MicroRNAs often occur in families whose members share an identical 5' terminal 'seed' sequence. The seed is a major determinant of miRNA activity, and family members are thought to act redundantly on target mRNAs with perfect seed matches, i.e. sequences complementary to the seed. However, recently sequences outside the seed were reported to promote silencing by individual miRNA family members. Here, we examine this concept and the importance of miRNA specificity for the robustness of developmental gene control. Using the let-7 miRNA family in Caenorhabditis elegans, we find that seed match imperfections can increase specificity by requiring extensive pairing outside the miRNA seed region for efficient silencing and that such specificity is needed for faithful worm development. In addition, for some target site architectures, elevated miRNA levels can compensate for a lack of complementarity outside the seed. Thus, some target sites require higher miRNA concentration for silencing than others, contrasting with a traditional binary distinction between functional and non-functional sites. We conclude that changing miRNA concentrations can alter cellular miRNA target repertoires. This diversifies possible biological outcomes of miRNA-mediated gene regulation and stresses the importance of target validation under physiological conditions to understand miRNA functions in vivo.

The "free radical theory of aging" suggests that reactive oxygen species (ROS) are responsible for age-related loss of cellular functions and, therefore, represent the main cause of aging. Redox regulation by thioredoxin-1 (TRX) plays a crucial role in responses to oxidative stress. We show that thioredoxin-interacting protein (TXNIP), a negative regulator of TRX, plays a major role in maintaining the redox status and, thereby, influences aging processes. This role of TXNIP is conserved from flies to humans. Age-dependent upregulation of TXNIP results in decreased stress resistance to oxidative challenge in primary human cells and in Drosophila. Experimental overexpression of TXNIP in flies shortens lifespan due to elevated oxidative DNA damage, whereas downregulation of TXNIP enhances oxidative stress resistance and extends lifespan.

The polar transport of auxin controls many aspects of plant development. However, the molecular mechanisms underlying auxin tranport regulation remain to be further elucidated. We identified a mutant named as usl1 (unflattened and small leaves) in a genetic screen in Arabidopsis thaliana. The usl1 displayed multiple aspects of developmental defects in leaves, embryogenesis, cotyledons, silique phyllotaxy and lateral roots in addition to abnormal leaves. USL1 encodes a protein orthologous to the yeast vacuolar protein sorting (Vps) 38p and human UV RADIATION RESISTANCE-ASSOCIATED GENE (UVRAG). Cell biology, Co-IP/MS and yeast two-hybrid were used to identify the function of USL1. USL1 colocalizes at the subcellular level with VPS29, a key factor of the retromer complex that controls auxin transport. The morphology of the VPS29-associated late endosomes (LE) is altered from small dots in the wild-type to aberrant enlarged circles in the usl1 mutants. The usl1 mutant synergistically interacts with vps29. We also found that USL1 forms a complex with AtVPS30 and AtVPS34. We propose that USL1 controls multiple aspects of plant development by affecting late endosome morphology and by regulating the PIN1 polarity. Our findings provide a new layer of the understanding on the mechanisms of plant development regulation.

To investigate the effects of sodium channel-blocking antiepileptic drugs (AEDs) on functional magnetic resonance imaging (fMRI) language network activations in patients with focal epilepsy.

BACKGROUND The aim of this retrospective study was to investigate the frequency of admissions, reason for admissions, and test socket satisfaction in patients who received a lower-limb prosthesis with or without a test socket in our unit. MATERIAL AND METHODS A total of 88 patients (54 men, 34 women) were included in the study. Patients were divided into 2 groups: the group with test socket (Group I, 44 patients) and the group without test socket (Group II, 44 patients). Variables related to the functional status, frequency of complaints, and test socket satisfaction were investigated in the 2 groups. The Trinity Amputation and Prosthesis Experience Scales (TAPES) and Beck Depression Inventory (BDI) were used to assess the level of patient satisfaction with their prosthesis. The VAS (Visual Analogue Scale) was used to assess pain at rest and during walking. RESULTS We found that the TAPES values were more significant in Group 1 in both transtibial and transfemoral amputations (P<0.05). However, prosthesis delivery time was more significant in Group 2 in both transtibial and transfemoral amputations (P<0.001) whereas the frequency of admissions within 3 months was more significant in Group 1 in both transtibial and transfemoral amputations (P<0.001). There was no statistically significant difference between the 2 groups in terms of other parameters (P>0.05). CONCLUSIONS Although the use of a test socket increases the cost of prosthesis units, we showed that patients with transtibial and transfemoral amputations have fewer complaints related to prosthesis increases patient functionality, and that it reduces pain and increases patient satisfaction with the prosthesis.

The thymectomy specimens from the "thymectomy trial in non-thymomatous myasthenia gravis patients receiving prednisone therapy" (MGTX) underwent rigid and comprehensive work-up, which permits analysis of the spatial distribution of histological and immunohistological features. This analysis revealed strong intra- and inter-case variability. While many histological features (e.g. median percent fat content among different specimens) can easily be correlated with clinical parameters, intra-case spatial variability of histological features has yet defied quantification and statistical evaluation. To overcome this gap in digital pathology, we here propose intra-case entropy of measured histological features in all available slides of a given thymectomy specimen as a quantitative marker of spatial histological heterogeneity. Calculation of entropy led to one value per specimen and histological feature. Through these 'entropy values' the so far neglected degree of spatial histological heterogeneity could be fed into statistical analyses, extending the scope of clinico-pathological correlations.

Chagas disease (CD) is a major public health concern in Latin America and a potentially serious emerging threat in non-endemic countries. Although the association between CD and cardiac abnormalities is widely reported, study design diversity, sample size and quality challenge the information, calling for its update and synthesis, which would be very useful and relevant for physicians in non-endemic countries where health care implications of CD are real and neglected. We performed to systematically review and meta-analyze population-based studies that compared prevalence of overall and specific ECG abnormalities between CD and non-CD participants in the general population.

Aggregation during diapause is a common phenomenon in arthropods that nevertheless remains poorly understood. The most commonly claimed benefit is that survival is higher in aggregations but animal aggregations could also be driven by sexual selection. In this perspective, we investigated whether aggregations in insects could be part of their mating system. We studied the overwintering aggregations of the ladybird Hippodamia undecimnotata (Schneider), an aphidophagous species from Southern and Eastern Europe as well as Asia. We collected ladybirds at three aggregation sites in Southwest France, during two overwintering periods (2013-2014 and 2014-2015). We checked their reproductive status by counting the viable sperm cells in the sperm storage organs of both males and females, and by assessing the ovarian status of females. We also investigated if mating behaviour occurred in these aggregations. We found that males have a high quantity of viable sperm cells (70-95%) in their reproductive organs throughout the overwintering periods. In contrast, although most females (85-95%) had empty spermatheca at the onset of the aggregations in autumn, the majority (65-91%) had numerous viable sperm in their spermatheca at the time of dispersal from the aggregation in early spring. Furthermore, frequent copulations were observed towards the end of the overwintering period, few weeks before dispersal. These results suggest that finding sexual mates may have been involved in overwintering aggregations in H. undecimnotata.

Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) and region (dorsal and ventral), (iii) distance from the geometric center, and (iv) follicular class. Ovaries from young and old mares (n = 8) were harvested in a slaughterhouse and submitted to histological processing for further evaluation. For data analyses, a novel methodology was developed according to the geometric center of each histological section for a precise determination of preantral follicle distribution. Results indicated that (i) equine preantral follicles are clustered and located near to the ovarian geometric center, and that aging induced their dispersion through the ovarian cortex; (ii) the distance from the geometric center was shorter for developing follicles than primordial; and (iii) secondary follicles were more distant from the geometric center but closer to the ovulation fossa. In conclusion, the spatial distribution of preantral follicles was successfully determined in the equine ovary and was affected by age, region, and portion.

Parasites of fishes have been shown to be effective bioindicators of the aquatic environment. Few investigations have been conducted on ectoparasite models and therefore little is known about the fate of trace elements and metals which they accumulate. In this study trace element sequestration was observed in the carapace of the fish louse, Argulus japonicus and found to relate to the sex of the parasite, as well as, the degree of sclerotization of the carapace. Adults of A. japonicus were collected from cyprinid hosts in the Vaal Dam, South Africa. Parasites were removed and flash frozen in liquid nitrogen before being sectioned with a cryomicrotome. Sections and whole mounts of parasites were prepared and treated with Phen-Green TM FL cell-permeant diacetate. Cryosections were assessed for trace elements and metals using a scanning electron microscope equipped with energy dispersive spectroscopy. Results indicated that in both male and female parasites, trace elements become bound to the carapace and produce more intense fluorescence than in soft tissues. Sexual dimorphic differences were further observed between male and female parasites. The intensity of the fluorescence signals was greater in the carapace of male parasites than in females, particularly when comparing the carapace of the ventral side of the thorax. In females, an amorphous layer of material surrounding the eggs was observed and produced an intense fluorescent signal. Levels of trace elements and metals detected were not significantly different between male and female parasites. Results observed serve as a demonstration for the first time of trace element sequestration in a freshwater crustacean parasite and possible mechanisms employed to reduce body burdens of trace elements and metals.

Genetic improvement for stress tolerance requires a solid understanding of biochemical processes involved with different physiological mechanisms and their relationships with different traits. The objective of this study was to demonstrate genetic variability in altered metabolic levels in a panel of six wheat genotypes in contrasting temperature regimes, and to quantify the correlation between those metabolites with different traits. In a controlled environment experiment, heat stress (35:28 ± 0.08°C) was initiated 10 days after anthesis. Flag leaves were collected 10 days after heat treatment to employ an untargeted metabolomics profiling using LC-HRMS based technique called IROA. High temperature stress produced significant genetic variations for cell and thylakoid membrane damage, and yield related traits. 64 known metabolites accumulated 1.5 fold of higher or lower due to high temperature stress. In general, metabolites that increased the most under heat stress (L-tryptophan, pipecolate) showed negative correlation with different traits. Contrary, the metabolites that decreased the most under heat stress (drummondol, anthranilate) showed positive correlation with the traits. Aminoacyl-tRNA biosysnthesis and plant secondary metabolite biosynthesis pathways were most impacted by high temperature stress. The robustness of metabolic change and their relationship with phenotypes renders those metabolites as potential bio-markers for genetic improvement.

Hepatic irradiation for the treatment of hepatobiliary malignancies often indirectly damages liver tissue and promotes the development of liver fibrosis. However, little is known concerning the effects of hepatic irradiation on the liver immune system, including natural killer (NK) cells. The aim of this study was therefore to investigate how hepatic irradiation influences the functions and characteristics of liver resident NK cells. An established murine hepatic irradiation model was used to examine the specific effects of hepatic irradiation on immune cell populations and metastasis. This analysis demonstrated that hepatic irradiation decreased the number of liver resident NK cells (DX5-TRAIL+), but did not affect the total NK number or proportions of NK cells in the liver or spleen. This effect was correlated with the hepatic irradiation dose. Surprisingly, the liver resident NK population had not recovered by two months after hepatic irradiation. We also found that hepatic irradiation limited the cytotoxic effects of liver-derived lymphocytes against a mouse hepatoma cell line and promoted hepatic metastases in an in vivo model, although adoptive transfer of activated NK cells could alleviate metastatic growth. Finally, we demonstrated that hepatic irradiation disrupted the development of liver-resident NK cells, even after the adoptive transfer of precursor cells from the bone marrow, liver, and spleen, suggesting that irradiation had altered the developmental environment of the liver. In summary, our data demonstrated that hepatic irradiation abolished the DX5-TRAIL+ liver-resident NK cell population and dampened antitumor activities in the liver for at least two months. Additionally, hepatic irradiation prevented differentiation of precursor cells into liver-resident NK cells.

Recent studies have highlighted the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior. Herein we report preclinical studies designed to characterize the effects of a novel JAK/STAT inhibitor on plantar flexor skeletal muscle function, morphology, and satellite cell content.

Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects in vivo and in vitro, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS). Particularly, high-resolution MS has identified the rBuLIF with 73% of sequence coverage with highest confidence parameters and with the search engine score of 4580. We determined the molecular weight of rBuLIF protein to be 58.99 kDa and 48.9 kDa with and without glycosylation, respectively. Moreover, the purified rBuLIF was verified to be functionally active by measuring the growth inhibition of M1 myeloid leukemia cells, revealing a maximum inhibition at 72 hours and half-maximal effective concentration (EC50) of 0.0555 ng/ml, corresponding to a specific activity of >1.6×10(7) units/mg. Next, we evaluated the effect of rBuLIF on buffalo mammary epithelial cell lines for its role in involution and also identified the IC50 value for BuMEC migrating cells to be 77.8 ng/ml. Additionally, the treatment of MECs (BuMEC and EpH4) displayed significant (P < 0.05) reduction in growth progression, as confirmed by qRT-PCR analysis, suggesting its strong involvement in the involution of the mammary gland in vivo. Thus, we conclude that the glycosylated rBuLIF, purified from COS-1 cells was found to be functionally active as its natural counterpart.

The translocator protein (TSPO) is an 18 kDa polytopic membrane protein of the outer mitochondrial membrane, abundantly present in the steroid-synthesising cells. TSPO has been linked to a number of disorders, and it is recognised as a promising drug target with a range of potential medical applications. Structural and biochemical characterisation of a mammalian TSPO requires expression and purification of the protein of high quality in sufficiently large quantities. Here we describe detailed procedures for heterologous expression and purification of mammalian TSPO in HEK293 cells. We demonstrate that the established procedures can be used for untagged TSPO as well as for C-terminally fused TSPO constructs. Our protocol can be routinely used to generate high-quality TSPO preparations for biochemical and structural studies.

Several studies have shown an increased accumulation of terminally differentiated T cells during HIV infection, suggestive of exhaustion/senescence, causing dysregulation of T cell homeostasis and function and rapid HIV disease progression. We have investigated whether long-term antiretroviral therapy (ART), which controls viremia and restores CD4 T cell counts, is correlated with reduction in terminally differentiated T cells, improved ratios of naïve to memory and function of T cells in 100 virologically controlled HIV-infected patients. We show that while the median frequencies of terminally differentiated CD4+ and CD8+ T cells (CD28-, CD27-, CD57+ and CD28-CD57+), were higher in the virologically controlled HIV-infected patients' cohort compared with uninfected individuals' cohort, the frequencies of these cells significantly decreased with increasing CD4 T cell counts in HIV-infected patients. Although, the naïve CD4+ and CD8+ T cells were lower in HIV patients' cohort than uninfected cohort, there was a significant increase in both naïve CD4+ and CD8+ T cells with increasing CD4 T cell counts in HIV-infected patients. The underlying mechanism behind this increased naïve CD4+ and CD8+ T cells in HIV-infected patients was due to an increase in recent thymic emigrants, CD4+CD31+, as compared to CD4+CD31-. The CD4+ T cells of HIV-infected patients produced cytokines, including IL-2, IL-10 and IFN-γ comparable to uninfected individuals. In conclusion, virologically controlled HIV-infected patients on long-term ART show a significant reduction in terminally differentiated T cells, suggestive of decreased exhaustion/senescence, and improvement in the ratios of naïve to memory and function of T cells.

1-Deoxynojirimycin (DNJ) is a potent α-glucosidase inhibitor and thus beneficial for prevention of diabetes. While we have succeeded in obtaining the culture supernatant extract (CSE) rich in DNJ from microorganism source, information regarding its anti-hyperglycemic effect and safety were still limited. Therefore, this study was aimed to evaluate the anti-hyperglycemic effect and safety of microorganism DNJ. Oral sucrose tolerance test was performed, and the result showed that CSE was able to significantly suppress the blood glucose elevation and suggested DNJ as the main active compound. To determine its safety, the absorption and excretion of microorganism DNJ were evaluated using 15N labeling method. Our findings investigated the recovery rate of 15N from DNJ reached 80% up to 48 hours after oral administration, suggesting its rapid excretion, suggesting the safety of DNJ. This study verified the functional properties and safety of DNJ from microorganisms, suggesting its potential use for functional purpose.

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between Phytophthora species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were β-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA+ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.

Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host's acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. Lactobacillus paracasei MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction in vivo. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA+ B cells and Tfh cells in Peyer's patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.

Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin.