Sirtuin 1 (Sirt1) is suppressed in non-alcoholic fatty liver disease (NAFLD), while its' stimulation or overexpression results in reduced disease severity in pre-clinical NAFLD models. Leucine allosterically activates Sirt1 and synergise with other Sirt/AMPK/NO pathway activators. We developed a triple combination of leucine, metformin and sildenafil (NS-0200), which was effective in a mouse model of non-alcoholic steatohepatitis (NASH).

Parallel corticostriatonigral circuits have been proposed that separately process motor, cognitive, and emotional-motivational information. Functional integration requires that interactions exist between neurons participating in these circuits. This makes it imperative to study the complex anatomical substrate underlying corticostriatonigral circuits. It has previously been proposed that dopaminergic neurons in the ventral mesencephalon may play a role in this circuit interaction. Therefore, we studied in rats convergence of basal ganglia circuits by depositing an anterograde neuroanatomical tracer into the ventral striatum together with a retrograde fluorescent tracer ipsilaterally in the dorsolateral striatum. In the mesencephalon, using confocal microscopy, we looked for possible appositions of anterogradely labeled fibers and retrogradely labeled neurons, "enhancing" the latter via intracellular injection of Lucifer Yellow. Tyrosine hydroxylase (TH) immunofluorescence served to identify dopaminergic neurons. In neurophysiological experiments, we combined orthodromic stimulation in the medial ventral striatum with recording from ventral mesencephalic neurons characterized by antidromic stimulation from the dorsal striatum. We observed terminal fields of anterogradely labeled fibers that overlap populations of retrogradely labeled nigrostriatal cell bodies in the substantia nigra pars compacta and lateral ventral tegmental area (VTA), with numerous close appositions between boutons of anterogradely labeled fibers and nigrostriatal, TH-immunopositive neurons. Neurophysiological stimulation in the medial ventral striatum caused inhibition of dopaminergic nigrostriatal neurons projecting to the ventrolateral striatal territory. Responding nigrostriatal neurons were located in the medial substantia nigra and adjacent VTA. Our results strongly suggest a functional link between ventromedial, emotional-motivational striatum, and the sensorimotor dorsal striatum via dopaminergic nigrostriatal neurons.

Brain metastases show a recurrence rate of about 50% after surgical resection. Adjuvant radiotherapy can prevent progression; however, whole-brain radiotherapy (WBRT) can be associated with significant side effects. Local hypofractionated stereotactic radiotherapy (HFSRT) is a good alternative to provide local control with minimal toxicity. In this multicenter analysis, we evaluated the treatment outcome of local HFSRT after resection brain metastases in 181 patients. Patient's characteristics, treatment data as well as follow-up data were collected and analyzed with special focus on local control, locoregional control and survival. After a median follow-up of 12.6 months (range 0.3-80.2 months), the crude rate for local control was 80.5%; 1- and 2-year local recurrence-free survival rates were 75% and 70% (median not reached). Resection cavity size was a significant predictor for local recurrence (P = 0.033). The median overall survival was 16.0 months. Both graded prognostic assessment score and recursive partitioning analysis were accurate predictors of survival. HFSRT leads to excellent local control and has a high potential to consolidate results after surgery; acute and late toxicity is low. Distant intracerebral metastases occur frequently during follow-up, and therefore, a close patient monitoring needs to be warranted if whole-brain radiotherapy is omitted.

Molecular imaging is advantageous for screening diseases such as breast cancer by providing precise spatial information on disease-associated biomarkers, something neither blood tests nor anatomical imaging can achieve. However, the high cost and risks of ionizing radiation for several molecular imaging modalities have prevented a feasible and scalable approach for screening. Clinical studies have demonstrated the ability to detect breast tumors using nonspecific probes such as indocyanine green, but the lack of molecular information and required intravenous contrast agent does not provide a significant benefit over current noninvasive imaging techniques. Here we demonstrate that negatively charged sulfate groups, commonly used to improve solubility of near-infrared fluorophores, enable sufficient oral absorption and targeting of fluorescent molecular imaging agents for completely noninvasive detection of diseased tissue such as breast cancer. These functional groups improve the pharmacokinetic properties of affinity ligands to achieve targeting efficiencies compatible with clinical imaging devices using safe, nonionizing radiation (near-infrared light). Together, this enables development of a "disease screening pill" capable of oral absorption and systemic availability, target binding, background clearance, and imaging at clinically relevant depths for breast cancer screening. This approach should be adaptable to other molecular targets and diseases for use as a new class of screening agents.

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5' ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that 'rewiring' of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

The Cancer Genome Atlas (TCGA) project is a public resource that provides transcriptomic, DNA sequence, methylation, and clinical data for 33 cancer types. Transforming the large size and high complexity of TCGA cancer genome data into integrated knowledge can be useful to promote cancer research. Alternative splicing (AS) is a key regulatory mechanism of genes in human cancer development and in the interaction with epigenetic factors. Therefore, AS-guided integration of existing TCGA data sets will make it easier to gain insight into the genetic architecture of cancer risk and related outcomes. There are already existing tools analyzing and visualizing alternative mRNA splicing patterns for large-scale RNA-seq experiments. However, these existing web-based tools are limited to the analysis of individual TCGA data sets at a time, such as only transcriptomic information.

Reliable detection of genome variations, especially insertions and deletions (indels), from single sample DNA sequencing data remains challenging, partially due to the inherent uncertainty involved in aligning sequencing reads to the reference genome. In practice a variety of ad hoc quality filtering methods are employed to produce more reliable lists of putative variants, but the resulting lists typically still include numerous false positives. Thus it would be desirable to be able to rigorously evaluate the degree to which each putative variant is supported by the data. Unfortunately, users who wish to do this, e.g. for the purpose of prioritizing validation experiments, have been faced with limited options.

It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects.

To compare embryo quality, fertilization, implantation, miscarriage and clinical pregnancy rates for embryos cultured in two different commercial culture media until D-2 or D-3.

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

Identifying the signals of artificial selection can contribute to further shaping economically important traits. Here, a chicken 600k SNP-array was employed to detect the signals of artificial selection using 331 individuals from 9 breeds, including Jingfen (JF), Jinghong (JH), Araucanas (AR), White Leghorn (WL), Pekin-Bantam (PB), Shamo (SH), Gallus-Gallus-Spadiceus (GA), Rheinlander (RH) and Vorwerkhuhn (VO). Per the population genetic structure, 9 breeds were combined into 5 breed-pools, and a 'two-step' strategy was used to reveal the signals of artificial selection. GA, which has little artificial selection, was defined as the reference population, and a total of 204, 155, 305 and 323 potential artificial selection signals were identified in AR_VO, PB, RH_WL and JH_JF, respectively. We also found signals derived from standing and de-novo genetic variations have contributed to adaptive evolution during artificial selection. Further enrichment analysis suggests that the genomic regions of artificial selection signals harbour genes, including THSR, PTHLH and PMCH, responsible for economic traits, such as fertility, growth and immunization. Overall, this study found a series of genes that contribute to the improvement of chicken breeds and revealed the genetic mechanisms of adaptive evolution, which can be used as fundamental information in future chicken functional genomics study.

Orientia tsutsugamushi (Ots) is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA) of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III) and three spacer conserved domains (SVDI, SVDII/III, SVDIII). The total number (152) of antigenic types (amino acid variants) varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435) of the predicted CD4 T-cell epitopes (TCEs) from all the six domains had high binding affinities (HBA) to at least one of the prevalent Indian human leukocyte antigen (HLA) alleles. A surprisingly high proportion (61%) of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT) than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.

The Onchidiidae family is ideal for studying the evolution of marine invertebrate species from sea to wetland environments. However, comparative studies of Onchidiidae species are rare. A total of 40 samples were collected from four species (10 specimens per onchidiid), and their histological and molecular differences were systematically evaluated to elucidate the morphological foundations underlying the adaptations of these species. A histological analysis was performed to compare the structures of respiratory organs (gill, lung sac, dorsal skin) among onchidiids, and transcriptome sequencing of four representative onchidiids was performed to investigate the molecular mechanisms associated with their respective habitats. Twenty-six SNP markers of Onchidium reevesii revealed some DNA polymorphisms determining visible traits. Non-muscle myosin heavy chain II (NMHC II) and myosin heavy chain (MyHC), which play essential roles in amphibian developmental processes, were found to be differentially expressed in different onchidiids and tissues. The species with higher terrestrial ability and increased integrated expression of Os-MHC (NMHC II gene) and the MyHC gene, illustrating that the expression levels of these genes were associated with the evolutionary degree. This study provides a comprehensive analysis of the adaptions of a diverse and widespread group of invertebrates, the Onchidiidae. Some onchidiids can breathe well through gills and skin when under seawater, and some can breathe well through lung sacs and skin when in wetlands. A histological comparison of respiratory organs and the relative expression levels of two genes provided insights into the adaptions of onchidiids that allowed their transition from shallow seas to wetlands. This work provides a valuable reference and might encourage further study.

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.

Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE) material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG) treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality <30. A larger baseline group of samples (n = 20) was examined to establish if there was a sample performance difference between the two DNA extraction methods, with/without UNG treatment. There was no statistical difference between sequencing performance of the two extraction methods when comparing read counts (raw, mapped, and filtered) and read quality (% mapped, % filtered). Analyzing mutation type, there was no significant difference between mutation calls until the 48 hour fixation treatment. At 48 hours there is a significant increase in C/G->T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (<2%). We do however recommend that suspected intratumoral heterogeneity events be verified by re-sequencing the same FFPE block.

The imidazole group is an ubiquitous chemical motif present in several key types of biomolecules. It is a structural moiety of purines, and plays a central role in biological catalysis as part of the side-chain of histidine, the amino acid most frequently found in the catalytic site of enzymes. Histidine biosynthesis starts with both ATP and the pentose phosphoribosyl pyrophosphate (PRPP), which is also the precursor for the de novo synthesis of purines. These two anabolic pathways are also connected by the imidazole intermediate 5-aminoimidazole-4-carboxamide ribotide (AICAR), which is synthesized in both routes but used only in purine biosynthesis. Rather surprisingly, the imidazole moieties of histidine and purines are synthesized by different, non-homologous enzymes. As discussed here, this phenomenon can be understood as a case of functional molecular convergence.

Diffusion kurtosis imaging (DKI) is a new promising MRI technique with microstructural sensitivity superior to conventional diffusion tensor (DTI) based methods. In stroke, considerable mismatch exists between the infarct lesion outline obtained from the two methods, kurtosis and diffusion tensor derived metrics. We aim to investigate if this mismatch can be examined in fixed tissue. Our investigation is based on estimates of mean diffusivity (MD) and mean (of the) kurtosis tensor (MKT) obtained using recent fast DKI methods requiring only 19 images. At 24 hours post stroke, rat brains were fixed and prepared. The infarct was clearly visible in both MD and MKT maps. The MKT lesion volume was roughly 31% larger than the MD lesion volume. Subsequent histological analysis (hematoxylin) revealed similar lesion volumes to MD. Our study shows that structural components underlying the MD/MKT mismatch can be investigated in fixed tissue and therefore allows a more direct comparison between lesion volumes from MRI and histology. Additionally, the larger MKT infarct lesion indicates that MKT do provide increased sensitivity to microstructural changes in the lesion area compared to MD.

Proposals to increase research reproducibility frequently call for focusing on effect sizes instead of p values, as well as for increasing the statistical power of experiments. However, it is unclear to what extent these two concepts are indeed taken into account in basic biomedical science. To study this in a real-case scenario, we performed a systematic review of effect sizes and statistical power in studies on learning of rodent fear conditioning, a widely used behavioral task to evaluate memory. Our search criteria yielded 410 experiments comparing control and treated groups in 122 articles. Interventions had a mean effect size of 29.5%, and amnesia caused by memory-impairing interventions was nearly always partial. Mean statistical power to detect the average effect size observed in well-powered experiments with significant differences (37.2%) was 65%, and was lower among studies with non-significant results. Only one article reported a sample size calculation, and our estimated sample size to achieve 80% power considering typical effect sizes and variances (15 animals per group) was reached in only 12.2% of experiments. Actual effect sizes correlated with effect size inferences made by readers on the basis of textual descriptions of results only when findings were non-significant, and neither effect size nor power correlated with study quality indicators, number of citations or impact factor of the publishing journal. In summary, effect sizes and statistical power have a wide distribution in the rodent fear conditioning literature, but do not seem to have a large influence on how results are described or cited. Failure to take these concepts into consideration might limit attempts to improve reproducibility in this field of science.

Human beings are exposed to compressed air or a nitrogen-oxygen mixture, they will produce signs and symptoms of nitrogen narcosis such as amnesia or even loss of memory, which may be disappeared once back to the normobaric environment. This study was designed to investigate the effect of nitrogen narcosis induced by repetitive hyperbaric nitrogen-oxygen mixture exposure on long-term cognitive function in newborn mice and the underlying mechanisms. The electroencephalogram frequency was decreased while the amplitude was increased in a pressure-dependent manner during 0.6, 1.2, 1.8 MPa (million pascal) nitrogen-oxygen mixture exposures in adult mice. Nitrogen narcosis in postnatal days 7-9 mice but not in adult mice induced by repetitive hyperbaric exposure prolonged the latency to find the platform and decreased the number of platform-site crossovers during Morris water maze tests, and reduced the time in the center during the open field tests. An increase in the expression of cleaved caspase-3 in the hippocampus and cortex were observed immediately on the first day after hyperbaric exposure, and this lasted for seven days. Additionally, nitrogen narcosis induced loss of the dendritic spines but not of the neurons, which may mainly account for the cognitive dysfunction. Nitrogen narcosis induced long-term cognitive and emotional dysfunction in the postnatal mice but not in the adult mice, which may result from neuronal apoptosis and especially reduction of dendritic spines of neurons.