Circadian coupling of mitochondria in a deep-diving mammal.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Biological timekeeping in polar environments: lessons from terrestrial vertebrates.

The polar regions receive less solar energy than anywhere else on Earth, with the greatest year-round variation in daily light exposure; this produces highly seasonal environments, with short summers and long, cold winters. Polar environments are also characterised by a reduced daily amplitude of solar illumination. This is obvious around the solstices, when the Sun remains continuously above (polar 'day') or below (polar 'night') the horizon. Even at the solstices, however, light levels and spectral composition vary on a diel basis. These features raise interesting questions about polar biological timekeeping from the perspectives of function and causal mechanism. Functionally, to what extent are evolutionary drivers for circadian timekeeping maintained in polar environments, and how does this depend on physiology and life history? Mechanistically, how does polar solar illumination affect core daily or seasonal timekeeping and light entrainment? In birds and mammals, answers to these questions diverge widely between species, depending on physiology and bioenergetic constraints. In the high Arctic, photic cues can maintain circadian synchrony in some species, even in the polar summer. Under these conditions, timer systems may be refined to exploit polar cues. In other instances, temporal organisation may cease to be dominated by the circadian clock. Although the drive for seasonal synchronisation is strong in polar species, reliance on innate long-term (circannual) timer mechanisms varies. This variation reflects differing year-round access to photic cues. Polar chronobiology is a productive area for exploring the adaptive evolution of daily and seasonal timekeeping, with many outstanding areas for further investigation.

Diversified regulation of circadian clock gene expression following whole genome duplication.

Across taxa, circadian control of physiology and behavior arises from cell-autonomous oscillations in gene expression, governed by a networks of so-called 'clock genes', collectively forming transcription-translation feedback loops. In modern vertebrates, these networks contain multiple copies of clock gene family members, which arose through whole genome duplication (WGD) events during evolutionary history. It remains unclear to what extent multiple copies of clock gene family members are functionally redundant or have allowed for functional diversification. We addressed this problem through an analysis of clock gene expression in the Atlantic salmon, a representative of the salmonids, a group which has undergone at least 4 rounds of WGD since the base of the vertebrate lineage, giving an unusually large complement of clock genes. By comparing expression patterns across multiple tissues, and during development, we present evidence for gene- and tissue-specific divergence in expression patterns, consistent with functional diversification of clock gene duplicates. In contrast to mammals, we found no evidence for coupling between cortisol and circadian gene expression, but cortisol mediated non-circadian regulated expression of a subset of clock genes in the salmon gill was evident. This regulation is linked to changes in gill function necessary for the transition from fresh- to sea-water in anadromous fish. Overall, this analysis emphasises the potential for a richly diversified clock gene network to serve a mixture of circadian and non-circadian functions in vertebrate groups with complex genomes.

Current practises and the future of robotic surgical training.

INTRODUCTION: This study reviews the current state of robotic surgery training for surgeons, including the various curricula, training methods, and tools available, as well as the challenges and limitations of these. METHODS: The authors carried out a literature search across PubMed, MEDLINE, and Google Scholar using keywords related to 'robotic surgery', 'computer-assisted surgery', 'simulation', 'virtual reality', 'surgical training', and 'surgical education'. Full text analysis was performed on 112 articles. TRAINING PROGRAMMES: The training program for robotic surgery should focus on proficiency, deliberation, and distribution principles. The curricula can be broadly split up into pre-console and console-side training. Pre-Console and Console-Side Training: Simulation training is an important aspect of robotic surgery training to improve technical skill acquisition and reduce mental workload, which helps prepare trainees for live procedures. OPERATIVE PERFORMANCE ASSESSMENT: The study also discusses the various validated assessment tools used for operative performance assessments. FUTURE ADVANCES: Finally, the authors propose potential future directions for robotic surgery training, including the use of emerging technologies such as AI and machine learning for real-time feedback, remote mentoring, and augmented reality platforms like Proximie to reduce costs and overcome geographic limitations. CONCLUSION: Standardisation in trainee performance assessment is needed. Each of the robotic curricula and platforms has strengths and weaknesses. The ERUS Robotic Curriculum represents an evidence-based example of how to implement training from novice to expert. Remote mentoring and augmented reality platforms can overcome the challenges of high equipment costs and limited access to experts. Emerging technologies offer promising advancements for real-time feedback and immersive training environments, improving patient outcomes.

Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.

Labeling Preferences of Diazirines with Protein Biomolecules.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Photoperiodic induction without light-mediated circadian entrainment in a High Arctic resident bird.

Organisms use changes in photoperiod to anticipate and exploit favourable conditions in a seasonal environment. While species living at temperate latitudes receive day length information as a year-round input, species living in the Arctic may spend as much as two-thirds of the year without experiencing dawn or dusk. This suggests that specialised mechanisms may be required to maintain seasonal synchrony in polar regions. Svalbard ptarmigan (Lagopus muta hyperborea) are resident at 74-81°N latitude. They spend winter in constant darkness (DD) and summer in constant light (LL); extreme photoperiodic conditions under which they do not display overt circadian rhythms. Here, we explored how Arctic adaptation in circadian biology affects photoperiodic time measurement in captive Svalbard ptarmigan. For this purpose, DD-adapted birds, showing no circadian behaviour, either remained in prolonged DD, were transferred into a simulated natural photoperiod (SNP) or were transferred directly into LL. Birds transferred from DD to LL exhibited a strong photoperiodic response in terms of activation of the hypothalamic thyrotropin-mediated photoperiodic response pathway. This was assayed through expression of the Eya3, Tshß and deiodinase genes, as well as gonadal development. While transfer to SNP established synchronous diurnal activity patterns, activity in birds transferred from DD to LL showed no evidence of circadian rhythmicity. These data show that the Svalbard ptarmigan does not require circadian entrainment to develop a photoperiodic response involving conserved molecular elements found in temperate species. Further studies are required to define how exactly Arctic adaptation modifies seasonal timer mechanisms.

Colour as a signal for entraining the mammalian circadian clock.

Twilight is characterised by changes in both quantity ("irradiance") and quality ("colour") of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue-yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.

The cost of circadian desynchrony: Evidence, insights and open questions.

Coordinated daily rhythms are evident in most aspects of our physiology, driven by internal timing systems known as circadian clocks. Our understanding of how biological clocks are built and function has grown exponentially over the past 20 years. With this has come an appreciation that disruption of the clock contributes to the pathophysiology of numerous diseases, from metabolic disease to neurological disorders to cancer. However, it remains to be determined whether it is the disruption of our rhythmic physiology per se (loss of timing itself), or altered functioning of individual clock components that drive pathology. Here, we review the importance of circadian rhythms in terms of how we (and other organisms) relate to the external environment, but also in relation to how internal physiological processes are coordinated and synchronized. These issues are of increasing importance as many aspects of modern life put us in conflict with our internal clockwork.

From Zero to Hero: The Cyanide-Free Formation of Amino Acids and Amides from Acetylene, Ammonia and Carbon Monoxide in Aqueous Environments in a Simulated Hadean Scenario.

Amino acids are one of the most important building blocks of life. During the biochemical process of translation, cells sequentially connect amino acids via amide bonds to synthesize proteins, using the genetic information in messenger RNA (mRNA) as a template. From a prebiotic perspective (i.e., without enzymatic catalysis), joining amino acids to peptides via amide bonds is difficult due to the highly endergonic nature of the condensation reaction. We show here that amides can be formed in reactions catalyzed by the transition metal sulfides from acetylene, carbon monoxide and ammonia under aqueous conditions. Some α- and ß-amino acids were also formed under the same conditions, demonstrating an alternative cyanide-free path for the formation of amino acids in prebiotic environments. Experiments performed with stable isotope labeled precursors, like 15NH4Cl and 13C-acetylene, enabled the accurate mass spectroscopic identification of the products formed from the starting materials and their composition. Reactions catalyzed using the transition metal sulfides seem to offer a promising alternative pathway for the formation of amides and amino acids in prebiotic environments, bypassing the challenges posed by the highly endergonic condensation reaction. These findings shed light on the potential mechanisms by which the building blocks of life could have originated on early Earth.

Methods of Sentinel Lymph Node Detection and Management in Urinary Bladder Cancer-A Narrative Review.

INTRODUCTION: Detection of lymph node status in bladder cancer significantly impacts clinical decisions regarding its management. There is a wide range of detection modalities for this task, including lymphoscintigraphy, computed tomography, magnetic resonance imaging, single-photon emission computed tomography, positron emission tomography, and fluoroscopy. We aimed to study the pre- and intraoperative detection modalities of sentinel lymph nodes in urinary bladder cancer. METHOD: This narrative review was performed by searching the PubMed and EMBASE libraries using the following search terms: ("Transitional cell carcinoma of the bladder" OR "urothelial cancer" OR "urinary bladder cancer" OR "bladder cancer") AND (("sentinel lymph node") OR ("lymphatic mapping") OR ("lymphoscintigraphy") OR ("lymphangiography") OR ("lymph node metastases")). Studies analysing the effectiveness and outcomes of sentinel lymph node detection in bladder cancer were included, while non-English language, duplicates, and non-article studies were excluded. After analysing the libraries and a further manual search of bibliographies, 31 studies were included in this paper. We followed the RAMESES publication standard for narrative reviews to produce this paper. RESULTS: Of the 31 studies included, 7 studies included multiple detection methods; 5 studies included lymphoscintigraphy; 5 studies included computed tomography and/or single-photon emission computed tomography; 5 studies included fluoroscopy; 4 studies included magnetic resonance imaging; and 5 studies included positron emission tomography. DISCUSSION: Anatomical, radioactive, and functional detection modalities have been studied independently and in combination. The consensus is that preoperative detection with imaging helps guide surgical management and intraoperative detection methods help capture any lymph nodes that may have been missed. Each of these types of detection represent their own set of benefits and drawbacks, but there is currently limited evidence to support any change in overall practice to replace conventional staging.

Is There a Relationship Between Frequency of Port-Care Maintenance and Related Complications in Patients With Cancer?

PURPOSE: Totally implantable ports require regular maintenance to prevent port-related complications. Manufacturers recommend monthly maintenance port flushes for patients for the life of the port. Previous studies show that extending intervals between maintenance port flushes up to 16 weeks does not increase incidence of port-related complications. To date, no prospective study has been conducted to evaluate the medical safety of extending flush intervals from monthly to every 12 weeks within a heterogeneous disease cohort. Research Question: Is it feasible and medically safe to extend intervals between maintenance port flushes to every 12 weeks in patients with cancer not on active treatment? PATIENTS AND METHODS: This study enrolled oncology and hematology patients who had retained their port following completion of anticancer treatment. Clinical data were extracted for 1,059 participants. The primary end points of this study were the overall number of ports removed and incidence of port-related complications reported between cohorts 1 and 2 (flushes every 4-8 weeks), and cohort 3 (flushes every 12 weeks). RESULTS: Data were allocated into three study cohorts on the basis of year and duration between port flushes. No difference was observed in the overall percentage of ports removed because of physician-reported complications across all cohorts (25%-30%). No change in the incidence of port-related complications including suspected infection and malfunction was observed between cohorts 1 and 2 (8%), or cohort 3 (5%). CONCLUSION: Our findings show that extending maintenance port flush intervals to 12 weeks does not increase the incidence of port-related adverse events and is medically safe.

Clinical outcomes of low-pressure pneumoperitoneum in minimally invasive urological surgery.

The adoption of minimally invasive laparoscopic techniques has revolutionised urological practice. This necessitates a pneumoperitoneum (PNP) and the impact the PNP pressure has on post-operative outcomes is uncertain. During the current COVID-19 era guidance has suggested the utilisation of lower PNP pressures to mitigate the risk of intra-operative viral transmission. Review the current literature regarding the impact of pneumoperitoneum pressure, within the field of urology, on post-operative outcomes. A search of the PubMed, Medline and EMBASE databases was undertaken to identify studies that met the inclusion criteria. The Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines were adhered to. Ten studies, that included both randomised controlled trials and retrospective case series reviews, were identified that met the inclusion criteria. The effect of PNP pressure on outcomes following prostatectomy, live donor nephrectomy, partial nephrectomy and a variety of benign upper tract procedures were discussed. Low pressure PNP appears safe when compared to high pressure PNP, potentially reducing post-operative pain and rates of ileus. When compared to general surgery, there is a lack of quality evidence investigating the impact of PNP pressures on outcomes within urology. Low pressure PNP appears non-inferior to high pressure PNP. More research is required to validate this finding, particularly post-cystectomy and nephrectomy.

Evidence for circadian-based photoperiodic timekeeping in Svalbard ptarmigan, the northernmost resident bird.

The high Arctic archipelago of Svalbard (74°-81° north) experiences extended periods of uninterrupted daylight in summer and uninterrupted night in winter, apparently relaxing the major driver for the evolution of circadian rhythmicity. Svalbard ptarmigan (Lagopus muta hyperborea) is the only year-round resident terrestrial bird species endemic to the high Arctic and is remarkably adapted to the extreme annual variation in environmental conditions.1 Here, we demonstrate that, although circadian control of behavior disappears rapidly upon transfer to constant light conditions, consistent with the loss of daily activity patterns observed during the polar summer and polar night, Svalbard ptarmigans nonetheless employ a circadian-based mechanism for photoperiodic timekeeping. First, we show the persistence of rhythmic clock gene expression under constant light within the mediobasal hypothalamus and pars tuberalis, the key tissues in the seasonal neuroendocrine cascade. We then employ a "sliding skeleton photoperiod" protocol, revealing that the driving force behind seasonal biology of the Svalbard ptarmigan is rhythmic sensitivity to light, a feature that depends on a functioning circadian rhythm. Hence, the unusual selective pressures of life in the high Arctic have favored decoupling of the circadian clock from organization of daily activity patterns, while preserving its importance for seasonal synchronization.

Prostate cancer and microfluids.

Microfluidic systems aim to detect sample matter quickly with high sensitivity and resolution, on a small scale. With its increased use in medicine, the field is showing significant promise in prostate cancer diagnosis and management due, in part, to its ability to offer point-of-care testing. This review highlights some of the research that has been undertaken in respect of prostate cancer and microfluidics. Firstly, this review considers the diagnosis of prostate cancer through use of microfluidic systems and analyses the detection of prostate specific antigen, proteins, and circulating tumor cells to highlight the scope of current advancements. Secondly, this review analyses progressions in the understanding of prostate cancer physiology and considers techniques used to aid treatment of prostate cancer, such as the creation of a micro-environment. Finally, this review highlights potential future roles of microfluidics in assisting prostate cancer, such as in exosomal analysis. In conclusion, this review shows the vast scope and application of microfluidic systems and how these systems will ensure advancements to future prostate cancer management.

Photoperiod-dependent developmental reprogramming of the transcriptional response to seawater entry in Atlantic salmon (Salmo salar).

The developmental transition of juvenile salmon from a freshwater resident morph (parr) to a seawater (SW) migratory morph (smolt), known as smoltification, entails a reorganization of gill function to cope with the altered water environment. Recently, we used RNAseq to characterize the breadth of transcriptional change which takes place in the gill in the FW phase of smoltification. This highlighted the importance of extended exposure to short, winter-like photoperiods (SP) followed by a subsequent increase in photoperiod for completion of transcriptional reprogramming in FW and efficient growth following transfer to SW. Here, we extend this analysis to examine the consequences of this photoperiodic history-dependent reprogramming for subsequent gill responses upon exposure to SW. We use RNAseq to analyze gill samples taken from fish raised on the photoperiod regimes we used previously and then challenged by SW exposure for 24 hours. While fish held on constant light (LL) throughout were able to hypo-osmoregulate during a 24 hours SW challenge, the associated gill transcriptional response was highly distinctive from that in fish which had experienced a 7-week period of exposure to SP followed by a return to LL (SPLL) and had consequently acquired the characteristics of fully developed smolts. Fish transferred from LL to SP, and then held on SP for the remainder of the study was unable to hypo-osmoregulate, and the associated gill transcriptional response to SW exposure featured many transcripts apparently regulated by the glucocorticoid stress axis and by the osmo-sensing transcription factor NFAT5. The importance of these pathways for the gill transcriptional response to SW exposure appears to diminish as a consequence of photoperiod mediated induction of the smolt phenotype, presumably reflecting preparatory developmental changes taking place during this process.

Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics.

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.

Correction: RNA profiling identifies novel, photoperiod-history dependent markers associated with enhanced saltwater performance in juvenile Atlantic salmon.

[This corrects the article DOI: 10.1371/journal.pone.0227496.].

RNA profiling identifies novel, photoperiod-history dependent markers associated with enhanced saltwater performance in juvenile Atlantic salmon.

Atlantic salmon migrate to sea following completion of a developmental process known as smolting, which establishes a seawater (SW) tolerant phenotype. Smolting is stimulated by exposure to long photoperiod or continuous light (LL) following a period of exposure to short photoperiod (SP), and this leads to major changes in gill ion exchange and osmoregulatory function. Here, we performed an RNAseq experiment to discover novel genes involved in photoperiod-dependent remodeling of the gill. This revealed a novel cohort of genes whose expression rises dramatically in fish transferred to LL following SP exposure, but not in control fish maintained continuously on LL or on SP. A follow-up experiment revealed that the SP-history dependence of LL induction of gene expression varies considerably between genes. Some genes were inducible by LL exposure after only 2 weeks exposure to SP, while others required 8 weeks prior SP exposure for maximum responsiveness to LL. Since subsequent SW growth performance is also markedly improved following 8 weeks SP exposure, these photoperiodic history-dependent genes may be useful predictive markers for full smolt development.

Misalignment with the external light environment drives metabolic and cardiac dysfunction.

Most organisms use internal biological clocks to match behavioural and physiological processes to specific phases of the day-night cycle. Central to this is the synchronisation of internal processes across multiple organ systems. Environmental desynchrony (e.g. shift work) profoundly impacts human health, increasing cardiovascular disease and diabetes risk, yet the underlying mechanisms remain unclear. Here, we characterise the impact of desynchrony between the internal clock and the external light-dark (LD) cycle on mammalian physiology. We reveal that even under stable LD environments, phase misalignment has a profound effect, with decreased metabolic efficiency and disrupted cardiac function including prolonged QT interval duration. Importantly, physiological dysfunction is not driven by disrupted core clock function, nor by an internal desynchrony between organs, but rather the altered phase relationship between the internal clockwork and the external environment. We suggest phase misalignment as a major driver of pathologies associated with shift work, chronotype and social jetlag.The misalignment between internal circadian rhythm and the day-night cycle can be caused by genetic, behavioural and environmental factors, and may have a profound impact on human physiology. Here West et al. show that desynchrony between the internal clock and the external environment alter metabolic parameters and cardiac function in mice.