The best of both worlds: Chemigenetic fluorescent sensors for biological imaging.

Synthetic-based fluorescent chemosensors and protein-based fluorescent biosensors are two well-established classes of tools for visualizing and monitoring biological processes in living tissues. Chemigenetic sensors, created using a combination of both synthetic parts and protein parts, are an emerging class of tools that aims to combine the strengths, and overcome the drawbacks, of traditional chemosensors and biosensors. This review will survey the landscape of strategies used for fluorescent chemigenetic sensor design. These strategies include: attachment of synthetic elements to proteins using in vitro protein conjugation; attachment of synthetic elements to proteins using autonomous protein labeling; and translational incorporation of unnatural amino acids.

Alterations in visible light exposure modulate platelet function and regulate thrombus formation.

BACKGROUND: Variations in light exposure are associated with changes in inflammation and coagulation. The impact of light spectra on venous (VT) and arterial thrombosis is largely unexplored. OBJECTIVES: To investigate the impact of altering light spectrum on platelet function in thrombosis. METHODS: Wild type (WT) C57BL/6J mice were exposed to ambient (micewhite, 400lux), blue (miceblue, 442nm, 1,400lux), or red light (micered, 617nm, 1,400lux) with 12:12 hour light:dark cycle for 72 hours. After 72 hours of light exposure, platelet aggregation, activation, transcriptomic, and metabolomic changes were measured. The ability of released products of platelet activation to induce thrombosis-generating NET formation was quantified. Subsequent thrombosis was measured using murine models of VT and stroke. To translate our findings to human patients, light filtering cataract patients were evaluated over an 8-year period for rate of VTE with multivariable logistic regression clustered by hospital. RESULTS: Exposure to long wavelength red light resulted in reduced platelet aggregation and activation. RNA-seq analysis demonstrated no significant transcriptomic changes between micered and micewhite. However, there were global metabolomic changes in platelets from micered compared to micewhite. Releasate from activated platelets resulted in reduced NET formation. Micered also had reduced VT weight and brain infarct size following stroke. On subgroup analysis of cataract patients, patients with a history of cancer have lower lifetime risk of VTE after implantation with lenses that filter low wavelength light. CONCLUSIONS: Light therapy may be a promising approach to thrombus prophylaxis by specifically targeting the intersection between innate immune function and coagulation.

Plant-derived compounds normalize platelet bioenergetics and function in hyperglycemia.

Background: Polyphenols have been shown to decrease oxidative stress and modulate glycemic response. Nevertheless, their effect on platelet bioenergetics and clot structure in diabetes and hyperglycemia is unknown. Objectives: To investigate the effect of polyphenols on human platelet bioenergetics and its subsequent effect on clot structure in normoglycemia vs acute hyperglycemia in vitro. Methods: Four polyphenols (resveratrol, hesperetin, epigallocatechin gallate [EGCG], and quercetin) were selected for initial analysis. Healthy volunteers' isolated platelets/platelet-rich plasma were treated with 5 or 25 mM glucose to represent normoglycemia and acute hyperglycemia, respectively. Platelet-derived reactive oxygen species (ROS), citrate synthase activity (mitochondrial density), mitochondrial calcium flux, and mitochondrial respiration were performed following exposure to polyphenols (20 µM, 1 hour) to determine their effects on platelet bioenergetics. Procoagulant platelets (annexin V) and fibrin fiber density (Alexa Fluor-488 fibrinogen; Invitrogen) were analyzed by laser scanning confocal microscopy, while clot porosity was determined using platelet-rich plasma following exposure to polyphenols (20 µM, 20 minutes). Results: Acute hyperglycemia increased ROS, mitochondrial calcium flux, maximal respiration, and procoagulant platelet number. Resveratrol, quercetin, and EGCG reduced platelet ROS in normoglycemic and acute hyperglycemic conditions. Mitochondrial density was decreased by quercetin and EGCG in normoglycemia. Resveratrol and EGCG reduced mitochondrial calcium flux in acute hyperglycemia. Resveratrol also decreased procoagulant platelet number and attenuated oxygen consumption rate in normoglycemia and acute hyperglycemia. No effect of hyperglycemia or polyphenols was observed on fibrin fiber density or clot pore size. Conclusion: Our results suggest polyphenols attenuate increased platelet activity stemming from hyperglycemia and may benefit thrombosis-preventative strategies in patients with diabetes.

Targeting the synthetic lethal relationship between FOCAD and TUT7 represents a potential therapeutic opportunity for TUT4/7 small molecule inhibitors in cancer.

Targeting synthetic lethal interactions between genes has emerged as a promising strategy for cancer therapy. This study explores the intricate interplay between terminal uridyltransferase 4 (TUT4) and terminal uridyltransferase 7 (TUT7), the 3'-5' exoribonuclease DIS3L2, and the SKI complex-interacting factor Focadhesin (FOCAD) in the context of cancer vulnerability. Using CRISPR and public functional genomics data, we show impairment of cell proliferation upon knockout of TUT7 or DIS3L2, but not TUT4, on cancer cells with FOCAD loss. Moreover, we report the characterization of the first potent and selective TUT4/7 inhibitors that substantially reduce uridylation and demonstrate in vitro and in vivo antiproliferative activity specifically in FOCAD-deleted cancer. FOCAD deficiency post-transcriptionally disrupts the stability of the SKI complex, whose role is to safeguard cells against aberrant RNA. Re-introduction of FOCAD restores the SKI complex and makes these cells less sensitive to TUT4/7 inhibitors, indicating that TUT7 dependency is FOCAD loss-driven. We propose a model where, in absence of FOCAD, TUT7 and DIS3L2 function as a salvage mechanism that degrades aberrant RNA, and genetic or pharmacological inhibition of this pathway leads to cell death. Our findings underscore the significance of FOCAD loss as a genetic driver of TUT7 vulnerability and provide insights into the potential utility of TUT4/7 inhibitors for cancer treatment.

Synchronous intracranial arteriovenous malformation and papillary glioneuronal tumour: hypothesis or reality?

Brain arteriovenous malformations (AVM) rarely occur with spatial and/or temporal co-localisation to intracranial neoplasms. Most prior reports describe this association with high-grade gliomas; however, reports of a co-occurrence with low grade gliomas are very rare. It is unclear whether such cases represent a true co-occurrence of separate pathologies or simply an unusually vascular phenotype of the neoplasm. Most such reports pre-date the era of molecularly defined gliomas. We present the first report of the spatial and temporal co-occurrence of an intracranial arteriovenous malformation traversing and within a papillary glioneuronal tumour, molecularly defined by the presence of SLC44A1::PRKCA fusion. This case was successfully managed by resection of both lesions adhering to the principles of AVM surgery. It is possible these exceptionally rare co-occurrences may have common underlying molecular drivers relating to the mitogen activated protein kinase (MAPK) pathway.

Surgeon- and hospital-level variation in wait times for scheduled non-urgent surgery in Ontario, Canada: A cross-sectional population-based study.

BACKGROUND: Canadian health systems fare poorly in providing timely access to elective surgical care, which is crucial for quality, trust, and satisfaction. METHODS: We conducted a cross-sectional analysis of surgical wait times for adults receiving non-urgent cataract surgery, knee arthroplasty, hip arthroplasty, gallbladder surgery, and non-cancer uterine surgery in Ontario, Canada, between 2013 and 2019. We obtained data from the Wait Times Information System (WTIS) database. Inter- and intra-hospital and surgeon variations in wait time were described graphically with caterpillar plots. We used non-nested 3-level hierarchical random effects models to estimate variation partition coefficients, quantifying the proportion of wait time variance attributable to surgeons and hospitals. RESULTS: A total of 942,605 procedures at 107 healthcare facilities, conducted by 1,834 surgeons, were included in the analysis. We observed significant intra- and inter-provider variations in wait times across all five surgical procedures. Inter-facility median wait time varied between six-fold for gallbladder surgery and 15-fold for knee arthroplasty. Inter-surgeon variation was more pronounced, ranging from a 17-fold median wait time difference for cataract surgery to a 216-fold difference for non-cancer uterine surgery. The proportion of variation in wait times attributable to facilities ranged from 6.2% for gallbladder surgery to 23.0% for cataract surgery. In comparison, surgeon-related variation ranged from 16.0% for non-cancer uterine surgery to 28.0% for cataract surgery. IMPLICATIONS: There is extreme variability in surgical wait times for five common, high-volume, non-urgent surgical procedures. Strategies to address surgical wait times must address the variation between service providers through better coordination of supply and demand. Approaches such as single-entry models could improve surgical system performance.

Minimally invasive far lateral tubular microdiscectomy: surgical technique and case series of 176 patients.

BACKGROUND: Far lateral (extraforaminal) disc herniations comprise approximately 10% of symptomatic lumbar disc herniations. They represent operative challenges due to accessibility and surgical unfamiliarity. Surgical strategies in the past have included open discectomy and posterior lumbar interbody fusion. Tubular microdiscectomies have gained traction due to their minimally invasive advantages, including reduced morbidity, pain and length of hospital stay. METHODS: We report our retrospective single institution consecutive case series of tubular far lateral microdiscectomies. One hundred and seventy-six patients were operated on over an eight-year period. Clinical outcomes were assessed after institutional ethics approval. We additionally describe our surgical technique with an illustrative video case. RESULTS: Over a mean follow-up of 21 weeks, 77% of patients had good or excellent clinical outcomes according to the MacNab criteria. 12% of patients underwent reoperation at the index level for symptom recurrence or persistence. Mean length of hospital stay was 1.3 days. There was a 1% rate of both postoperative haematoma and infection. Mean operation duration was 86 minutes. CONCLUSION: This case series represents the largest currently reported in the literature. Minimally invasive microdiscectomies performed through tubes allow for precise localisation, reduced tissue disruption and favourable clinical outcomes. Our results appear consistent with a review of the literature, demonstrating the safety and efficacy of this approach.

Ten-year statewide cross-sectional review of pediatric sinogenic intracranial abscess and empyema in Queensland, Australia: microbial profile before and after COVID-19.

OBJECTIVE: Sinogenic intracranial infections in children, such as subdural empyema or intracranial abscess, are a rare disease process with significant associated morbidity. Recent literature has suggested that there may have been an increase in frequency of these infections following the COVID-19 pandemic, but the literature has been conflicting, perhaps related to the heterogenous management of COVID-19 lockdowns in various states and differences in data capture between methods. The collection of statewide Australian data overcomes these limitations by capturing a comprehensive sample though the public healthcare system of patients who were subject to a homogeneous statewide approach to public health policy during the COVID-19 pandemic (population 5.6 million, including 1.3 million children). The objective of this study was to present population-level data to address the question of whether the incidence of intracranial infections changed in pediatric patients before and after the COVID-19 pandemic. METHODS: The authors present a retrospective 10-year statewide description of sinogenic intracranial infections in Queensland, Australia. A comparison was made between the incidence and microbiological profile before and after the onset of COVID-19 lockdowns on March 22, 2020. RESULTS: Forty-four pediatric intracranial infections undergoing neurosurgical intervention were identified within the review period. After exclusion of postsurgical and cardioembolic causes, 33 sinogenic intracranial infections were included (16 before and 17 after 2020, with a mean annualized incidence of 0.25 vs 0.37 cases per 100,000 children, respectively; p > 0.05). The most frequent organisms identified were Streptococcus milleri (n = 19), polymicrobial (n = 4), and S. aureus (n = 3). No significant differences in antimicrobial profile, susceptibility, parenchymal involvement, or clinical outcome were identified between the pre- and post-COVID-19 groups. CONCLUSIONS: No statistically significant differences in the epidemiology of pediatric intracranial infection have occurred in the state of Queensland, Australia, before and after March 22, 2020, and the COVID-19 pandemic.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice.

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of D-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter D-serine, by its precursor L-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards L-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

Public funding for private for-profit centres and access to cataract surgery by patient socioeconomic status: an Ontario population-based study.

BACKGROUND: Public funding of cataract surgery provided in private, for-profit surgical centres increased to help mitigate surgical backlogs during the COVID-19 pandemic in Ontario, Canada. We sought to compare the socioeconomic status of patients who underwent cataract surgery in not-for-profit public hospitals with those who underwent this surgery in private for-profit surgical centres and to evaluate whether differences in access by socioeconomic status decreased after the infusion of public funding for private, for-profit centres. METHODS: We conducted a population-based study of all cataract operations in Ontario, Canada, between January 2017 and March 2022. We analyzed differences in socioeconomic status among patients who accessed surgery at not-for-profit public hospitals versus those who accessed it at private for-profit surgical centres before and during the period of expanded public funding for private for-profit centres. RESULTS: Overall, 935 729 cataract surgeries occurred during the study period. Within private for-profit surgical centres, the rate of cataract surgeries rose 22.0% during the funding change period for patients in the highest socioeconomic status quintile, whereas, for patients in the lowest socioeconomic status quintile, the rate fell 8.5%. In contrast, within public hospitals, the rate of surgery decreased similarly among patients of all quintiles of socioeconomic status. During the funding change period, 92 809 fewer cataract operations were performed than expected. This trend was associated with socioeconomic status, particularly within private for-profit surgical centres, where patients with the highest socioeconomic status were the only group to have an increase in cataract operations. INTERPRETATION: After increased public funding for private, for-profit surgical centres, patient socioeconomic status was associated with access to cataract surgery in these centres, but not in public hospitals. Addressing the factors underlying this incongruity is vital to ensure access to surgery and maintain public confidence in the cataract surgery system.

Prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: communication from the SSC of the ISTH.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbß3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Special Section Guest Editorial: Molecular Neurophotonics.

The editorial introduces the Special Section on Molecular Neurophotonics.

Aging-related alterations in mechanistic target of rapamycin signaling promote platelet hyperreactivity and thrombosis.

BACKGROUND: Aging is an independent risk factor for the development of cardiovascular, thrombotic, and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. OBJECTIVES: Here, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. METHODS: Quantitative mass spectrometry with tandem mass tag labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mechanistic target of rapamycin (mTOR) in aging-induced platelet hyperreactivity was assessed using pharmacologic mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt-/-). RESULTS: Quantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase, and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared with that in young controls. Inhibition of mTOR with either Torin 1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen-epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster than young controls, while time to death of aged mTORplt-/- mice was similar to that of young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. CONCLUSION: Aging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation to enhance thromboxane generation, platelet hyperactivity, and thrombosis.

Improving Clinical Registry Data Quality via Linkage With Survival Data From State-Based Population Registries.

PURPOSE: Real-world data (RWD) collected on patients treated as part of routine clinical care form the basis of cancer clinical registries. Capturing accurate death data can be challenging, with inaccurate survival data potentially compromising the integrity of registry-based research. Here, we explore the utility of data linkage (DL) to state-based registries to enhance the capture of survival outcomes. METHODS: We identified consecutive adult patients with brain tumors treated in the state of Victoria from the Brain Tumour Registry Australia: Innovation and Translation (BRAIN) database, who had no recorded date of death and no follow-up within the last 6 months. Full name and date of birth were used to match patients in the BRAIN registry with those in the Victorian Births, Deaths and Marriages (BDM) registry. Overall survival (OS) outcomes were compared pre- and post-DL. RESULTS: Of the 7,346 clinical registry patients, 5,462 (74%) had no date of death and no follow-up recorded within the last 6 months. Of the 5,462 patients, 1,588 (29%) were matched with a date of death in BDM. Factors associated with an increased number of matches were poor prognosis tumors, older age, and social disadvantage. OS was significantly overestimated pre-DL compared with post-DL for the entire cohort (pre- v post-DL: hazard ratio, 1.43; P < .001; median, 29.9 months v 16.7 months) and for most individual tumor types. This finding was present independent of the tumor prognosis. CONCLUSION: As revealed by linkage with BDM, a high proportion of patients in a brain cancer clinical registry had missing death data, contributed to by informative censoring, inflating OS calculations. DL to pertinent registries on an ongoing basis should be considered to ensure accurate reporting of survival data and interpretation of RWD outcomes.

An Experimental Infection Model in Sheep and Goats to Evaluate Salmonella Colonization in Deep Tissue Lymph Nodes and after Carcass Vascular Rinsing with Bacteriophages in Goats.

An animal infection model was evaluated on sheep and goats to confirm which species infected with Salmonella enterica serovar Enteritidis C StR (SE13) would provide a consistent and high frequency of Salmonella colonization in lymph nodes (LNs) without causing undue animal morbidity. Sheep and goats (n = 5) were intradermally inoculated with Salmonella, postincubated for 7 days, and euthanized. Superficial cervical, medial iliac, subiliac, mammary, and popliteal LNs were excised from each carcass. Goat LNs had approximately 53% greater Salmonella level compared to sheep. Also, Salmonella was inconsistently recovered from the sheep LNs. Thus, goats were selected to determine the ability of carcass vascular rinsing (with and without bacteriophages) to reduce Salmonella in infected LNs. Goats with similar characteristics were grouped together before being randomly assigned to 3 postharvest treatments; control (CN, not vascularly rinsed; n = 10), vascularly rinsed with a standard Rinse & Chill® solution (RC; 98.5% water and a blend of saccharides and phosphates; n = 10), or vascularly rinsed with a standard Rinse & Chill® solution plus the addition of bacteriophages (BP; n = 10). Rinse & Chill® system was able to successfully deliver a mean 7.0 log PFU/g to the S. Enteritidis-infected LNs (mean 3.5 log CFU/g). However, neither Rinse & Chill® without bacteriophages nor with bacteriophages caused Salmonella reduction (P > 0.05) compared to the nonrinsed goat carcasses.

Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype in Women.

We created the c.1286C>G stop-gain mutation found in a family with primary ovarian insufficiency (POI) at age 30 years. The Eif4enif1 C57/Bl6 transgenic mouse model contained a floxed exon 10-19 cassette with a conditional knock-in cassette containing the c.1286C>G stop-gain mutation in exon 10. The hybrid offspring of CMV- Cre mice with Eif4enif1 WT/flx mice were designated Eif4enif1 WT/ Δ for simplicity. A subset of female heterozygotes ( Eif4enif1 WT/ Δ ) had no litters. In those with litters, the final litter was earlier (5.4±2.6 vs. 10.5±0.7 months; p=0.02). Heterozygous breeding pair ( Eif4enif1 WT/ Δ x Eif4enif1 WT/ Δ ) litter size was 60% of WT litter size (3.9±2.0 vs. 6.5±3.0 pups/litter; p <0.001). The genotypes were 35% Eif4enif1 WT/flx and 65% Eif4enif1 WT/ Δ , with no homozygotes. Homozygote embryos did not develop beyond the 4-8 cell stage. The number of follicles in ovaries from Eif4enif1 WT/ Δ mice was lower starting at the primordial (499±290 vs. 1445±381) and primary follicle stage (1069±346 vs. 1450±193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213±86 vs. 522±227; p<0.01). Examination of ribosome protected mRNAs (RPR) demonstrated altered mRNA expression. The Eif4enif1 stop-gain mice replicate the POI phenotype in women. The unique mouse model provides a platform to study regulation of protein translation across oocyte and embryo development in mammals.