Subtractional Heterogeneity: A Crucial Step toward Defining Specialized Ribosomes.

In this issue of Molecular Cell, Shi et al. (2017) identify translating ribosomes which lack specific proteins and associate with specific classes of mRNAs. This challenges the popular conception of "the ribosome" as a homogeneous, monolithic molecular machine.

Programmed Ribosomal Frameshifting Generates a Copper Transporter and a Copper Chaperone from the Same Gene.

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo.

Formation of Type II Silicon Clathrate with Lithium Guests through Thermal Diffusion.

At low guest atom concentrations, Si clathrates can be viewed as semiconductors, with the guest atoms acting as dopants, potentially creating alternatives to diamond Si with exciting optoelectronic and spin properties. Studying Si clathrates with different guest atoms would not only provide insights into the electronic structure of the Si clathrates but also give insights into the unique properties that each guest can bring to the Si clathrate structure. However, the synthesis of Si clathrates with guests other than Na is challenging. In this study, we have developed an alternative approach, using thermal diffusion into type II Si clathrate with an extremely low Na concentration, to create Si clathrate with Li guests. Using time-of-flight secondary-ion mass spectroscopy, X-ray diffraction, and Raman scattering, thermal diffusion of Li into the nearly empty Si clathrate framework is detected and characterized as a function of the diffusion temperature and time. Interestingly, the Si clathrate exhibits reduced structural stability in the presence of Li, converting to polycrystalline or disordered phases for anneals at temperatures where the starting Na guest Si clathrate is quite stable. The Li atoms inserted into the Si clathrate lattice contribute free carriers, which can be detected in Raman scattering through their effect on the strength of Si-Si bonds in the framework. These carriers can also be observed in electron paramagnetic resonance (EPR). EPR shows, however, that Li guests are not simple analogues of Na guests. In particular, our results suggest that Li atoms, with their smaller size, tend to doubly occupy cages, forming "molecular-like" pairs with other Li or Na atoms. Results of this work provide a deeper insight into Li guest atoms in Si clathrate. These findings are also relevant to understanding how Li moves through and interacts with Si clathrate anodes in Li-ion batteries. Additionally, techniques presented in this work demonstrate a new method for filling the Si clathrate cages, enabling studies of a broad range of other guests in Si clathrates.

Forensic Assessments of Alcohol, Cannabis and Methamphetamine Intoxication in Cases of Violent Offending.

Whether a person was voluntarily or intentionally intoxicated at the time of commission of a violent offence is a common question in forensic contexts. While a person who was intoxicated may not be able to form the requisite specific intent to commit some offences, voluntary intoxication usually disentitles a person from an insanity or "mental impairment" defence. However, a person may also consume alcohol or use a substance without becoming intoxicated and the presence of alcohol, substances or metabolites of substances in a person's urine or blood is not conclusive when the question of intoxication is relevant. A jury (or a judge sitting without a jury) may require expert opinion evidence when cannabis or methamphetamine intoxication are implicated in the alleged offending.

Frequency noise stabilisation of a 1550 nm external cavity diode laser with hybrid feedback for next generation gravitational wave interferometry.

Longer wavelength lasers will be needed for future gravitational wave detectors that use cryogenic cooling of silicon based test-mass optics. Diode lasers with a 1550 nm wavelength output are potential seed light sources for such a detector, however diode laser devices have a different spectral profile and higher frequency noise than the solid state lasers used in current detectors. We present a frequency stabilisation system for a 1550 nm external cavity diode laser capable of reducing the laser frequency noise to a level of 0.1HzHz up to 1 kHz with a unity gain frequency of 535 kHz using a hybrid analogue-digital servo with in-loop cancellation of resonant features. In addition, a method of high speed digital filter optimisation and automated design is demonstrated.

Prolonged Solitary Confinement (Administrative Segregation) and the Human Rights of a Serving Prisoner.

Solitary confinement is the harshest method of social control that can be applied to a prisoner. There is considerable research which establishes that prolonged solitary confinement may have profoundly negative effects. The United Nations Standard Minimum Rules for the Treatment of Prisoners (the "Mandela Rules") stipulate that solitary confinement should only be used "in exceptional circumstances as a last resort for as short a time as possible" and that solitary confinement should be "subject to independent review and only pursuant to the authorization by a competent authority". In Owen-D'Arcy v Chief Executive, Queensland Corrective Services [2021] QSC 273, a prisoner had spent over six and a half years in continuous solitary confinement. The Queensland Supreme Court held that the decision to continue the solitary confinement was not compatible with the prisoner's human rights pursuant to s 30 (humane treatment when deprived of liberty) of the Human Rights Act 2019 (Qld).

Murder (Infanticide) in Post-partum Depression: The Case of Akon Guode.

In 2015, 35-year-old Sudanese refugee Akon Guode had post-traumatic stress disorder and a post-partum depression when she drove her vehicle into a lake in a murder (infanticide, filicide)-suicide attempt. In 2017, Ms Guode pleaded guilty to two counts of murder, one count of attempted murder and one count of infanticide and was sentenced to 26 years' imprisonment. In August 2019, the Victorian Court of Appeal found the original sentence was "manifestly excessive". In March 2020, a majority of the High Court found that the Court of Appeal erred by taking into account that the Crown had accepted Ms Guode's plea of guilty to the charge of infanticide. The High Court quashed the sentence . In September 2020, the Court of Appeal imposed the same 18-year sentence and the same non-parole period as in August 2019. This commentary considers the application of the defences of "infanticide" and "mental impairment" and "fitness for trial" in post-partum depression and PTSD.

A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism.

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.

'Rape myths' and a 'reasonable belief' of consent R v Lazarus [2017] NSWCCA 279.

Judicial directions in rape trials are designed to emphasise to jury members the importance of negating consent or that the accused believed on reasonable grounds that the complainant consented. After a jury convicted the accused in R v Lazarus [2015], the NSW Court of Appeal in R v Lazarus [2016] NSWCCA 52 found that the trial judge misdirected the jury on the question of the state of mind of the accused at the time of the alleged rape. After a judge sitting without a jury acquitted the accused, the NSW Court of Appeal in R v Lazarus [2017] NSWCCA 279 found that the judge in the re-trial failed to direct herself in relation to making a finding about the steps taken by the accused to establish whether the complainant was consenting. As well as reviewing the reasoning in the decisions, this article discusses rape myths and the justice gap and considers law reform on the issue of consent in rape cases.

Functional and structural characterization of the chikungunya virus translational recoding signals.

Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed -1 ribosomal frameshift (-1 PRF) signal located toward the 3' end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and -1 PRF. Analyses of RNA chemical modification data with selective 2'-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV -1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.

Murine osteosarcoma primary tumour growth and metastatic progression is maintained after marked suppression of serum insulin-like growth factor I.

The insulin-like growth factor I (IGF-I) signaling pathway has been shown to play an important role in several aspects of cancer biology, including metastasis. The aim of this study was to define the contribution of serum (endocrine) and local (tumour microenvironment) IGF-I on osteosarcoma tumour growth and metastasis, a cancer that is known to be dependent on the IGF-I axis. To test this hypothesis, we evaluated the primary tumour growth and metastatic progression of K7M2 murine osteosarcoma cells injected to a genetically engineered mouse [liver-specific IGF-I deficient (LID)] in which serum IGF-I levels are reduced by 75%, while maintaining expression of IGF-I in normal tissues. We first demonstrated that IGF-I in the tumour and the tumour-microenvironment were maintained in the LID mice. Within this designed model, there was no difference in primary tumour growth or in pulmonary metastasis in LID mice compared to control mice. Furthermore, there was no difference in the number or localization of single metastatic cells immediately after their arrival in the lungs of LID mice and control mice, as analysed by single cell video microscopy. Collectively, these data suggest that marked reduction in serum IGF-I is not sufficient to slow the progression of either primary or metastatic models of osteosarcoma.

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

Transcriptional upregulation of SPARC, in response to c-Jun overexpression, contributes to increased motility and invasion of MCF7 breast cancer cells.

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotype changes related to malignant progression including increased motility and invasion. Concurrent with these phenotypic effects are changes in the expression of multiple gene targets. We previously demonstrated that expression of the SPARC/osteonectin gene, while undetectable in the MCF7 cell line, is highly induced in response to stable c-Jun overexpression (c-Jun/MCF7). Because the SPARC gene product is associated with tumor cell invasion in a variety of different cancers, we have examined its role in mediating the phenotypic changes induced by c-Jun in MCF7 cells. We found that antisense mediated suppression of SPARC dramatically inhibits both motility and invasion in this c-Jun/MCF7 model. In contrast, stable overexpression of SPARC in the parental MCF7 cell line is not sufficient to stimulate cell motility or invasion. Examination of the promoter region of the human SPARC gene reveals three non-canonical AP-1 sites. We demonstrate that one of these sites binds c-Jun/Fra1 heterodimers in vitro, but that this and the other AP-1 like sites are dispensable with respect to c-Jun stimulated SPARC promoter activation. Deletion analysis identified a region between -120 and -70 as a c-Jun responsive element sufficient to induce maximal promoter activation. This region does not contain any AP-1 sites but does mediate binding by SP1 'like' complexes. Furthermore, this region is necessary for SP1/SP3 responsiveness in Drosophila SL2 cells. These results demonstrate that SPARC plays an important role in stimulating motility and the invasive behavior of c-Jun/MCF7 cells and that SPARC promoter activation by c-Jun appears to occur through an indirect mechanism.

v-Jun downregulates the SPARC target gene by binding to the proximal promoter indirectly through Sp1/3.

Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a downregulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Repression of SPARC contributes to the oncogenic process by facilitating tumor development in vivo. A proximal promoter fragment, designated -124/+16, is responsible for high constitutive activity of the SPARC gene and is the target of repression by v-Jun. In this paper, using electrophoretic mobility shift and pull-down assays in vitro, and transient transfections and chromatin immunoprecipitation assays in Sp1/3-deficient Drosophila SL2 cells and in chick embryo fibroblasts, we show that (i) Sp1 and/or Sp3 is required for constitutive activation of SPARC transcription, by binding directly to the GGA-rich -92/-57 fragment; and (ii) v-Jun does not bind -124/+16 directly, but binds to the GGA-rich fragment indirectly, most likely through a physical interaction with Sp1/3. Moreover, a transactivation-proficient v-Jun derivative, designated v-Jun/cebp/glz, which cannot bind Jun DNA motifs anymore and cannot heterodimerize, is still capable of downregulating SPARC efficiently. Taken together, these data strongly suggest that v-Jun downregulates SPARC through the formation of a DNA-Sp1/3-v-Jun, chromatin-associated complex.

Characterization of the metastatic phenotype of a panel of established osteosarcoma cells.

Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metastatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models.

Dysregulation of ezrin phosphorylation prevents metastasis and alters cellular metabolism in osteosarcoma.

Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers; however, the precise mechanistic basis for its role remains unknown. Here, we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biologic characteristics to full-length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism.

The ezrin metastatic phenotype is associated with the initiation of protein translation.

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.

A compendium of canine normal tissue gene expression.

BACKGROUND: Our understanding of disease is increasingly informed by changes in gene expression between normal and abnormal tissues. The release of the canine genome sequence in 2005 provided an opportunity to better understand human health and disease using the dog as clinically relevant model. Accordingly, we now present the first genome-wide, canine normal tissue gene expression compendium with corresponding human cross-species analysis. METHODOLOGY/PRINCIPAL FINDINGS: The Affymetrix platform was utilized to catalogue gene expression signatures of 10 normal canine tissues including: liver, kidney, heart, lung, cerebrum, lymph node, spleen, jejunum, pancreas and skeletal muscle. The quality of the database was assessed in several ways. Organ defining gene sets were identified for each tissue and functional enrichment analysis revealed themes consistent with known physio-anatomic functions for each organ. In addition, a comparison of orthologous gene expression between matched canine and human normal tissues uncovered remarkable similarity. To demonstrate the utility of this dataset, novel canine gene annotations were established based on comparative analysis of dog and human tissue selective gene expression and manual curation of canine probeset mapping. Public access, using infrastructure identical to that currently in use for human normal tissues, has been established and allows for additional comparisons across species. CONCLUSIONS/SIGNIFICANCE: These data advance our understanding of the canine genome through a comprehensive analysis of gene expression in a diverse set of tissues, contributing to improved functional annotation that has been lacking. Importantly, it will be used to inform future studies of disease in the dog as a model for human translational research and provides a novel resource to the community at large.

Modeling metastasis biology and therapy in real time in the mouse lung.

Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.