Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.     

Moths complement bumblebee pollination of red clover: a case for day-and-night insect surveillance

Recent decades have seen a surge in awareness about insect pollinator declines. Social bees receive the most attention, but most flower-visiting species are lesser known, non-bee insects. Nocturnal flower visitors, e.g. moths, are especially difficult to observe and largely ignored in pollination studies. Clearly, achieving balanced monitoring of all pollinator taxa represents a major scientific challenge. Here, we use time-lapse cameras for season-wide, day-and-night pollinator surveillance of Trifolium pratense (L.; red clover) in an alpine grassland. We reveal the first evidence to suggest that moths, mainly Noctua pronuba (L.; large yellow underwing), pollinate this important wildflower and forage crop, providing 34% of visits (bumblebees: 61%). This is a remarkable finding; moths have received no recognition throughout a century of T. pratense pollinator research. We conclude that despite a non-negligible frequency and duration of nocturnal flower visits, nocturnal pollinators of T. pratense have been systematically overlooked. We further show how the relationship between visitation and seed set may only become clear after accounting for moth visits. As such, population trends in moths, as well as bees, could profoundly affect T. pratense seed yield. Ultimately, camera surveillance gives fair representation to non-bee pollinators and lays a foundation for automated monitoring of species interactions in future.     

Local Recurrence of Soft Tissue Sarcoma Revisited: Is there a Role for “Selective” Radiation?

BackgroundRadiation therapy (RT) is often utilized in cases of high-grade soft tissue sarcoma (STS), but there remain situations where treatment is with surgical excision alone. Our goals were to determine (1) the local recurrence (LR) rate with and without perioperative RT and (2) associations between local recurrence, patient, tumor, and treatment variables.MethodsWe performed a retrospective review of 165 consecutive STS patients. A Cox proportional hazards model was used to investigate variables associated with local recurrence.ResultsLR occurred in 15/78 (19%) without RT, 4/29 (14%) with postoperative RT, and 0/58 with preoperative RT (p=0.002). We found increased rates of local recurrence at 24 months for myxofibrosarcoma (p=0.001) and no-RT (p=0.003). Myxofibrosarcoma accounted for 33 (20%) of the study patients and 12 (63%) of the local recurrences.ConclusionThe LR rate in patients treated with surgery alone was disproportionately attributable to myxofibrosarcoma (11/23 cases, 48%). Other subtypes demonstrated a lower rate of LR in the absence of RT (4/55 cases, 7%), and no LR occurred when final margins were >2 mm. In certain circumstances treatment with a negative margin surgical resection followed by close observation is justifiable. RT is effective and should continue to be considered routinely in myxofibrosarcoma or when surgical margins are inadequate. Level of Evidence: III     

Intrathecal Morphine Use in Adolescent Idiopathic Scoliosis Surgery is Associated with Decreased Opioid Use and Decreased Length of Stay

BackgroundLength of stay (LOS) in the hospital following posterior spinal fusion (PSF) for adolescent idiopathic scoliosis (AIS) has decreased over the past decade due to well-defined postoperative clinical pathways, earlier mobilization, and improved pain control methods. Historically, liberal use of parenteral and oral opioids for pain control caused side effects, resulting in delayed discharge. Intraoperative intrathecal morphine (ITM) has been posited to reduce the need for postoperative opioids and to expedite the discharge process. This study examines the relationship between the use of ITM with average required postoperative opioid usage and with average LOS.MethodsThis IRB-approved retrospective cohort study examined 105 patients with AIS who received PSF with instrumentation split into two cohorts. One cohort underwent PSF via standard surgical protocol (n=40) while the other cohort received intraoperative ITM with the standard surgical protocol (n=65). Power analysis demonstrated a study power of 0.8. LOS and total postoperative opioid analgesic medication (morphine milligram equivalent, MME) data were collected. Age at surgery, gender, number of spinal levels fused, estimated intraoperative blood loss (EBL), preoperative Cobb angle, and any complications related to the use of ITM were also recorded. Continuous variables were analyzed with Student’s t-test and categorical variables were analyzed with chi-square independent-sample tests using SAS 9.4 (α = 0.05).ResultsPatients who were treated with ITM displayed shorter LOS (p<0.0001) and reduced postoperative analgesic requirement (p<0.0001). Patients who received ITM spent an average of 1.8 fewer midnights in the hospital and received an average of 221.2 MME less than patients who received standard protocol (57% decrease). There were no significant differences between the two groups for any other variable.ConclusionIntraoperative ITM is a simple and effective treatment for scoliosis surgeons to better control postoperative pain in patients, reduce the risk of dependency, and achieve earlier discharge from the hospital. Shortened LOS reduces the overall cost of care, benefitting patients, hospitals, and insurance companies. Based on the results of this study and several earlier studies, the authors recommended that scoliosis surgeons consider incorporating use of ITM into their standard operative protocols. Level of Evidence: IV     

Evidence for widespread genomic methylation in the migratory locust, Locusta migratoria (Orthoptera: Acrididae)

The importance of DNA methylation in mammalian and plant systems is well established. In recent years there has been renewed interest in DNA methylation in insects. Accumulating evidence, both from mammals and insects, points towards an emerging role for DNA methylation in the regulation of phenotypic plasticity. The migratory locust (Locusta migratoria) is a model organism for the study of phenotypic plasticity. Despite this, there is little information available about the degree to which the genome is methylated in this species and genes encoding methylation machinery have not been previously identified. We therefore undertook an initial investigation to establish the presence of a functional DNA methylation system in L. migratoria. We found that the migratory locust possesses genes that putatively encode methylation machinery (DNA methyltransferases and a methyl-binding domain protein) and exhibits genomic methylation, some of which appears to be localised to repetitive regions of the genome. We have also identified a distinct group of genes within the L. migratoria genome that appear to have been historically methylated and show some possible functional differentiation. These results will facilitate more detailed research into the functional significance of DNA methylation in locusts.     

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .