ABSTRACT
Secreted proteins regulate the balance between cellular proliferation and G0 arrest and therefore play important roles in tumour dormancy. Tumour dormancy presents a significant clinical challenge for breast cancer patients, where non-proliferating, G0-arrested cancer cells remain at metastatic sites, below the level of clinical detection, some of which can re-enter proliferation and drive tumour relapse. Knowing which secreted proteins can regulate entry into and exit from G0 allows us to manipulate their signalling to prevent tumour relapse. To identify novel secreted proteins that can promote breast cancer G0 arrest, we performed a secretome-wide, image-based screen for proteins that increase the fraction of cells in G0 arrest. From a secretome library of 1282 purified proteins, we identified 29 candidates that promote G0 arrest in non-transformed and transformed breast epithelial cells. The assay we have developed can be adapted for use in other perturbation screens in other cell types. All datasets have been made available for re-analysis and our candidate proteins are presented for alternative bioinformatic refinement or further experimental follow up.
Subject(s)
Breast Neoplasms , Humans , Breast Neoplasms/pathology , Female , Cell Cycle Checkpoints , Resting Phase, Cell Cycle , Secretome , Cell Line, TumorABSTRACT
Background: The increasing incidence and prevalence of breast malignancies have led to increasing numbers of surgical interventions performed on the axilla and breast, including axillary lymph node dissection (ALND), sentinel lymph node biopsy (SLNB), and mastectomy. The risk of postoperative complications, like breast cancer-related lymphoedema (BCRL), can have significant deleterious cosmetic and quality of life effects. National guidelines and cancer councils publish recommendations to avoid skin puncturing procedures, such as venepuncture and intravenous (IV) cannulation, on arms ipsilateral to the surgical site to prevent BCRL occurrence. The initial trials that established a link between BCRL and skin puncture were conducted in the 1950s and 1960s; the evolution of surgical management of breast cancer has likely led to large decreases in complication rates. Methods: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, four databases were systematically searched for relevant articles. Eleven relevant articles were identified for inclusion in the final analysis. Updated Australian and New Zealand College of Anaesthetists & Faculty of Pain Medicine (ANZCA) guidelines were included in the analysis following their publication after the initial search had been completed. Results: The overall quality and quantity of evidence in this field is sufficient to conclude that skin puncturing procedures on ipsilateral arms should not be avoided in patients with previous breast or axillary surgery. The highest-quality and most recent available evidence does not support an association between BCRL and skin puncturing procedures. Policies and practices that advocate avoiding skin puncture procedures to prevent BCRL may lead to delays in clinical care. The 2023 ANZCA guidelines recommend against avoiding affected arms for peripheral access and suggest the removal of institutional policies preventing this practice. Conclusions: In patients that have undergone breast surgery or axillary procedures, venous access procedures can be safely performed on the ipsilateral arm. The evidence does not support overarching restrictions on using the ipsilateral arm without pre-existing lymphoedema.
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Lassa fever is a zoonotic disease identified by the World Health Organization (WHO) as having pandemic potential. This study estimates the health-economic burden of Lassa fever throughout West Africa and projects impacts of a series of vaccination campaigns. We also model the emergence of "Lassa-X" - a hypothetical pandemic Lassa virus variant - and project impacts of achieving 100 Days Mission vaccination targets. Our model predicted 2.7M (95% uncertainty interval: 2.1M-3.4M) Lassa virus infections annually, resulting over ten years in 2.0M (793.8K-3.9M) disability-adjusted life years (DALYs). The most effective vaccination strategy was a population-wide preventive campaign primarily targeting WHO-classified "endemic" districts. Under conservative vaccine efficacy assumptions, this campaign averted $20.1M ($8.2M-$39.0M) in lost DALY value and $128.2M ($67.2M-$231.9M) in societal costs (International dollars 2021). Reactive vaccination in response to local outbreaks averted just one-tenth the health-economic burden of preventive campaigns. In the event of Lassa-X emerging, spreading throughout West Africa and causing approximately 1.2M DALYs within two years, 100 Days Mission vaccination averted 22% of DALYs given a vaccine 70% effective against disease, and 74% of DALYs given a vaccine 70% effective against both infection and disease. These findings suggest how vaccination could alleviate Lassa fever's burden and assist in pandemic preparedness.
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The retrosplenial cortex (RSC) plays an important role in spatial cognition. RSC neurons exhibit a variety of spatial firing patterns and lesion studies have found that the RSC is necessary for spatial working memory tasks. However, little is known about how RSC neurons might encode spatial memory during a delay period. In the present study, we trained control rats and rats with excitotoxic lesions of the RSC on spatial alternation task with varying delay durations and in a separate group of rats, we recorded RSC neuronal activity as the rats performed the alternation task. We found that RSC lesions significantly impaired alternation performance, particularly at the longest delay duration. We also found that RSC neurons exhibited reliably different firing patterns throughout the delay periods preceding left and right trials, consistent with a working memory signal. These differential firing patterns were absent during the delay periods preceding errors. We also found that many RSC neurons exhibit a large spike in firing rate leading up to the start of the trial. Many of these trial start responses also differentiated left and right trials, suggesting that they could play a role in priming the 'go left' or 'go right' behavioral responses. Our results suggest that these firing patterns represent critical memory information that underlies the RSC role in spatial working memory.
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Plant organelle transcription has been studied for decades. As techniques advanced, so did the fields of mitochondrial and plastid transcriptomics. The current view is that organelle genomes are pervasively transcribed, irrespective of their size, content, structure, and taxonomic origin. However, little is known about the nature of organelle noncoding transcriptomes, including pervasively transcribed noncoding RNAs (ncRNAs). Next-generation sequencing data have uncovered small ncRNAs in the organelles of plants and other organisms, but long ncRNAs remain poorly understood. Here, we argue that publicly available third-generation long-read RNA sequencing data from plants can provide a fine-tuned picture of long ncRNAs within organelles. Indeed, given their bloated architectures, plant mitochondrial genomes are well suited for studying pervasive transcription of ncRNAs. Ultimately, we hope to showcase this new avenue of plant research while also underlining the limitations of the proposed approach.
Subject(s)
RNA, Antisense , RNA, Long Noncoding , RNA, Plant , High-Throughput Nucleotide Sequencing/methods , Organelles/genetics , Organelles/metabolism , Plants/genetics , RNA, Antisense/genetics , RNA, Long Noncoding/genetics , RNA, Plant/genetics , RNA-Seq/methods , Sequence Analysis, RNA/methods , Transcriptome/geneticsABSTRACT
OBJECTIVES: Metastatic prostate cancer (mPCa) patients with BRCA mutations benefit from targeted treatments (e.g., olaparib). Additionally, family members of affected patients have increased risk of hereditary cancers and benefit from early detection and prevention. International guidelines recommend genetic testing in mPCa, however, the value for money of testing mPCa patients and cascade testing of blood-related family members has not been assessed. In this context we evaluated the cost-effectiveness of germline BRCA testing in mPCa patients followed by cascade testing of first-degree relatives (FDRs) of mutation carriers. METHODS: We conducted a cost-utility analysis of germline BRCA testing using two scenarios: 1) testing mPCa patients only; 2) testing mPCa patients and first-degree relatives (FDRs) of those who test positive. A semi-Markov multi-health-state transition model was constructed using a lifetime time horizon. The analyses were performed from an Australian payer perspective. Decision uncertainty was characterized using probabilistic analyses. RESULTS: Compared with no testing, BRCA testing in mPCa was associated with an incremental cost of AU$3,731 and a gain of 0.014 quality-adjusted life years (QALYs), resulting in an incremental cost-effectiveness ratio (ICER) of AU$265,942/QALY. Extending testing to FDRs of variant positive patients resulted in an ICER of AU$16,392/QALY. Probability of cost-effectiveness at a willingness-to-pay of AU$75,000/QALY was 0% in the standalone mPCa analysis and 100% in the cascade testing analysis. CONCLUSION: BRCA testing when performed as a standalone strategy in patients with mPCa may not be cost-effective but demonstrates significant value for money after the inclusion of cascade testing of FDRs of mutation carriers.
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Improved soft tissue integration (STI) around dental implants is key for implant success. The formation of an early and long-lasting transmucosal seal around the implant abutment might help to prevent peri-implantitis, one of the major causes of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicular to the gingival tissue. In contrast, around dental implants, collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Previous studies have shown that nanostructured Ti surfaces improve gingival cell response in monolayer cell cultures. Here, we aimed at evaluating the implant-tissue interface using a 3D gingival tissue equivalent (GTE). First, we evaluated the GTE response to a nanostructured (NN) and machined Ti surface after the stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS), to simulate peri-implantitis conditions. Thus, GTE viability, through MTT assay, the release of metalloproteinase-1 (MMP1) and its inhibitor (TIMP1) through ELISA, and the gene expression of extracellular matrix turnover genes by real-time RT-PCR were analyzed. Second, GTE-implant interaction was characterized by serial block face scanning electron microscopy, and collagen-1 orientation at the tissue-implant interface was analyzed by immunofluorescence. While a similar GTE response to LPS stimulation was found for both implant surfaces, a higher proportion of collagen oriented perpendicular to the implant was observed on the NN implant surface. Thus, our results indicate that the nanostructuration of titanium dental implant abutments could allow the correct orientation of collagen fibers and greater soft tissue sealing, while keeping biocompatibility levels and LPS response comparable.
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Heterozygous de novo or inherited gain-of-function mutations in the MTOR gene cause Smith-Kingsmore syndrome (SKS). SKS is a rare autosomal dominant condition, and individuals with SKS display macrocephaly/megalencephaly, developmental delay, intellectual disability, and seizures. A few dozen individuals are reported in the literature. Here, we report a cohort of 28 individuals with SKS that represent nine MTOR pathogenic variants. We conducted a detailed natural history study and found pathophysiological deficits among individuals with SKS in addition to the common neurodevelopmental symptoms. These symptoms include sleep-wake disturbance, hyperphagia, and hyperactivity, indicative of homeostatic imbalance. To characterize these variants, we developed cell models and characterized their functional consequences. We showed that these SKS variants display a range of mechanistic target of rapamycin (mTOR) activities and respond to the mTOR inhibitor, rapamycin, differently. For example, the R1480_C1483del variant we identified here and the previously known C1483F are more active than wild-type controls and less responsive to rapamycin. Further, we showed that SKS mutations dampened circadian rhythms and low-dose rapamycin improved the rhythm amplitude, suggesting that optimal mTOR activity is required for normal circadian function. As SKS is caused by gain-of-function mutations in MTOR, rapamycin was used to treat several patients. While higher doses of rapamycin caused delayed sleep-wake phase disorder in a subset of patients, optimized lower doses improved sleep. Our study expands the clinical and molecular spectrum of SKS and supports further studies for mechanism-guided treatment options to improve sleep-wake behavior and overall health.
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PURPOSE: An early genetic diagnosis can guide the time-sensitive treatment of individuals with genetic epilepsies. However, most genetic diagnoses occur long after disease onset. We aimed to identify early clinical features suggestive of genetic diagnoses in individuals with epilepsy through large-scale analysis of full-text electronic medical records (EMR). METHODS: We extracted 89 million time-stamped standardized clinical annotations using Natural Language Processing from 4,572,783 clinical notes from 32,112 individuals with childhood epilepsy, including 1,925 individuals with known or presumed genetic epilepsies. We applied these features to train random forest models to predict SCN1A-related disorders and any genetic diagnosis. RESULTS: We identified 47,774 age-dependent associations of clinical features with genetic etiologies a median of 3.6 years prior to molecular diagnosis. Across all 710 genetic etiologies identified in our cohort, neurodevelopmental differences between 6-9 months increased the likelihood of a later molecular diagnosis fivefold (P<0.0001, 95% CI=3.55-7.42). A later diagnosis of SCN1A-related disorders (AUC=0.91) or an overall positive genetic diagnosis (AUC=0.82) could be reliably predicted using random forest models. CONCLUSION: Clinical features predictive of genetic epilepsies precede molecular diagnoses by up to several years in conditions with known precision treatments. An earlier diagnosis facilitated by automated EMR analysis has the potential for earlier targeted therapeutic strategies in the genetic epilepsies.
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BACKGROUND: Incomplete revascularization (ICR) has been associated with a worse prognosis after a percutaneous coronary intervention (PCI). In NSTEMI patients with multivessel disease (MVD) however, the benefit of a complete revascularization (CR) remains unclear. METHODS: Patients presenting with an NSTEMI and MVD were selected from the global e-ULTIMASTER registry and grouped according to completeness of revascularization at index hospitalization discharge. The primary endpoint was the patient oriented composite endpoint (POCE) defined as all death, any myocardial infarction, and any revascularization at 1 year. Target lesion failure (TLF) was defined as the composite of cardiac death, target vessel related myocardial infarction and clinically driven target lesion revascularization. Inverse propensity score weighting (IPSW) was performed to harmonize the patient's baseline characteristics between the groups. RESULTS: CR was achieved in 1800 patients (47.0 %) and ICR in 2032 patients (53.0 %). The incidence of POCE at 1 year was lower in the CR group compared to the ICR group: 7.0 % vs. 12.9 %, p < 0.0001. Similarly for TLF at 1 year: 3.6 % vs. 5.5 %, p < 0.01. After IPSW, the incidence of POCE was 7.7 % vs. 12.0 %, p < 0.0001, due to a lower all-cause mortality: 2.7 % vs. 4.2 %, p = 0.02 and less revascularizations: 4.9 % vs. 7.9 %, p < 0.001. The incidence of TLF was no longer statistically significant: CR 3.9 % vs. IR 5.0 %, p = 0.10. CONCLUSIONS: Patients with a NSTEMI and multi vessel disease undergoing a percutaneous coronary revascularization with a complete revascularization during index hospitalization have better 1-year clinical outcomes. Randomized studies are warranted to confirm these results.
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A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.
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Heavy nuclides like uranium and their decay products are commonly found in nuclear industries and can pose a significant health risk to humans due to their alpha-emitting properties. Traditional alpha detectors require close contact with the contaminated surface, which can be time-consuming, labour-intensive, and put personnel at risk. Remote detection is urgently needed but very challenging. To this end, a candidate detection mechanism is alpha-induced radio-luminescence. This approach uses the emission of photons from radio-ionised excited nitrogen molecules to imply the presence of alpha emitters from a distance. Herein, the use of this phenomenon to remotely image various alpha emitters with unparalleled levels of sensitivity and spatial accuracy is demonstrated. Notably, the system detected a 29 kBq Am-241 source at a distance of 3 m within 10 min. Furthermore, it demonstrated the capability to discern a 29 kBq source positioned 7 cm away from a 3 MBq source at a 2 m distance. Additionally, a 'sandwich' filter structure is described that incorporates an absorptive filter between two interference filters to enhance the ambient light rejection. The testing of the system is described in different lighting environments, including room light and inside a glovebox. This method promises safer and more efficient alpha monitoring, with applications in nuclear forensics, waste management and decommissioning.
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The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were tested and found to directly interact with the proteasome. ProEnd's extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer. Link: http://proend.org/.
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Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, and transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1 , while germline GATA1s mutations in a non-T21 context lead to congenital cytopenias without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically, but this interaction has been challenging to study in part due to limited human cell and murine models. To dissect the developmental impacts of GATA1s on hematopoiesis in euploid and T21 cells, we performed a single-cell RNA-sequencing timecourse on hematopoietic progenitors (HPCs) derived from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. These HPCs were surprisingly heterogeneous and displayed spontaneous lineage skew apparently dictated by T21 and/or GATA1s. In euploid cells, GATA1s nearly eliminated erythropoiesis, impaired MK maturation, and promoted an immature myelopoiesis, while in T21 cells, GATA1s appeared to compete with the enhanced erythropoiesis and suppressed megakaryopoiesis driven by T21 to give rise to immature erythrocytes, MKs, and myeloid cells. T21 and GATA1s both disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. These findings in an isogenic system can thus be attributed specifically to T21 and GATA1s and suggest that these genetic changes together enhance HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.
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Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding proteasome activation mechanisms. Most proteasome activator complexes use the C-terminal HbYX motif to bind and trigger gate-opening in the 20S proteasome. This study defines a critical molecular interaction in the HbYX mechanism that triggers gate opening. Here, we focus on the Hb site interaction and find it plays a surprisingly central and crucial role in driving the allosteric conformational changes that induce gate opening in the archaeal 20S. We examined the cryo-EM structure of two mutant archaeal proteasomes, αV24Y T20S and αV24F T20S. These two mutants were engineered to place a bulky aromatic residue in the HbYX hydrophobic pocket and both mutants are highly active, though their mechanisms of activation are undefined. Collectively, our findings indicate that the interaction between the Hb group of the HbYX motif and its corresponding hydrophobic pocket is sufficient to induce gate opening in a mechanistically similar way to the HbYX motif. The involved activation mechanism appears to involve expansion of this hydrophobic binding site affecting the state of the IT switch to triggering gate-opening. Furthermore, we show that the canonical αK66 residue, understood to be critical for proteasome activator binding, plays a key role in stabilizing the open gate, irrespective of activator binding. This study differentiates between the residues in the HbYX motif that support binding interactions ("YX") versus those that allosterically contribute to gate opening (Hb). The insights reported here will guide future drug development efforts, particularly in designing small molecule proteasome activators, by targeting the identified hydrophobic pocket.
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40 years ago, organelle genomes were assumed to be streamlined and, perhaps, unexciting remnants of their prokaryotic past. However, the field of organelle genomics has exposed an unparallel diversity in genome architecture (i.e. genome size, structure, and content). The transcription of these eccentric genomes can be just as elaborate - organelle genomes are pervasively transcribed into a plethora of RNA types. However, while organelle protein-coding genes are known to produce polycistronic transcripts that undergo heavy posttranscriptional processing, the nature of organelle noncoding transcriptomes is still poorly resolved. Here, we review how wet-lab experiments and second-generation sequencing data (i.e. short reads) have been useful to determine certain types of organelle RNAs, particularly noncoding RNAs. We then explain how third-generation (long-read) RNA-Seq data represent the new frontier in organelle transcriptomics. We show that public repositories (e.g. NCBI SRA) already contain enough data for inter-phyla comparative studies and argue that organelle biologists can benefit from such data. We discuss the prospects of using publicly available sequencing data for organelle-focused studies and examine the challenges of such an approach. We highlight that the lack of a comprehensive database dedicated to organelle genomics/transcriptomics is a major impediment to the development of a field with implications in basic and applied science.
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Photostasis is the light-dependent maintenance of energy balance associated with cellular homeostasis in photoautotrophs. We review evidence that illustrates how photosynthetic adaptation in polar photoautrophs such as aquatic green algae, cyanobacteria, boreal conifers as well as terrestrial angiosperms exhibit an astonishing plasticity in structure and function of the photosynthetic apparatus. This plasticity contributes to the maintenance of photostasis, which is essential for the long-term survival in the seemingly inhospitable Antarctic and Arctic habitats. However, evidence indicates that polar photoautrophic species exhibit different functional solutions for the maintenance of photostasis. We suggest that this reflects, in part, the genetic diversity symbolized by inherent genetic redundancy characteristic of polar photoautotrophs which enhances their survival in a thermodynamically challenging environment.
Subject(s)
Adaptation, Physiological , Photosynthesis , Photosynthesis/physiology , Arctic Regions , Antarctic Regions , Cyanobacteria/physiology , Cyanobacteria/genetics , Chlorophyta/physiology , Chlorophyta/genetics , Ecosystem , Light , Magnoliopsida/physiology , Magnoliopsida/genetics , Tracheophyta/physiology , Tracheophyta/geneticsABSTRACT
Background: The abdominal donor site is the most common flap used for breast reconstruction, with flap necrosis a feared complication. The technique of surgical 'delay' involves the inducing of relative ischemia to promote neovascularisation, amongst other metabolic adaptations, and has been used to augment flap vascularity and reduce this complication. There is significant variability in the manner in which flap surgery and surgical delay may be performed, such as the vessels ligated, the presence and degree of flap elevation, and the decision to harvest muscle with the flap, amongst other factors. A formal review of techniques, however, has not yet been performed, and there is no consensus as to the optimal technique for surgical delay. Methods: A scoping review of the current literature was undertaken to determine the optimal surgical delay technique in abdominal-based flap surgery. A literature search was conducted across PubMed, Embase, Cochrane, and Medline databases. Data regarding the type of flap surgery, delay techniques, and corresponding clinical outcomes was collected and categorised by technique type. Results: Nine studies met the inclusion criteria and were included for review. Levels of evidence and rates of complications were compared. The range of surgical delay techniques reported in the literature was described. Surgical delay was found to reduce overall complication rates, and the available data suggests it may be particularly beneficial in high-risk patients. Conclusions: The current data support delay as a viable method for reducing rates of complications. Further studies and data are required to compare surgical delay techniques and determine the benefit delay may pose to patients with risk factors.