Recurrent Glioblastoma-Molecular Underpinnings and Evolving Treatment Paradigms.

Glioblastoma is the most common and lethal central nervous system malignancy with a median survival after progression of only 6-9 months. Major biochemical mechanisms implicated in glioblastoma recurrence include aberrant molecular pathways, a recurrence-inducing tumor microenvironment, and epigenetic modifications. Contemporary standard-of-care (surgery, radiation, chemotherapy, and tumor treating fields) helps to control the primary tumor but rarely prevents relapse. Cytoreductive treatment such as surgery has shown benefits in recurrent glioblastoma; however, its use remains controversial. Several innovative treatments are emerging for recurrent glioblastoma, including checkpoint inhibitors, chimeric antigen receptor T cell therapy, oncolytic virotherapy, nanoparticle delivery, laser interstitial thermal therapy, and photodynamic therapy. This review seeks to provide readers with an overview of (1) recent discoveries in the molecular basis of recurrence; (2) the role of surgery in treating recurrence; and (3) novel treatment paradigms emerging for recurrent glioblastoma.

Spatial Proteomics of Single Hepatocytes with Multiplexed Data-Independent Acquisition (mDIA).

Spatially resolved mass spectrometry-based proteomics at single-cell resolution promises to provide insights into biological heterogeneity. We describe a protocol based on multiplexed data-independent acquisition (mDIA) with dimethyl labeling to enhance proteome depth, accuracy, and throughput while minimizing costs. It enables high-quality proteome analysis of single isolated hepatocytes and utilizes liver zonation for single-cell proteomics benchmarking. This adaptable, modular protocol will promote the use of single-cell proteomics in spatial biology.

The Case for Neurosurgical Intervention in Cancer Neuroscience.

The emerging field of cancer neuroscience reshapes our understanding of the intricate relationship between the nervous system and cancer biology; this new paradigm is likely to fundamentally change and advance neuro-oncological care. The profound interplay between cancers and the nervous system is reciprocal: Cancer growth can be induced and regulated by the nervous system; conversely, tumors can themselves alter the nervous system. Such crosstalk between cancer cells and the nervous system is evident in both the peripheral and central nervous systems. Recent advances have uncovered numerous direct neuron-cancer interactions at glioma-neuronal synapses, paracrine mechanisms within the tumor microenvironment, and indirect neuroimmune interactions. Neurosurgeons have historically played a central role in neuro-oncological care, and as the field of cancer neuroscience is becoming increasingly established, the role of neurosurgical intervention is becoming clearer. Examples include peripheral denervation procedures, delineation of neuron-glioma networks, development of neuroprostheses, neuromodulatory procedures, and advanced local delivery systems. The present review seeks to highlight key cancer neuroscience mechanisms with neurosurgical implications and outline the future role of neurosurgical intervention in cancer neuroscience.

A fast thermal desorption unit for micro thermal desorption tubes, Part I: Development of the system and proof of concept measurements with hyper-fast gas chromatography.

A system consisting of a thermal desorption unit (TDU) and micro thermal desorption tubes (µTD-tubes, 1.4 mm I.D., 10mg Tenax TA) for fast desorption of analytes was developed for the efficient combination of hyper fast gas chromatography with thermal desorption. The fast desorption is achieved by a significantly reduced thermal mass compared to conventional thermal desorption tubes. Therefore, extremely fast heating and cooling cycles are possible. Proof of concept measurements combining the new setup with a flow-field thermal gradient gas chromatograph (FF-TG-GC) and FID detection show good precision and linearity with R2≥0.995 in the analysis of an n-alkane mix (C8-C20). Thermal desorption occurs within 12s. The impact of reduced µTD-tube dimensions on desorption time, full width at half maximum (FWHM), breakthrough volumes, tube flow rates ergo linear velocities, porosity and back pressure is discussed.

AI-based opportunistic quantitative image analysis of lung cancer screening CTs to reduce disparities in osteoporosis screening.

Osteoporosis is underdiagnosed, especially in ethnic and racial minorities who are thought to be protected against bone loss, but often have worse outcomes after an osteoporotic fracture. We aimed to determine the prevalence of osteoporosis by opportunistic CT in patients who underwent lung cancer screening (LCS) using non-contrast CT in the Northeastern United States. Demographics including race and ethnicity were retrieved. We assessed trabecular bone and body composition using a fully-automated artificial intelligence algorithm. ROIs were placed at T12 vertebral body for attenuation measurements in Hounsfield Units (HU). Two validated thresholds were used to diagnose osteoporosis: high-sensitivity threshold (115-165 HU) and high specificity threshold (<115 HU). We performed descriptive statistics and ANOVA to compare differences across sex, race, ethnicity, and income class according to neighborhoods' mean household incomes. Forward stepwise regression modeling was used to determine body composition predictors of trabecular attenuation. We included 3708 patients (mean age 64 ± 7 years, 54 % males) who underwent LCS, had available demographic information and an evaluable CT for trabecular attenuation analysis. Using the high sensitivity threshold, osteoporosis was more prevalent in females (74 % vs. 65 % in males, p < 0.0001) and Whites (72 % vs 49 % non-Whites, p < 0.0001). However, osteoporosis was present across all races (38 % Black, 55 % Asian, 56 % Hispanic) and affected all income classes (69 %, 69 %, and 91 % in low, medium, and high-income class, respectively). High visceral/subcutaneous fat-ratio, aortic calcification, and hepatic steatosis were associated with low trabecular attenuation (p < 0.01), whereas muscle mass was positively associated with trabecular attenuation (p < 0.01). In conclusion, osteoporosis is prevalent across all races, income classes and both sexes in patients undergoing LCS. Opportunistic CT using a fully-automated algorithm and uniform imaging protocol is able to detect osteoporosis and body composition without additional testing or radiation. Early identification of patients traditionally thought to be at low risk for bone loss will allow for initiating appropriate treatment to prevent future fragility fractures. CLINICALTRIALS.GOV IDENTIFIER: N/A.

Ultrastructure Expansion Microscopy applied to C. elegans embryos.

Visualization of organelles using expansion microscopy has been previously applied to Caenorhadbitis elegans adult gonads or worms. However, its application to embryos has remained a challenge due to the protective eggshell barrier. Here, by combining freeze-cracking and ultrastructure expansion microscopy (U-ExM), we demonstrate a four-time isotropic expansion of C. elegans embryos. As an example structure, we chose the nuclear pore and demonstrate that we achieve sufficient resolution to distinguish them individually. Our work provides proof of principle for U-ExM in C. elegans embryos, which will be applicable for imaging a wide range of cellular structures in this model system.

Clinical Outcome following Management of Severe Osteomyelitis due to Pin Site Infection.

Aim: This study has investigated cases of pin site infection (PSI) which required surgery for persistent osteomyelitis (OM) despite pin removal. Materials and methods: Patients requiring surgery for OM after PSI between 2011 and 2021 were included in this retrospective cohort study. Single-stage surgery was performed in accordance with a protocol at one institution. This involved deep sampling, debridement, implantation of local antibiotics, culture-specific systemic antibiotics and soft tissue closure. A successful outcome was defined as an infection-free interval of at least 24 months following surgery. Results: Twenty-seven patients were identified (the sites were 22 tibias, 2 humeri, 2 calcanei, 1 radius); about 85% of them were males with a median age of 53.9 years. The majority of infections (21/27) followed fracture treatment. Fifteen patients were classified as BACH uncomplicated and 12 were BACH complex. Staphylococci were the most common pathogens, polymicrobial infections were detected in five cases (19%). Seven patients required flap coverage which was performed in the same operation.After a median of 3.99 years (2.00-8.05) follow-up, all patients remained infection free at the site of the former OM. Wound leakage after local antibiotic treatment was seen in 3/27 (11.1%) cases but did not require further treatment. Conclusion: Osteomyelitis after PSI is uncommon but has major implications for the patient as 7 patients needed flap coverage. This reinforces the need for careful pin placement and pin site care to prevent deep infection. These infections were treated in accordance with a protocol and were not managed simply by curettage. All patients treated in this manner remained infection-free after a minimum follow-up of 2 years suggesting that this protocol is effective. Clinical significance: Pin site infection is a very common complication in external fixation. The sequela of a chronic pin site OM is rare but the implications to the patient are huge. In this series, more than a quarter of patients required flap coverage as part of the treatment of the deep infection. How to cite this article: Frank FA, Pomeroy E, Hotchen AJ, et al. Clinical Outcome following Management of Severe Osteomyelitis due to Pin Site Infection. Strategies Trauma Limb Reconstr 2024;19(1):21-25.

mitoBKCa is functionally expressed in murine and human breast cancer cells and potentially contributes to metabolic reprogramming.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

Acute physiological, biomechanical, and perceptual responses of runners wearing downward-curved carbon fiber insoles.

In a randomized controlled cross-over study ten male runners (26.7 ± 4.9 years; recent 5-km time: 18:37 ± 1:07 min:s) performed an incremental treadmill test (ITT) and a 3-km time trial (3-km TT) on a treadmill while wearing either carbon fiber insoles with downwards curvature or insoles made of butyl rubber (control condition) in light road racing shoes (Saucony Fastwitch 9). Oxygen uptake, respiratory exchange ratio, heart rate, blood lactate concentration, stride frequency, stride length and time to exhaustion were assessed during ITT. After ITT, all runners rated their perceived exertion, perceived shoe comfort and perceived shoe performance. Running time, heart rate, blood lactate levels, stride frequency and stride length were recorded during, and shoe comfort and shoe performance after, the 3-km TT. All parameters obtained during or after the ITT did not differ between the two conditions [range: p = 0.188 to 0.948 (alpha value: 0.05); Cohen's d = 0.021 to 0.479] despite the rating of shoe comfort showing better scores for the control insoles (p = 0.001; d = -1.646). All parameters during and after the 3-km TT showed no differences (p = 0.200 to 1.000; d = 0.000 to 0.501) between both conditions except for shoe comfort showing better scores for control insoles (p = 0.017; d = -0.919). Running with carbon fiber insoles with downwards curvature did not change running performance or any submaximal or maximal physiological or biomechanical parameter and perceived exertion compared to control condition. Shoe comfort is impaired while running with carbon fiber insoles. Wearing carbon fiber insoles with downwards curvature during treadmill running is not beneficial when compared to running with control insoles.

Tools for Measuring Photosynthesis at Different Scales.

Measurements of in vivo photosynthesis are powerful tools that probe the largest fluxes of carbon and energy in an illuminated leaf, but often the specific techniques used are so varied and specialized that it is difficult for researchers outside the field to select and perform the most useful assays for their research questions. The goal of this chapter is to provide a broad overview of the current tools available for the study of photosynthesis, both in vivo and in vitro, so as to provide a foundation for selecting appropriate techniques, many of which are presented in detail in subsequent chapters. This chapter will also organize current methods into a comparative framework and provide examples of how they have been applied to research questions of broad agronomical, ecological, or biological importance. This chapter closes with an argument that the future of in vivo measurements of photosynthesis lies in the ability to use multiple methods simultaneously and discusses the benefits of this approach to currently open physiological questions. This chapter, combined with the relevant methods chapters, could serve as a laboratory course in methods in photosynthesis research or as part of a more comprehensive laboratory course in general plant physiology methods.

Using Carbon Stable Isotopes to Study C3 and C4 Photosynthesis: Models and Calculations.

Stable carbon isotopes are a powerful tool to study photosynthesis. Initial applications consisted of determining isotope ratios of plant biomass using mass spectrometry. Subsequently, theoretical models relating C isotope values to gas exchange characteristics were introduced and tested against instantaneous online measurements of 13C photosynthetic discrimination. Beginning in the twenty-first century, laser absorption spectroscopes with sufficient precision for determining isotope mixing ratios became commercially available. This has allowed collection of large data sets at lower cost and with unprecedented temporal resolution. More data and accompanying knowledge have permitted refinement of 13C discrimination model equations, but often at the expense of increased model complexity and difficult parametrization. This chapter describes instantaneous online measurements of 13C photosynthetic discrimination, provides recommendations for experimental setup, and presents a thorough compilation of equations available to researchers. We update our previous 2018 version of this chapter by including recently improved descriptions of (photo)respiratory processes and associated fractionations. We discuss the capabilities and limitations of the diverse 13C discrimination model equations and provide guidance for selecting the model complexity needed for different applications.

Safety and effectiveness of glycoprotein IIb/IIIa inhibitors in acute coronary syndromes: insights from the SPUM-ACS study.

AIMS: Data on Glycoprotein IIb/IIIa inhibitors (GPI) use in real world ACS patients following the introduction of potent P2Y12 inhibitors and newer generation stents are scant. Here, we aimed to assess the utilization, effectiveness, and safety of GPI in a large prospective multi-centre cohort of contemporary ACS patients. METHODS AND RESULTS: SPUM-ACS prospectively recruited patients presenting with ACS between 2009 and 2017. The primary endpoint of the present study was major adverse cardiovascular events (MACE), a composite of all-cause death, non-fatal myocardial infarction (MI) and non-fatal stroke at one year. Secondary endpoints were defined as any bleeding events, BARC 3-5 bleeding, and net adverse cardiovascular events (NACE). A total of 4395 ACS patients were included in the analysis. GPI-treated patients had more total coronary artery occlusion (56% vs 35%, p<0.001) and thrombus (60% vs 35%, p<0.001) at angiography. Among the propensity score matched (PSM) population (1992 patients equally split into two groups), GPI-treated patients showed lower risk of MACE (PSM adjusted HR 0.70, 95% CI 0.49-0.99), but a higher risk of any (PSM adj HR 1.46, 95% CI 1.06-1.99) and major bleedings (PSM adj HR 1.73, 95% CI 1.09-2.76), resulting in a neutral effect on NACE (PSM adj HR 0.87, 95% CI 0.65-1.17). These results remained consistent across all subgroups. CONCLUSION: In patients with ACS undergoing PCI and receiving potent P2Y12 inhibitors, we observed a reduced risk of MACE and an increased risk of major bleedings at 1 year in patients treated with GPI. Although the routine use of GPI is currently not recommended, they might be considered in selected patients following a personalized balancing between ischaemic and bleeding risks.

MAPK-mediated PHGDH induction is essential for melanoma formation and represents an actionable vulnerability.

Overexpression of PHGDH, the rate-limiting enzyme in the serine synthesis pathway, promotes melanomagenesis, melanoma cell proliferation, and survival of metastases in serine-low environments such as the brain. While PHGDH amplification explains PHGDH overexpression in a subset of melanomas, we find that PHGDH levels are universally increased in melanoma cells due to oncogenic BRAFV600E promoting PHGDH transcription through mTORC1-mediated translation of ATF4. Importantly, PHGDH expression was critical for melanomagenesis as depletion of PHGDH in genetic mouse models blocked melanoma formation. Despite BRAFV600E-mediated upregulation, PHGDH was further induced by exogenous serine restriction. Surprisingly, BRAFV600E inhibition diminished serine restriction-mediated PHGDH expression by preventing ATF4 induction, creating a potential vulnerability whereby melanoma cells could be specifically starved of serine by combining BRAFV600E inhibition with exogenous serine restriction. Indeed, we show that this combination promoted cell death in vitro and attenuated melanoma growth in vivo. This study identified a melanoma cell-specific PHGDH-dependent vulnerability.

Photosynthetic Gas Exchange in Land Plants at the Leaf Level.

Leaf-level gas exchange enables insights into the physiology and in vivo biochemical processes of plants. Advances in infrared gas analysis have resulted in user-friendly off-the-shelf gas exchange systems that allow researchers to collect physiological measurements with the push of a few buttons. Here, I describe how to set up the gas exchange equipment, what to pay attention to while making measurements, and provide some guidelines on how to analyze and interpret the data obtained.

Dynamic Covalent Amphiphilic Polymer Conetworks Based on End-Linked Pluronic F108: Preparation, Characterization, and Evaluation as Matrices for Gel Polymer Electrolytes.

We present the development of a platform of well-defined, dynamic covalent amphiphilic polymer conetworks (APCN) based on an α,ω-dibenzaldehyde end-functionalized linear amphiphilic poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (PEG-b-PPG-b-PEG, Pluronic) copolymer end-linked with a triacylhydrazide oligo(ethylene glycol) triarmed star cross-linker. The developed APCNs were characterized in terms of their rheological (increase in the storage modulus by a factor of 2 with increase in temperature from 10 to 50 °C), self-healing, self-assembling, and mechanical properties and evaluated as a matrix for gel polymer electrolytes (GPEs) in both the stretched and unstretched states. Our results show that water-loaded APCNs almost completely self-mend, self-organize at room temperature into a body-centered cubic structure with long-range order exhibiting an aggregation number of around 80, and display an exceptional room temperature stretchability of ∼2400%. Furthermore, ionic liquid-loaded APCNs could serve as gel polymer electrolytes (GPEs), displaying a substantial ion conductivity in the unstretched state, which was gradually reduced upon elongation up to a strain of 4, above which it gradually increased. Finally, it was found that recycled (dissolved and re-formed) ionic liquid-loaded APCNs could be reused as GPEs preserving 50-70% of their original ion conductivity.

Endovascular Applications for the Management of High-Grade Gliomas in the Modern Era.

High-grade gliomas (HGGs) have a poor prognosis and are difficult to treat. This review examines the evolving landscape of endovascular therapies for HGGs. Recent advances in endovascular catheter technology and delivery methods allow for super-selective intra-arterial cerebral infusion (SSIACI) with increasing precision. This treatment modality may offer the ability to deliver anti-tumoral therapies directly to tumor regions while minimizing systemic toxicity. However, challenges persist, including blood-brain barrier (BBB) penetration, hemodynamic complexities, and drug-tumor residence time. Innovative adjunct techniques, such as focused ultrasound (FUS) and hyperosmotic disruption, may facilitate BBB disruption and enhance drug penetration. However, hemodynamic factors that limit drug residence time remain a limitation. Expanding therapeutic options beyond chemotherapy, including radiotherapy and immunobiologics, may motivate future investigations. While preclinical and clinical studies demonstrate moderate efficacy, larger randomized trials are needed to validate the clinical benefits. Additionally, future directions may involve endovascular sampling for peri-tumoral surveillance; changes in drug formulations to prolong residence time; and the exploration of non-pharmaceutical therapies, like radioembolization and photodynamic therapy. Endovascular strategies hold immense potential in reshaping HGG treatment paradigms, offering targeted and minimally invasive approaches. However, overcoming technical challenges and validating clinical efficacy remain paramount for translating these advancements into clinical care.

BioPrev-C - development and validation of a contemporary prostate cancer risk calculator.

Objectives: To develop a novel biopsy prostate cancer (PCa) prevention calculator (BioPrev-C) using data from a prospective cohort all undergoing mpMRI targeted and transperineal template saturation biopsy. Materials and methods: Data of all men who underwent prostate biopsy in our academic tertiary care center between 11/2016 and 10/2019 was prospectively collected. We developed a clinical prediction model for the detection of high-grade PCa (Gleason score ≥7) based on a multivariable logistic regression model incorporating age, PSA, prostate volume, digital rectal examination, family history, previous negative biopsy, 5-alpha-reductase inhibitor use and MRI PI-RADS score. BioPrev-C performance was externally validated in another prospective Swiss cohort and compared with two other PCa risk-calculators (SWOP-RC and PBCG-RC). Results: Of 391 men in the development cohort, 157 (40.2%) were diagnosed with high-grade PCa. Validation of the BioPrev C revealed good discrimination with an area under the curve for high-grade PCa of 0.88 (95% Confidence Interval 0.82-0.93), which was higher compared to the other two risk calculators (0.71 for PBCG and 0.84 for SWOP). The BioPrev-C revealed good calibration in the low-risk range (0 - 0.25) and moderate overestimation in the intermediate risk range (0.25 - 0.75). The PBCG-RC showed good calibration and the SWOP-RC constant underestimation of high-grade PCa over the whole prediction range. Decision curve analyses revealed a clinical net benefit for the BioPrev-C at a clinical meaningful threshold probability range (≥4%), whereas PBCG and SWOP calculators only showed clinical net benefit above a 30% threshold probability. Conclusion: BiopPrev-C is a novel contemporary risk calculator for the prediction of high-grade PCa. External validation of the BioPrev-C revealed relevant clinical benefit, which was superior compared to other well-known risk calculators. The BioPrev-C has the potential to significantly and safely reduce the number of men who should undergo a prostate biopsy.

Interdependence of movement amplitude and tempo during self-paced finger tapping: evaluation of a preferred velocity hypothesis.

This study examined the relation between movement amplitude and tempo during self-paced rhythmic finger tapping to test a preferred velocity account of the preferred tempo construct. Preferred tempo refers to the concept that individuals have preferences for the pace of actions or events in their environment (e.g., the desired pace of walking or tempo of music). The preferred velocity hypothesis proposes that assessments of preferred tempo do not represent a pure time preference independent of spatial movement characteristics, but rather reflects a preference for an average movement velocity, predicting that preferred tempo will depend on movement amplitude. We tested this by having participants first perform a novel spontaneous motor amplitude (SMA) task in which they repetitively tapped their finger at their preferred amplitude without instructions about tapping tempo. Next, participants completed the spontaneous motor tempo (SMT) task in which they tapped their finger at their preferred tempo without instructions about tapping amplitude. Finally, participants completed a target amplitude version of the SMT task where they tapped at their preferred tempo at three target amplitudes (low, medium, and high). Participants (1) produced similar amplitudes and tempi regardless of instructions to produce either their preferred amplitude or preferred tempo, maintaining the same average movement velocity across SMA and SMT tasks and (2) altered their preferred tempo for different target amplitudes in the direction predicted by their estimated preferred velocity from the SMA and SMT tasks. Overall, results show the interdependence of movement amplitude and tempo in tapping assessments of preferred tempo.

The interplay of short-term mesophyll and stomatal conductance responses under variable environmental conditions.

Understanding the short-term responses of mesophyll conductance (gm ) and stomatal conductance (gsc ) to environmental changes remains a challenging yet central aspect of plant physiology. This review synthesises our current knowledge of these short-term responses, which underpin CO2 diffusion within leaves. Recent methodological advances in measuring gm using online isotopic discrimination and chlorophyll fluorescence have improved our confidence in detecting short-term gm responses, but results need to be carefully evaluated. Environmental factors like vapour pressure deficit and CO2 concentration indirectly impact gm through gsc changes, highlighting some of the complex interactions between the two parameters. Evidence suggests that short-term responses of gm are not, or at least not fully, mechanistically linked to changes in gsc , cautioning against using gsc as a reliable proxy for gm . The overarching challenge lies in unravelling the mechanistic basis of short-term gm responses, which will contribute to the development of accurate models bridging laboratory insights with broader ecological implications. Addressing these gaps in understanding is crucial for refining predictions of gm behaviour under changing environmental conditions.