Tuning tRNAs for improved translation.

Transfer RNAs have been extensively explored as the molecules that translate the genetic code into proteins. At this interface of genetics and biochemistry, tRNAs direct the efficiency of every major step of translation by interacting with a multitude of binding partners. However, due to the variability of tRNA sequences and the abundance of diverse post-transcriptional modifications, a guidebook linking tRNA sequences to specific translational outcomes has yet to be elucidated. Here, we review substantial efforts that have collectively uncovered tRNA engineering principles that can be used as a guide for the tuning of translation fidelity. These principles have allowed for the development of basic research, expansion of the genetic code with non-canonical amino acids, and tRNA therapeutics.

Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility.

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H2 (a strong product inhibitor), is greatly enhanced relative to H2 oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H2 production catalysts. Second, an inflection (an S-shaped "twist" in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton-electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton-electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.

Late dumping syndrome preceded by Coxsackievirus B4 infection and cholecystectomy.

A 44-year-old female patient presented with weight loss, diarrhea and intermittent episodes of left upper quadrant (LUQ) pain lasting for 3 years, accompanied by acute episodes of focal LUQ pain, dizziness, tachycardia, borborygmi and bloating, occurring approximately 60 min after meals. The patient developed chronic acalculous cholecystitis and transient exocrine pancreatic insufficiency after infection with Coxsackievirus B4 (CVB4), which resolved following laparoscopic cholecystectomy 2 years before the current presentation. Although imaging and functional investigation studies were unremarkable, a gastric transit study revealed rapid clearance of radiolabeled food, and the patient's symptomatology and gastrointestinal studies supported the diagnosis of late dumping syndrome. The patient's symptoms significantly improved with adherence to recommended dietary changes, including an increase in protein intake, abstinence from simple carbohydrates and avoidance of simultaneous consumption of beverages with food, following consultation with a dietitian.

The beavertail modified radial forearm free flap: Retrospective review of a versatile technique to increase flap bulk in the head and neck.

OBJECTIVE: Utilization of free tissue transfers in head and neck reconstruction has greatly increased due to their dependability and reliability. Anterolateral thigh (ALT) and rectus abdominus (RA) free flaps may provide too much soft tissue bulk, especially in patients with a large body habitus. A radial forearm free flap (RFFF) may be modified with a "beaver tail" (BT), which provides a flap whose bulk may be tailored to a defect. The purpose of this paper is to describe the technique, how it can be used for a variety of defects and the outcomes of these reconstructions. METHODS: A retrospective review of prospectively collected data was performed at single tertiary care center between 2012 and 2022. BT-RFFF was designed by leaving a fibroadipose tail vascularized to branches of the radial artery or separated from the vascular pedicle and left attached to the proximal portion of the skin paddle. Functional outcomes, tracheostomy dependence, and gastrostomy tube (G-tube) dependence as well as complications were determined. RESULTS: Fifty-eight consecutive patients undergoing BTRFFF were included. Defects reconstructed included: oral tongue and/or floor of mouth 32 (55%), oropharynx 10 (17%), parotid 6 (10%), orbit 6 (10%), lateral temporal bone 3 (5%), and mentum 1 (2%). Indications for BTRFF were: need for bulk when the ALT and RA were too thick (53%) and need for a separate subcutaneous flap for contouring or deep defect lining (47%). Complications directly related to beavertail included a widened forearm scar (100%), wrist contracture (2%) partial flap loss (2%), and flap loss requiring a revision flap (3%). Ninety-three percent of patients with oral/oropharyngeal defects and 12-month follow-up tolerated oral intake without aspiration and 76% were tube-independent. Ninety-three percent were tracheostomy-free at last follow-up. CONCLUSION: The BTRFF is a useful tool for reconstructing complex 3D defects requiring bulk where an ALT or rectus would otherwise provide too much bulk.

Neurodegeneration in a novel invertebrate model system: Failed microtubule-mediated cell adhesion and unraveling of macroglia.

Neurodegenerative diseases are among the main causes of death in the United States, leading to irreversible disintegration of neurons. Despite intense international research efforts, cellular mechanisms that initiate neurodegeneration remain elusive, thus inhibiting the development of effective preventative and early onset medical treatment. To identify underlying cellular mechanisms that initiate neuron degeneration, it is critical to identify histological and cellular hallmarks that can be linked to underlying biochemical processes. Due to the poor tissue preservation of degenerating mammalian brain tissue, our knowledge regarding histopathological hallmarks of early to late degenerative stages is only fragmentary. Here, we introduce a novel model organism to study histological hallmarks of neurodegeneration, the spider Cupiennius salei. We utilized toluidine blue-stained 0.9-µm serial semithin and 50-nm ultrathin sections of young and old spider nervous tissue. Our findings suggest that the initial stages of neurodegeneration in spiders may be triggered by (1) dissociation of neuron- and glia-derived microtubules, and (2) the weakening of microtubule-associated desmosomal junctions that lead to the unraveling of neuron-insulating macroglia, compromising the structural integrity of affected neurons. The involvement of macroglia in the disposal of neuronal debris described here-although different in the proposed transport mechanisms-shows resemblance to the mammalian glymphatic system. We propose that this model system is highly suitable to investigate invertebrate neurodegenerative processes from early onset to scar formation and that this knowledge may be useful for the study of neurodegeneration in mammalian tissue.

Shifting the focus of zebrafish toward a model of the tumor microenvironment.

Cancer cells exist in a complex ecosystem with numerous other cell types in the tumor microenvironment (TME). The composition of this tumor/TME ecosystem will vary at each anatomic site and affects phenotypes such as initiation, metastasis, and drug resistance. A mechanistic understanding of the large number of cell-cell interactions between tumor and TME requires models that allow us to both characterize as well as genetically perturb this complexity. Zebrafish are a model system optimized for this problem, because of the large number of existing cell-type-specific drivers that can label nearly any cell in the TME. These include stromal cells, immune cells, and tissue resident normal cells. These cell-type-specific promoters/enhancers can be used to drive fluorophores to facilitate imaging and also CRISPR cassettes to facilitate perturbations. A major advantage of the zebrafish is the ease by which large numbers of TME cell types can be studied at once, within the same animal. While these features make the zebrafish well suited to investigate the TME, the model has important limitations, which we also discuss. In this review, we describe the existing toolset for studying the TME using zebrafish models of cancer and highlight unique biological insights that can be gained by leveraging this powerful resource.

Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Case report: chronic acalculous cholecystitis preceded by Coxsackievirus B4 infection.

A 41-year-old female presented with an 8-month history of right upper quadrant pain, exacerbated by ingestion of saturated fats. The patient was positive for antibodies to Coxsackievirus serotype B4, established by an investigation incited by an acute episode of pleurodynia 8 months before the current presentation. Imaging studies including a hepatobiliary iminodiacetic acid scan showed no gallbladder structural or functional abnormalities. Laboratory studies indicated pancreatic enzyme insufficiency associated with below-normal lipase and amylase levels. Patient symptomology was consistent with cholecystitis with positive Murphy's sign, so cholecystectomy was recommended. Post-surgery pathological report confirmed chronic acalculous cholecystitis. Patient demonstrated full recovery, indicated by return of normal pancreatic enzymes levels and resolution of abdominal pain.

Spatially resolved transcriptomics reveals the architecture of the tumor-microenvironment interface.

During tumor progression, cancer cells come into contact with various non-tumor cell types, but it is unclear how tumors adapt to these new environments. Here, we integrate spatially resolved transcriptomics, single-cell RNA-seq, and single-nucleus RNA-seq to characterize tumor-microenvironment interactions at the tumor boundary. Using a zebrafish model of melanoma, we identify a distinct "interface" cell state where the tumor contacts neighboring tissues. This interface is composed of specialized tumor and microenvironment cells that upregulate a common set of cilia genes, and cilia proteins are enriched only where the tumor contacts the microenvironment. Cilia gene expression is regulated by ETS-family transcription factors, which normally act to suppress cilia genes outside of the interface. A cilia-enriched interface is conserved in human patient samples, suggesting it is a conserved feature of human melanoma. Our results demonstrate the power of spatially resolved transcriptomics in uncovering mechanisms that allow tumors to adapt to new environments.

Developmental chromatin programs determine oncogenic competence in melanoma.

Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cell­derived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.

An in vivo reporter for tracking lipid droplet dynamics in transparent zebrafish.

Lipid droplets are lipid storage organelles found in nearly all cell types from adipocytes to cancer cells. Although increasingly implicated in disease, current methods to study lipid droplets in vertebrate models rely on static imaging or the use of fluorescent dyes, limiting investigation of their rapid in vivo dynamics. To address this, we created a lipid droplet transgenic reporter in whole animals and cell culture by fusing tdTOMATO to Perilipin-2 (PLIN2), a lipid droplet structural protein. Expression of this transgene in transparent casper zebrafish enabled in vivo imaging of adipose depots responsive to nutrient deprivation and high-fat diet. Simultaneously, we performed a large-scale in vitro chemical screen of 1280 compounds and identified several novel regulators of lipolysis in adipocytes. Using our Tg(-3.5ubb:plin2-tdTomato) zebrafish line, we validated several of these novel regulators and revealed an unexpected role for nitric oxide in modulating adipocyte lipid droplets. Similarly, we expressed the PLIN2-tdTOMATO transgene in melanoma cells and found that the nitric oxide pathway also regulated lipid droplets in cancer. This model offers a tractable imaging platform to study lipid droplets across cell types and disease contexts using chemical, dietary, or genetic perturbations.

Organisms need fat molecules as a source of energy and as building blocks, but these 'lipids' can also damage cells if they are present in large amounts. Cells guard against such toxicity by safely sequestering lipids in specialized droplets that participate in a range of biological processes. For instance, these structures can quickly change size to store or release lipids depending on the energy demands of a cell. It is possible to image lipid droplets ­ using, for example, dyes that preferentially stain fat ­ but often these methods can only yield a snapshot: tracking lipid droplet dynamics over time remains difficult. Lumaquin, Johns et al. therefore set out to develop a new method that could label lipid droplets and monitor their behaviour 'live' in the cells of small, transparent zebrafish larvae. First, the fish were genetically manipulated so that a key protein found in lipid droplets would carry a fluorescent tag: this made the structures strongly fluorescent and easy to track over time. And indeed, Lumaquin, Johns et al. could monitor changes in the droplets depending on the fish diet, with the structures getting bigger when the animal received rich food, and shrinking when resources were scarce. Finally, experiments were conducted to screen for compounds that could lead to lipids being released in fat cells. The new imaging technique was then used to confirm the effect of these molecules in live cells, revealing an unexpected role for a signalling molecule known as nitric oxide, which also turned out to be regulating lipid droplets in cancerous cells. Further work then showed that drugs affecting nitric oxide could modulate lipid droplet size in both normal and tumor cells. This work has validated a new method to study the real-time behavior of lipid droplets and their responses to different stimuli in living cells. In the future, Lumaquin, Johns et al. hope that the technique will help to shed new light on how lipids are involved in both healthy and abnormal biological processes.

Microbiome Analysis of Cholesteatoma by Gene Sequencing.

OBJECTIVE: To compare the microbial flora of cholesteatoma and normal middle ears using gene-based sequencing analysis. STUDY DESIGN: Controlled ex vivo human study. SETTING: Academic, tertiary medical center. SUBJECTS AND METHODS: Brush, swab, and tissue samples were each taken from cholesteatoma matrix and uninvolved tissue in patients with previously untreated, acquired cholesteatoma (n = 19) or middle ear mucosa from patients undergoing cochlear implantation with no history of cholesteatoma or previous middle ear surgery (control; n = 12). DNA was isolated from specimens then 16S rRNA gene sequencing was performed. RESULTS: There was no difference in microbial yield between the sampling methods. Cholesteatoma specimens had lower relative abundance of 14 bacterial species compared with controls including Acidovorax sp., Bacillus sp., Masillia sp., Moraxella osloensis, Phenylobacterium conjunctum, Sphingomonas sp., and Staphylococcus epidermidis (all p < 0.05). Alternaria sp. were present on nearly all the specimens. Alternaria sp. and Cladosporium herbarum (both p ≤ 0.05) were lower in the cholesteatoma compared with control group. There was no difference in the relative abundance of any bacteria or fungi between the cholesteatoma matrix and uninvolved middle ear mucosa. CONCLUSIONS: Microbiome of cholesteatoma matrix is largely similar to adjacent mucosa. This differs from healthy ears. Further study is needed to understand if middle ear microbiome may impact cholesteatoma pathogenesis or treatment.

Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis.

Tumor-secreted extracellular vesicles (EVs) are critical mediators of intercellular communication between tumor cells and stromal cells in local and distant microenvironments. Accordingly, EVs play an essential role in both primary tumor growth and metastatic evolution. EVs orchestrate multiple systemic pathophysiological processes, such as coagulation, vascular leakiness, and reprogramming of stromal recipient cells to support pre-metastatic niche formation and subsequent metastasis. Clinically, EVs may be biomarkers and novel therapeutic targets for cancer progression, particularly for predicting and preventing future metastatic development.

Update on mandibular condylar fracture management.

PURPOSE OF REVIEW: Fractures of the mandibular condyle have provided a lasting source of controversy in the field of facial trauma. Concerns regarding facial nerve injury as well as reasonable functional outcomes with closed management led to a reluctance to treat with an open operative intervention. This article reviews how incorporating new technologies and surgical methods have changed the treatment paradigm. RECENT FINDINGS: Multiple large studies and meta-analyses continue to demonstrate superior outcomes for condylar fractures when managed surgically. Innovations, including endoscopic techniques, three-dimensional miniplates, and angled drills provide increased options in the treatment of condylar fractures. The literature on pediatric condylar fractures is limited and continues to favor a more conservative approach. SUMMARY: There continues to be mounting evidence in radiographic, quality of life, and functional outcome studies to support open reduction with internal fixation for the treatment of condylar fractures in patients with malocclusion, significant displacement, or dislocation of the temporomandibular joint. The utilization of three-dimensional trapezoidal miniplates has shown improved outcomes and theoretically enhanced biomechanical properties when compared with traditional fixation with single or double miniplates. Endoscopic-assisted techniques can decrease surgical morbidity, but are technically challenging, require skilled assistants, and utilize specialized equipment.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Validation of the HCR-20 Scale for Assessing Risk of Violent Behavior in Israeli Psychiatric Inpatients.

BACKGROUND: Assessment of risk of violent behavior in forensic psychiatric practice is a complex and responsible clinical task and the use of a valid instrument can make the expert's work more effective. The Historical Clinical and Risk Management scale 20 (HCR-20) is a widely accepted measure of the risk of violence, sexual and criminal behavior. The aim of this study was to validate the HCR-20 in Israeli psychiatric inpatient settings. METHOD: In a prospective design, data were collected on 150 male patients aged 15-65, diagnosed with ICD-10 schizophrenia, who were hospitalized in three wards: an acute psychiatric ward (n=50), a high security ward (n=50), and an open ward (n=50). The HCR-20, as the predictor measure, and the Positive and Negative Syndrome Scale, as a concurrent measure, werecompleted at baseline, and the Violence Assessment Scale, as the outcome measure, was completed at 6-, 12- and 18-month follow-up points. RESULTS: Internal consistency reliability was good for the total HCR-20 scale, satisfactory for the H-subscale, but low for the C- and R-subscales. Concurrent validity was good for the C-subscale, and discriminative validity was reasonable for the C- and H-subscales. The total scale as well as the Historical and Clinical subscales predicted the risk of physical as well as physical/sexual violent behavior at both 6- and 18-month follow-up points. CONCLUSIONS: Appropriate psychometric properties of the HCR-20 suggest that it can serve as a useful measure of the risk of violent behavior in psychiatric settings in Israel. Further research is necessary to confirm norms and cut-off scores, using a larger representative sample.