Development and Evaluation of a Connective Tissue Phantom Model for Subsurface Visualization of Cancers Requiring Wide Local Excision

Wide local excision (WLE) of tumors with negative margins remains a challenge because surgeons cannot directly visualize the mass. Fluorescence-guided surgery (FGS) may improve surgical accuracy; however, conventional methods with direct surface tumor visualization are not immediately applicable, and properties of tissues surrounding the cancer must be considered. We developed a phantom model for sarcoma resection with the near-infrared fluorophore IRDye 800CW and used it to iteratively define the properties of connective tissues that typically surround sarcoma tumors. We then tested the ability of a blinded surgeon to resect fluorescent tumor-simulating inclusions with ∼1-cm margins using predetermined target fluorescence intensities and a Solaris open-air fluorescence imaging system. In connective tissue-simulating phantoms, fluorescence intensity decreased with increasing blood concentration and increased with increasing intralipid concentrations. Fluorescent inclusions could be resolved at ≥1-cm depth in all inclusion concentrations and sizes tested. When inclusion depth was held constant, fluorescence intensity decreased with decreasing volume. Using targeted fluorescence intensities, a blinded surgeon was able to successfully excise inclusions with ∼1-cm margins from fat- and muscle-simulating phantoms with inclusion-to-background contrast ratios as low as 2∶1. Indirect, subsurface FGS is a promising tool for surgical resection of cancers requiring WLE

Employing machine learning for reliable miRNA target identification in plants

<p>Abstract</p> <p>Background</p> <p>miRNAs are ~21 nucleotide long small noncoding RNA molecules, formed endogenously in most of the eukaryotes, which mainly control their target genes post transcriptionally by interacting and silencing them. While a lot of tools has been developed for animal miRNA target system, plant miRNA target identification system has witnessed limited development. Most of them have been centered around exact complementarity match. Very few of them considered other factors like multiple target sites and role of flanking regions.</p> <p>Result</p> <p>In the present work, a Support Vector Regression (SVR) approach has been implemented for plant miRNA target identification, utilizing position specific dinucleotide density variation information around the target sites, to yield highly reliable result. It has been named as p-TAREF (plant-Target Refiner). Performance comparison for p-TAREF was done with other prediction tools for plants with utmost rigor and where p-TAREF was found better performing in several aspects. Further, p-TAREF was run over the experimentally validated miRNA targets from species like <it>Arabidopsis</it>, <it>Medicago</it>, Rice and Tomato, and detected them accurately, suggesting gross usability of p-TAREF for plant species. Using p-TAREF, target identification was done for the complete Rice transcriptome, supported by expression and degradome based data. miR156 was found as an important component of the Rice regulatory system, where control of genes associated with growth and transcription looked predominant. The entire methodology has been implemented in a multi-threaded parallel architecture in Java, to enable fast processing for web-server version as well as standalone version. This also makes it to run even on a simple desktop computer in concurrent mode. It also provides a facility to gather experimental support for predictions made, through on the spot expression data analysis, in its web-server version.</p> <p>Conclusion</p> <p>A machine learning multivariate feature tool has been implemented in parallel and locally installable form, for plant miRNA target identification. The performance was assessed and compared through comprehensive testing and benchmarking, suggesting a reliable performance and gross usability for transcriptome wide plant miRNA target identification.</p

Genome sequencing and molecular characterisation of Staphylococcus aureus ST772-MRSA-V, “Bengal Bay Clone”

Background: The PVL-positive ST772-MRSA-V is an emerging community-associated (CA-) MRSA clone that has been named Bengal Bay Clone since most patients have epidemiological connections to the Indian subcontinent. It is found increasingly common in other areas of the world. Methods: One isolate of ST772-MRSA-V was sequenced using the Illumina Genome Analyzer System. After initial assembling the multiple sequence contigs were analysed using different in-house annotation scripts. Results were compared to microarray hybridisation results of clinical isolates of ST772-MRSA-V, of related strains and to another ST772-MRSA-V genome sequence. Results: According to MLST e-burst analysis, ST772-MRSA-V belongs to Clonal Complex (CC)1, differing from ST1 only in one MLST allele (pta-22). However, there are several additional differences including agr alleles (group II rather than III), capsule type (5 rather than 8), the presence of the egc enterotoxin gene cluster and of the enterotoxin homologue ORF CM14 as well as the absence of the enterotoxin H gene seh. Enterotoxin genes sec and sel are present. ST772-MRSA-V harbours the genes encoding enterotoxin A (sea) and PVL (lukS/F-PV). Both are located on the same prophage. Conclusions: ST772-MRSA-V may have emerged from the same lineage as globally spread CC1 and CC5 strains. It has acquired a variety of virulence factors, and for a CA-MRSA strain it has an unusually high number of genes associated with antibiotic resistance

MiR-RACE, a New Efficient Approach to Determine the Precise Sequences of Computationally Identified Trifoliate Orange (Poncirus trifoliata) MicroRNAs

BACKGROUND: Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. METHODS: In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5' and 3' miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5' RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. RESULTS: The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. CONCLUSION: The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor

Impairment of the mitochondrial one-carbon metabolism enzyme SHMT2 causes a novel brain and heart developmental syndrome

Inborn errors of metabolism cause a wide spectrum of neurodevelopmental and neurodegenerative conditions [15]. A pivotal enzyme located at the intersection of the amino acid and folic acid metabolic pathways is SHMT2, the mitochondrial form of serine hydroxymethyltransferase. SHMT2 performs the first step in a series of reactions that provide one-carbon units covalently bound to folate species in mitochondria: it transfers one-carbon units from serine to tetrahydrofolate (THF), generating glycine and 5,10-methylene-THF. Using whole exome sequencing (WES), we identified biallelic SHMT2 variants in five individuals from four different families. All identified variants were located in conserved residues, either absent or extremely rare in control databases (gnomAD, ExAC), and cosegregated based on a recessive mode of inheritance (pRec = 0.9918 for this gene). In family F1, a homozygous missense variant present in two affected siblings was located in a region without heterozygosity (~ 10 Mb, the only region > 1 Mb shared by both siblings) in which no other candidate variants were found, providing a strong genetic evidence of causality for these variants. The missense/in-frame deletion nature of these variants, and the absence of loss-of-function homozygous individuals in control databases, combined with the fact that complete loss of SHMT2 is embryonic lethal in the mouse, suggested that these variants may cause hypomorphic effects. Using 3D molecular dynamics models of the SHMT2 protein, we concluded that these candidate variants probably alter the SHMT2 oligomerization process, and/or disrupt the conformation of the active site, thus inducing deleterious effects on SHMT2 enzymatic function

Ketamine induces a robust whole-brain connectivity pattern that can be differentially modulated by drugs of different mechanism and clinical profile

Ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, has been studied in relation to the glutamate hypothesis of schizophrenia and increases dissociation, positive and negative symptom ratings. Ketamine effects brain function through changes in brain activity; these activity patterns can be modulated by pre-treatment of compounds known to attenuate the effects of ketamine on glutamate release. Ketamine also has marked effects on brain connectivity; we predicted that these changes would also be modulated by compounds known to attenuate glutamate release. Here, we perform task-free pharmacological magnetic resonance imaging (phMRI) to investigate the functional connectivity effects of ketamine in the brain and the potential modulation of these effects by pre-treatment of the compounds lamotrigine and risperidone, compounds hypothesised to differentially modulate glutamate release. Connectivity patterns were assessed by combining windowing, graph theory and multivariate Gaussian process classification. We demonstrate that ketamine has a robust effect on the functional connectivity of the human brain compared to saline (87.5 % accuracy). Ketamine produced a shift from a cortically centred, to a subcortically centred pattern of connections. This effect is strongly modulated by pre-treatment with risperidone (81.25 %) but not lamotrigine (43.75 %). Based on the differential effect of these compounds on ketamine response, we suggest the observed connectivity effects are primarily due to NMDAR blockade rather than downstream glutamatergic effects. The connectivity changes contrast with amplitude of response for which no differential effect between pre-treatments was detected, highlighting the necessity of these techniques in forming an informed view of the mechanistic effects of pharmacological compounds in the human brain

Identification of CRISPR and riboswitch related RNAs among novel noncoding RNAs of the euryarchaeon Pyrococcus abyssi

<p>Abstract</p> <p>Background</p> <p>Noncoding RNA (ncRNA) has been recognized as an important regulator of gene expression networks in Bacteria and Eucaryota. Little is known about ncRNA in thermococcal archaea except for the eukaryotic-like C/D and H/ACA modification guide RNAs.</p> <p>Results</p> <p>Using a combination of <it>in silico </it>and experimental approaches, we identified and characterized novel <it>P</it>. <it>abyssi </it>ncRNAs transcribed from 12 intergenic regions, ten of which are conserved throughout the Thermococcales. Several of them accumulate in the late-exponential phase of growth. Analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel <it>P</it>. <it>abyssi </it>ncRNA families. The CRISPR family is comprised of crRNAs expressed from two of the four <it>P</it>. <it>abyssi </it>CRISPR cassettes. The 5'UTR derived family includes four conserved ncRNAs, two of which have features similar to known bacterial riboswitches. Several of the novel ncRNAs have sequence similarities to orphan OrfB transposase elements. Based on RNA secondary structure predictions and experimental results, we show that three of the twelve ncRNAs include Kink-turn RNA motifs, arguing for a biological role of these ncRNAs in the cell. Furthermore, our results show that several of the ncRNAs are subjected to processing events by enzymes that remain to be identified and characterized.</p> <p>Conclusions</p> <p>This work proposes a revised annotation of CRISPR loci in <it>P</it>. <it>abyssi </it>and expands our knowledge of ncRNAs in the Thermococcales, thus providing a starting point for studies needed to elucidate their biological function.</p