediterranean water shrews and its specificity was confirmed. The reliability of the method was further assessed by PCR sequencing of mitochondrial Cyb and d-loop, resulting fully compatible with the RT-PCR approach. Intraspecific phylogenetic relationship was reported to improve knowledge about mtDNA variability in the desman from the Central System.ConclusionsWe demonstrated that RT-PCR gives a gold opportunity to further map the species using faeces which minimizes disturbance and reports both population status and individual presence. Cost-effective RT-PCR combined with field-collected faeces allows us to better investigate the full range of occurrence of the species.

Unraveling Impacts of Chamber-Specific Differences in Intercalated Disc Ultrastructure and Molecular Organization on Cardiac Conduction

BackgroundPropagation of action potentials through the heart coordinates the heartbeat. Thus, intercalated discs, specialized cell–cell contact sites that provide electrical and mechanical coupling between cardiomyocytes, are an important target for study. Impaired propagation leads to arrhythmias in many pathologies, where intercalated disc remodeling is a common finding, hence the importance and urgency of understanding propagation dependence on intercalated disc structure. Conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, because of lack of quantitative structural data at subcellular through nano scales. ObjectivesThis study sought to quantify intercalated disc structure at these spatial scales in the healthy adult mouse heart and relate them to chamber-specific properties of propagation as a precursor to understanding the effects of pathological intercalated disc remodeling. MethodsUsing super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. ResultsBy incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by interchamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. ConclusionsThese data provide the first stepping stone to elucidating chamber-specific effects of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.

Glutathione S-transferase Mu 3 is associated to in vivo fertility, but not sperm quality, in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.

CCR3 antagonist impairs estradiol-induced eosinophil migration to the uterus in ovariectomized mice.

Eosinophils are abundant in the reproductive tract, contributing to the remodeling and successful implantation of the embryo. However, the mechanisms by which eosinophils migrate into the uterus and their relationship to edema are still not entirely clear, since there are a variety of chemotactic factors that can cause migration of these cells. Therefore, to evaluate the role of CCR3 in eosinophil migration, ovariectomized C57BL/6 mice were treated with CCR3 antagonist SB 328437 and 17β-estradiol. The hypothesis that the CCR3 receptor plays an important role in eosinophil migration to the mouse uterus was confirmed, because we observed reduction in eosinophil peroxidase activity in these antagonist-treated uteruses. The antagonist also influenced uterine hypertrophy, inhibiting edema formation. Finally, histological analysis of the orcein-stained uteruses showed that the antagonist reduced eosinophil migration together with edema. These data showed that the CCR3 receptor is an important target for studies that seek to clarify the functions of these cells in uterine physiology.

Biochemical characterization of Plasmodiumfalciparum parasite specific helicase 1 (PfPSH1).

Malaria, a disease caused by infection with parasites of the genus Plasmodium, causes millions of deaths worldwide annually. Of the five Plasmodium species that can infect humans, Plasmodium falciparum causes the most serious parasitic infection. The emergence of drug resistance and the ineffectiveness of old therapeutic regimes against malaria mean there is an urgent need to better understand the basic biology of the malaria parasite. Previously, we have reported the presence of parasite‐specific helicases identified through genome‐wide analysis of the P. falciparum (3D7) strain. Helicases are involved in various biological pathways in addition to nucleic acid metabolism, making them an important target of study. Here, we report the detailed biochemical characterization of P. falciparum parasite‐specific helicase 1 (PfPSH1) and the effect of phosphorylation on its biochemical activities. The C‐terminal of PfPSH1 (PfPSH1C) containing all conserved domains was used for biochemical characterization. PfPSH1C exhibits DNA‐ or ribonucleic acid (RNA)‐stimulated ATPase activity, and it can unwind DNA and RNA duplex substrates. It shows bipolar directionality because it can translocate in both (3′–5′ and 5′–3′) directions. PfPSH1 is mainly localized to the cytoplasm during early stages (including ring and trophozoite stages of intraerythrocytic development), but at late stages, it is partially located in the cytoplasm. The biochemical activities of PfPSH1 are upregulated after phosphorylation with PKC. The detailed biochemical characterization of PfPSH1 will help us understand its functional role in the parasite and pave the way for future studies.

MA14.02 Evaluation of PD1/PDL1 Expression on Peripheral Blood Cells Subpopulations in Patients with Non-Small Cell Lung Cancer

Currently the immune system is considered an important target of study within the therapeutic alternatives for many tumors that have developed resistance in lung cancer. Many molecules called checkpoints regulate antitumor immunity as PD-L1 it is expressed in tumor cells and is a biomarker for anti PD-L1/PD-1 therapy. PD-1/PD-L1 is expressed on exhausted activated T cells. This signaling pathway is involved in tumor evasion of the immune system. It has recently been demonstrated that the blockade of PD-1 or its ligand PD-L1 and PD-L2, restore the antitumor immune response leading to a durable tumor regression. However, the expression of PD-1/PD-L1 in T cells from peripheral blood of patients with non-small cell lung cancer has not been widely studied. We investigated the expression of PD-1 and its ligands PD-L1 and PD-L2 on peripheral blood T cells subpopulations (CD3+ CD4+ / CD8+) of patients with non-small cell lung cancer. We included 50 NSCLC patients (stage IIIB and IV) naive to treatment and 10 healthy subjects. Immunophenotyping was performed using multiparametric flow cytometry. Analyzing its prognostic significance regarding outcome analysis as well as its potential biomarker. Our results showed that the percentage of PD-1, PD-L1 and PD-L2 expression in peripheral blood cells in NSCLC patients was lower compared to healthy subjects [P<0.005] and the Mean Fluorescence Intensity (MFI) was higher in patients compared to the control group [P<0.001]; The expression of PD-1 in T-helper or CD4+ of NSCLC patients was significantly higher than in cells from control subjects [P<0.001]. Similarly, the expression of PD-1 in T cytotoxic cells or CD8+ patients was significantly higher than in controls [P<0.001]. In the clinical analysis, we found that a higher percentage of PD-1+ CD3+ cells was statistically associated with tobacco exposure [P=0.0160], and de MFI was associated with the non-adenocarcinoma histology [P=0.0001] additionally, the presence of 3 or more metastases was associated to a higher MFI of PD-1 on CD3+ CD8+ [P=0.0490]. In the overall survival (OS) analysis the percentage of CD3+/CD4+/PD-1+ ≤20.91 was associated with a higher median OS [P= 0.045]. Several studies demonstrate the importance of infiltrating PD-1+ T cells within tumors; however these results showed that the PD-1/PD-L1/PDL-2 expression in peripheral blood cells could be used also as a potential biomarkers in NSCLC patients.

The P5 disulfide switch: taming the aging unfolded protein response.

Aging cells are characterized by a loss of proteostasis and a decreased ability to survive under environmental stress. Regulation of the UPR in aging cells has been under much scrutiny, and studies have shown that the UPR in these cells differs considerably from younger cells with regard to the induction of apoptosis and chaperone activity. The role of IRE-1 and PERK in UPR-associated apoptosis makes the regulation of these signaling cascades an important target of study. The seemingly contradictory findings regarding the role of P5 in activating and deactivating these responses warrant further investigation and may hold the key to unlocking the role of this protein in various pathological conditions. Another important target for study with regard to P5 is the effects of the localization of this protein in the mitochondria and the consequences, if any, of these effects on the activation of the UPR.

The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses

16S ribosomal RNA currently represents the most important target of study in bacterial ecology. Its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. Here we use the information from sequenced bacterial genomes to explore the variability of 16S rRNA sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome and DNA abundances. In total, 7,081 16S rRNA sequences were in silico extracted from 1,690 available bacterial genomes (1–15 per genome). While there are several phyla containing low 16S rRNA copy numbers, in certain taxa, e.g., the Firmicutes and Gammaproteobacteria, the variation is large. Genome sizes are more conserved at all tested taxonomic levels than 16S rRNA copy numbers. Only a minority of bacterial genomes harbors identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers. While certain taxa harbor dissimilar 16S rRNA genes, others contain sequences common to multiple species. Sequence identity clusters (often termed operational taxonomic units) thus provide an imperfect representation of bacterial taxa of a certain phylogenetic rank. We have demonstrated that the information on 16S rRNA copy numbers and genome sizes of genome-sequenced bacteria may be used as an estimate for the closest related taxon in an environmental dataset to calculate alternative estimates of the relative abundance of individual bacterial taxa in environmental samples. Using an example from forest soil, this procedure would increase the abundance estimates of Acidobacteria and decrease these of Firmicutes. Using the currently available information, alternative estimates of bacterial community composition may be obtained in this way if the variation of 16S rRNA copy numbers among bacteria is considered.

Probing circulating tumor cells in microfluidics.

Circulating tumor cells (CTCs) are important targets for study as we strive to better understand, diagnose, and treat cancers. However, CTCs are found in blood at extremely low concentrations; this makes isolation, enrichment, and characterization of CTCs technically challenging. Recently, the development of CTC separation devices has grown rapidly in both academia and industry. Part of this development effort centered on microfluidic platforms, exploiting the advantages of microfluidics to improve CTC separation performance and device integration. In this Focus article, we highlight some of the recent work in microfluidic CTC separation and detection systems and discuss our appraisal of what the field should do next.

Abstract 4570: Functional analysis of gene expression profiling of primary and metastatic melanoma: Possible implications from BRAF mutant and wild-type cell lines

Abstract Metastatic melanoma is largely refractory to existing therapies and has a very poor prognosis. Despite the recent development of new therapies, the median survival rate is only 6-9 months and 5-year survival is less than 5%. Better understanding of melanoma biology, specifically the gene expression profile between primary and metastatic melanoma, may provide useful information for developing new therapeutic approaches to this disease. The gene expression profile from 89 primary and metastatic melanoma tissues was investigated by cDNA microarray. 8261 differentially expressed genes were identified between primary and metastatic melanoma at a statistical level p &amp;lt;0.01 and false discovery rate (FDR) of 5%. Panther Pathway Classification showed that the 8261 differentially expressed genes were enriched with genes which were involved in cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathways, Notch signalling pathways, p53 and angiogenesis pathways. The primary melanoma microarrays were analyzed according to the thickness of tumour. Group A: the thickness of a tumour less than 2.0 mm, 20 microarrays. Group B: the thickness of a tumour was equal or more than 2.0 mm, 11 microarrays. 981 genes were identified as differentially expressed between Group A and Group B at statistical level p&amp;lt;0.01 and FDR=5%. 657 out of 981 differentially expressed genes were shared with the 8261 differentially expressed genes. We believed these 657 genes were related to malignant potential of melanoma; they were enriched with genes which were involved in apoptosis, cell adhesion, cell cycle regulation and immune system processing. 218 out of the 657 genes overlapped with a published gene list (308 genes) which have been implicated in melanoma metastases. We further evaluated the expression of the transcription factor gene: ETV1. Ets variant 1 (ETV1) gene expression was significantly increased in metastatic melanoma and in primary melanoma with tumour thickness equal or more than 2.0 mm. We investigated the ETV1 expression in human melanoma cell lines. ETV1 mRNA level in 7 BRAF inhibitor sensitive cell lines was significantly lower than that in 6 BRAF inhibitor resistant cell lines (0.230±0.057 vs 0.927±0.6, p&amp;lt;0.0001). The ETV1 expression in BRAF-induced resistant melanoma cell lines was significantly (p&amp;lt;0.05) higher than in their parental cell lines. While a variety of genes are differentially expressed in primary and metastatic melanoma, the transcription factor–ETV1 gene may be an important target for study and as a therapeutic target as its expression may be associated with BRAF inhibitor resistance in melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4570. doi:1538-7445.AM2012-4570

Properties of gamma frequency oscillatory activity induced in hippocampal slices from the adult GAD67-GFP (Δneo) mouse

The green fluorescent protein (GFP)-linked expression of protein in transgenic mice provides an ideal tool for the correlation of structure and function in the CNS. An important target of study is the role of GABAergic neurons in oscillatory activity in the hippocampus, and this would be facilitated with transgenic mice in which GFP is linked to the expression of GABA markers. One such mouse is the GAD67-GFP (Δneo), and here we compare the properties of kainate- and carbachol-induced oscillatory activity generated in CA3 of hippocampal slices from heterozygous GAD67-GFP (Δneo) mice and wild type litter mates. For both paradigms and in both mouse preparations oscillations were generated in the 20–30 Hz range, and for the kainate-, but not the carbachol-induced oscillations, there was a small but significant difference in peak frequency of the oscillations between GAD67-GFP (Δneo) mice (28.4 ± 2.2 Hz) and wild type mice (25 ± 1.6 Hz). For both oscillatory paradigms there was no significant difference between mouse strains in area power of the oscillatory activity in the stratum oriens lamina of CA3, but for the kainate-induced oscillations, area power became significantly diminished in the stratum radiatum lamina of the GAD67-GFP (Δneo) mouse compared with the wild type mouse after prolonged exposure to kainate. This gradual reduction in area power in CA3 of the transgenic mouse was rescued by inclusion of Guvacine, a GABA uptake inhibitor, suggesting that the reported lower levels of GABA in the GAD67-GFP (Δneo) mouse brain during development and in the adult may contribute to a reduction in the efficiency of GABA neurotransmission after prolonged stimulation of the GABAergic circuitry.

Nine Things You Might Not Say or Hear in Transplantation

As an exercise, the editorial board of the AJT put together a long list of important, hot or cutting edge areas in transplantation research. As we examined the topics, categorized them and played with their relative merits, it became apparent that this process of analyzing science and research sometimes disguises some uncomfortable truths and realities. In the spirit of the late George Carlin and his ‘seven words you can never say on television’, we now offer some uncomfortable conclusions about the current state of transplantation. These topics represent our personal favorite bete noires among a large list of many important issues, some of which truly cannot be said or heard.

Gamblers Anonymous: A critical review of the literature

This study surveys existing literature on Gamblers Anonymous (GA) and issues that help to contextualise our understanding of this mutual aid association. While GA has been the subject of investigation by social scientists, it is still understudied, with a notable shortage of research on issues facing women and ethnic minorities. A need exists for large-scale assessments of GA's effectiveness, more detailed accounts of GA beliefs and practices, increased knowledge of the ways in which GA attendance interacts with both formal treatment and attendance at other mutual aid organisations, and a better understanding of the profiles of gamblers best (and least) suited to GA, along with a clearer grasp of what GA was able to offer those gamblers that it seems to have helped. This assessment of the current state of knowledge underscores the embryonic state of our collective inquiry into the nature of GA, and the authors emphasise that significant advances have been made. Notably, important targets for study are being identified.

Cholangiocyte biology

Cholangiocytes are of considerable intrinsic biologic interest, particularly with regard to their roles in the transport of water, ions, and solutes, and to their heterogeneity and proliferative capacity. Cholangiocytes represent an important target of study in the cholangiopathies, a group of genetic developmental and acquired diseases of the liver. New biologic concepts continue to evolve through the use of experimental models (eg, knockout mice and selective gene silencing) and enhanced approaches to three-dimensional modeling and microscopy. The role of the cholangiocyte cytoskeleton in transport and intracellular trafficking has been recently recognized. These paradigms provide a framework for further understanding the mechanisms modulating normal cholangiocyte growth, transport, and signaling, and the abnormalities that result in disease.