Label-free imaging of lipid dynamics using Coherent Anti-stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy

The recently developed Coherent Anti-stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy have provided new methods to visualize the localization and regulation of biological molecules without the use of invasive and potentially perturbative labels. They allow rapid imaging of specific molecules with high resolution and sensitivity. These tools have been effectively applied to the study of lipid metabolism using Caenorhabditis elegans as a genetic model, unraveling new lipid storage phenotypes and their regulatory mechanisms. Here we review the underlying principle of CARS and SRS microscopy, as well as their recent applications in lipid biology research in C. elegans.     

The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.     

Deciphering information encoded in birdsong: male songbirds with fertile mates respond most strongly to complex, low-amplitude songs used in courtship

Research on the function of acoustic signals has focused on high-amplitude long-range songs (LRS) and largely ignored low-amplitude songs produced by many species during close-proximity, conspecific interactions. Low-amplitude songs can be structurally identical to LRS (soft LRS), or they can be widely divergent, sharing few spectral and temporal attributes with LRS (short-range song [SRS]). SRS is often more complex than LRS and is frequently sung by males during courtship. To assess function, we performed two playback experiments on males of a socially monogamous songbird. We compared responses of males whose mates were fertile or nonfertile with differences in song structure (SRS vs. LRS and soft LRS), amplitude (SRS and soft LRS vs. LRS), and tempo (slow SRS vs. fast SRS). Males responded more strongly to SRS than to LRS or soft LRS, indicating that song structure had a greater effect on response than song amplitude. SRS tempo did not detectably affect male response. Importantly, males responded more strongly to SRS when their mates were fertile, presumably because hearing SRS can indicate that a male's mate is being courted by an intruding male and a strong response can deter extrapair competitors. We conclude that low-amplitude songs can function in both inter- and intrasexual communication and should receive greater attention in future studies of mate choice and male-male competition.     

Arylsulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is ome evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Release of slow reacting substance (SRS) from rat basophilic leukemia (RBL-1) cells.

When rat basophilic leukemia (RBL-1) cells were exposed to the ionophore A23187, a substance was released that produced a prolonged contraction of guinea pig ileum resembling that seen with slow reacting substances (SRSs) from various sources. The response was temperature, dose, and the time dependent with no activity being demonstrated in unstimulated cells. Several lines of evidence indicated that the RBL-1 product was markedly similar or identical to SRSs obtained from non-neoplastic tissues: 1) appropriate behavior in seven different chromatographic systems, 2) an appropriate profile of activity on various smooth muscle preparations, 3) an ability of low concentrations of the selective SRS inhibitor FPL 55712 to block the guinea pig ileal response, 4) failure of chymotrypsin to destroy activity, 5) loss of the activity after incubation with arylsulfatase, and 6) an ability to release activity from cells preincubated with indomethacin. Since RBL-1 cells can be grown in considerable guantity and under optimal conditions an average of 1500 SRS units/10(7) cells can be obtained, these cells should be useful as a biosynthetic source in further attempts to purify and characterize the SRS molecule.     

A concise review of the efficacy of stereotactic radiosurgery in the management of melanoma and renal cell carcinoma brain metastases

ABSTRACT: Melanoma and renal cell carcinoma have a well-documented tendency to develop metastases to the brain. Treating these lesions has traditionally been problematic, because chemotherapy has difficulty crossing the blood brain barrier and whole brain radiation therapy (WBRT) is a relatively ineffective treatment against these radioresistant tumor histologies. In recent years, stereotactic radiosurgery (SRS) has emerged as an effective and minimally-invasive treatment modality for irradiating either single or multiple intracranial structures in one clinical treatment setting. For this reason, we conducted a review of modern literature analyzing the efficacy of SRS in the management of patients with melanoma and renal cell carcinoma brain metastases. In our analysis we found SRS to be a safe, effective and attractive treatment modality for managing radioresistant brain metastases and highlighted the need for randomized trials comparing WBRT alone vs. SRS alone vs. WBRT plus SRS in treating patients with radioresistant brain metastases.     

Slow reacting substance (SRS) from ionophore A23187-stimulated peritoneal mast cells of the normal rat. II. Evidence for a precursor role of arachidonic acid and further purification.

The generation of slow reacting substance (SRS) from ionophore A23187-stimulated rat peritoneal mast cells was enhanced by arachidonic acid (AA). This SRS generation was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analogue of AA and an inhibitor of both fatty acid cyclooxygenase and lipoxygenase. Indomethacin, a fatty acid cyclooxgenase inhibitor, had an enhancing effect upon SRS generation. This suggests SRS generation occurred through an ETYA sensitive step--perhaps a lipoxygenase. Radiolabel from [14C]-AA was incorporated into SRS with comigration of radioactivity and bioreactivity in silicic acid and thin layer chromatographies. Upon silicic acid chromatography, the active principle was eluted in the methanol fraction. Two-dimensional thin layer chromatography revealed chromatographic separation from other known spasmogenic substances and phospholipids. Mast cell SRS was found to display physiochemical properties similar to those of rat basophilic leukemia cell SRS, namely: that mast cell SRS generation was 1) enhanced by arachidonic acid; 2) inhibited by ETYA but not by indomethacin; 3) incorporation of [14C]-AA into the active principle; and 4) similar behavior during purification in silicic acid and thin layer chromatographies.     

Models and detection of spontaneous recurrent seizures in laboratory rodents

Epilepsy,characterized by spontaneous recurrent seizures (SRS),is a serious and common neurological disorder afflicting an estimated 1％ of the population worldwide.Animal experiments,especially those utilizing small laboratory rodents,remain essential to understanding the fundamental mechanisms underlying epilepsy and to prevent,diagnose,and treat this disease.While much attention has been focused on epileptogenesis in animal models of epilepsy,there is little discussion on SRS,the hallmark of epilepsy.This is in part due to the technical difficulties of rigorous SRS detection.In this review,we comprehensively summarize both genetic and acquired models of SRS and discuss the methodology used to monitor and detect SRS in mice and rats.     

The SMALL AND ROUND SEED1 (SRS1/DEP2) gene is involved in the regulation of seed size in rice

The causal gene of a novel small and round seed mutant 1 (srs1) was identified in rice by map-based cloning and named SMALL AND ROUND SEED 1 (SRS1). The SRS1 gene is identical to the previously identified DENSE AND ERECT PANICLE 2 (DEP2). The SRS1/DEP2 gene encodes a novel protein of 1365 amino acids residues without known functional domains. In the longitudinal direction of the lemma, both cell length and cell number are reduced in srs1-1 compared to the wild type, whereas in the lateral cross section of the lemma, cell length in srs1-1 is greater than that in the wild type, but the cell number in srs1-1 is the same as that in wild type. These results suggest that the small and round seed phenotype of srs1-1 is due to the reduction in both cell length and cell number in the longitudinal direction, and the elongation of the cells in the lateral direction of the lemma. The SRS1 mRNA and proteins are abundant in wild type rice specifically in young organs, namely young leaves, internodes and panicles. Interestingly, the tissues expressing SRS1 are closely related to the tissues that exhibit abnormalities in the srs1 mutants.     

Radiosurgery dose reduction for brain metastases on immunotherapy (RADREMI): A prospective phase I study protocol

IntroductionUp to 20% of patients with brain metastases treated with immune checkpoint inhibitor (ICI) therapy and concomitant stereotactic radiosurgery (SRS) suffer from symptomatic radiation necrosis. The goal of this study is to evaluate Radiosurgery Dose Reduction for Brain Metastases on Immunotherapy (RADREMI) on six-month symptomatic radiation necrosis rates.MethodsThis study is a prospective single arm Phase I pilot study which will recruit patients with brain metastases receiving ICI delivered within 30 days before SRS. All patients will be treated with RADREMI dosing, which involves SRS doses of 18 Gy for 0−2 cm lesions, 14 Gy for 2.1−3 cm lesions, and 12 Gy for 3.1−4 cm lesions. All patients will be monitored for six-month symptomatic radiation necrosis (defined as a six-month rate of clinical symptomatology requiring steroid administration and/or operative intervention concomitant with imaging findings consistent with radiation necrosis) and six-month local control. We expect that RADREMI dosing will significantly reduce the symptomatic radiation necrosis rate of concomitant SRS + ICI without significantly sacrificing the local control obtained by the present RTOG 90−05 SRS dosing schema. Local control will be defined according to the Response Assessment in Neuro-Oncology (RANO) criteria.DiscussionThis study is the first prospective trial to investigate the safety of dose-reduced SRS in treatment of brain metastases with concomitant ICI. The findings should provide fertile soil for future multi-institutional collaborative efficacy trials of RADREMI dosing for this patient population.Trial RegistrationClinicaltrials.gov identifier: NCT04047602 (registration date: July 25, 2019).     

Duplication of 7p12.1-p13, including GRB10 and IGFBP1, in a mother and daughter with features of Silver-Russell syndrome

Silver-Russell syndrome (SRS) has been associated with maternal uniparental disomy (UPD) of chromosome 7 in approximately 10% of cases, suggesting that at least one imprinted gene on chromosome 7 is involved in the pathogenesis of the disease. We report a proximal 7p interstitial inverted duplication in a mother and daughter both of whom have features of SRS, including marked short stature, low birth weight, facial asymmetry and 5th finger clinodactyly. Fluorescence in situ hybridisation (FISH) with YAC probes enabled delineation of the duplicated region to 7p12.1-p13. This region of proximal chromosome 7 is known to be homologous to an imprinted region in the mouse chromosome 11 and contains the growth-related genes GRB10 (growth factor receptor-bound protein 10), EGFR (epidermal growth factor receptor) and IGFBP1 (insulin-like growth factor binding protein 1), all of which have been suggested as candidate genes for SRS. Molecular analysis showed that the duplication in both mother and daughter spanned a distance of approximately 10 cM and included GRB10 and IGFBP1 but not EGFR. The de novo duplication in the proband's mother was shown to be of paternal origin. In order to test the hypothesis that sub-microscopic duplications of 7p, whether maternal or paternal in origin, are responsible for at least some cases of SRS, we screened a further eight patients referred to our laboratory for SRS. None were found to have duplications of either GRB10 or IGFBP1. The hypothesis that sub-microscopic duplications including GRB10 and IGFBP1 is a cause of SRS remains a possibility and warrants further investigation. Importantly, in contrast to current thinking, our results suggest that imprinted genes may not underlie the SRS phenotype, and we propose an alternative hypothesis to explain the occurrence of maternal UPD 7 seen in some cases of SRS.     

Wrapping SRS with CORBA: from textual data to distributed objects.

MOTIVATION: Biological data come in very different shapes. Databanks are maintained and used by distinct organizations. Text is the de facto Standard exchange format. The SRS system can integrate heterogeneous textual databanks but it was lacking a way to structure the extracted data. RESULTS: This paper presents a CORBA interface to the SRS system which manages databanks in a flat file format. SRS Object Servers are CORBA wrappers for SRS. They allow client applications (visualisation tools, data mining tools, etc.) to access and query SRS servers remotely through an Object Request Broker (ORB). They provide loader objects that contain the information extracted from the databanks by SRS. Loader objects are not hard-coded but generated in a flexible way by using loader specifications which allow SRS administrators to package data coming from distinct databanks. AVAILABILITY: The prototype may be available for beta-testing. Please contact the SRS group (http://srs.ebi.ac.uk).     

Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.     

Stochastic roadmap simulation for the study of ligand-protein interactions

Understanding the dynamics of ligand-protein interactions is indispensable in the design of novel therapeutic agents. In this paper, we establish the use of Stochastic Roadmap Simulation (SRS) for the study of ligand-protein interactions through two studies. In our first study, we measure the effects of mutations on the catalytic site of a protein, a process called computational mutagenesis. In our second study, we focus on distinguishing the catalytic site from other putative binding sites. SRS compactly represents many Monte Carlo (MC) simulation paths in a compact graph structure, or roadmap. Furthermore, SRS allows us to analyze all the paths in this roadmap simultaneously. In our application of SRS to the domain of ligand-protein interactions, we consider a new parameter called escape time, the expected number of MC simulation steps required for the ligand to escape from the 'funnel of attraction' of the binding site, as a metric for analyzing such interactions. Although computing escape times would probably be infeasible with MC simulation, these computations can be performed very efficiently with SRS. Our results for six mutant complexes for the first study and seven ligand-protein complexes for the second study, are very promising: In particular, the first results agree well with the biological interpretation of the mutations, while the second results show that escape time is a good metric to distinguish the catalytic site for five out of seven complexes.     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome p450 monooxygenase

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.     

The Medium-Chain Dehydrogenase/reductase Engineering Database: a systematic analysis of a diverse protein family to understand sequence-structure-function relationship

The Medium-Chain Dehydrogenase/Reductase Engineering Database (MDRED, http://www.mdred.uni-stuttgart.de) has been established to serve as an analysis tool for a systematic investigation of sequence-structure-function relationships. It includes sequence and structure information of 2684 and 42 medium-chain dehydrogenases/reductases (MDRs), respectively. Although MDRs are very diverse in sequence, they have a conserved tertiary structure. MDRs are assigned to 199 homologous families and 29 superfamilies. For each family, annotated multiple sequence alignments are provided, and functionally relevant residues are annotated. Twenty-five superfamilies were classified as zinc-containing MDRs, four as non-zinc-containing MDRs. For the zinc-containing MDRs, three subclasses were identified by systematic analysis of a variable loop region, the quaternary structure determining loop (QSDL): the class of short, medium, and long QSDL, which include 11, 3, and 5 superfamilies, respectively. The length of the QSDL is predictive for tetramer (short QSDL) and dimer (long QSDL) formation. The class of medium QSDL includes both tetrameric and dimeric MDRs. The shape of the substrate-binding site is highly conserved in all zinc-containing MDRs with the exception of two variable regions, the substrate recognition sites (SRS): two residues located on the QSDL (SRS1) and, for the class of long QSDL, one residue located in the catalytic domain (SRS2). The MDRED is the first online-accessible resource of MDRs that integrates information on sequence, structure, and function. Annotation of functionally relevant residues assist the understanding of sequence-structure-function relationships. Thus, the MDRED serves as a valuable tool to identify potential hotspots for engineering properties such as substrate specificity.     

Biparental expression of IGFBP1 and IGFBP3 renders their involvement in the etiology of Silver-Russell syndrome unlikely.

Maternal uniparental disomy (UPD) of chromosome 7 has recently been reported in about 10% of Silver-Russell (SRS) patients. It can therefore be concluded that at least one gene on chromosome 7 is imprinted and mutations in this gene/these genes might contribute to the phenotype of the disease. Two genes which are involved in growth and localised in 7p12-13 are the insulin-like growth factor binding proteins 1 and 3 (IGFBP1; IGFBP3). Comparison to the mouse genome shows that the syntenic region on mouse chromosome 11 is imprinted, UPD of this region leads to deviations in growth in mice. In the present study we investigated whether the genes for IGFBP1 and IGFBP3 might be involved in the etiology of SRS: after exclusion of SRS specific mutations we could demonstrate biparental expression of both genes in lymphocytes of an SRS patient without UPD7 as well as expression in a patient with maternal UPD7. Our results as well as those from other groups show biparental expression of IGFBP1 in fetal tissues and expression of IGFBP3 in nearly every tissue during puberty and adult life. Thus, no evidence is given for an involvement of the two genes in SRS.     

A case of Silver-Russell syndrome (SRS): multiple pituitary hormone deficiency,lack of H19 hypomethylation and favourable growth hormone (GH) treatment response

Hypomethylation of the imprinting control region 1 (ICR1) at the IGF2/H19 locus on 11p15 is linked to Silver-Russell syndrome (SRS) and/or hemihypertrophy. This SRS patient was born in term with weight of 3500 g (50 percentile) and length 48 cm (>1 SD below the mean). He was first noticed at the age of 10 years for short stature (114.5 cm, −3.85 SD), relatively normal head circumference, a classic facial phenotype, hemihypertrophy (2.5 cm thinner left arm and leg in comparison to the right, asymmetric face), moderate clinodactyly and striking thinness (BMI of 15.3). At the age of 30, the body asymmetry ameliorated (1 cm thinner left arm and leg than the right), and BMI normalized (20.5 cm). Methylation analysis was performed by bisulphate treatment of DNA samples, radiolabelled PCR amplification, and digestion of the PCR products using restriction enzymes. The patient had normomethylation, and in addition hypopituitarism, with low levels of growth hormone (GH) (provocative testing before the start and after termination of GH treatment), thyroxin, TSH, FSH, LH and testosterone. The GH was given for six years, growth response was satisfactory and he reached an adult height of 166 cm. This is a first report of hypopituitarism in a patient with SRS without H19 hypomethylation. It seems that the lack of hypomethylation in this hypopituitary SRS patient is responsible, at least partly, for the favourable final adult height under GH treatment.     

The Macaque Social Responsiveness Scale (mSRS): A Rapid Screening Tool for Assessing Variability in the Social Responsiveness of Rhesus Monkeys (Macaca mulatta)

Understanding the biological mechanisms underlying human neuropsychiatric disorders, such as autism spectrum disorder (ASD), has been hindered by the lack of a robust, translational animal model. Rhesus monkeys (Macaca mulatta) display many of the same social behaviors that are affected in ASD, making them an excellent animal species in which to model social impairments. However, the social impairments associated with ASD may reflect extreme ends of a continuous distribution of traits. Thus, to validate the rhesus monkey as an animal model for studying social impairments that has strong translational relevance for ASD, researchers need an easily-implemented measurement tool that can quantify variation in social behavior dimensionally. The Social Responsiveness Scale (SRS) is a 65-item survey that identifies both typical and atypical social behaviors in humans that covary with ASD symptom severity. A chimpanzee SRS has already been validated and the current study adapted this tool for use in the rhesus monkey (mSRS). Fifteen raters completed the mSRS for 105 rhesus monkeys living at the Yerkes National Primate Research Center. The mSRS scores showed a unimodal distribution with a positive skew that identified 6 statistical outliers. Inter-rater reliability was very strong, but only 17 of the 36 questions showed positive intra-item reliability. The results of an exploratory factor analysis identified 3 factors that explained over 60% of the variance, with 12 items significantly loading onto the primary factor. These items reflected behaviors associated with social avoidance, social anxiety or inflexibility and social confidence. These initial findings are encouraging and suggest that variability in the social responsiveness of rhesus monkeys can be quantified using the mSRS: a tool that has strong translational relevance for human disorders. With further modification, the mSRS may provide an promising new direction for research on the biological mechanisms underlying social impairments.