Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Retinal Tear and Retinal Detachment Following Cataract Surgery in Eyes with a Previous History of Treated Phakic Retinal Tears.

OBJECTIVE: To investigate the incidence and outcomes of retinal tear (RT) and retinal detachment (RD) after cataract extraction in patients with a history of previous phakic RT. DESIGN: Retrospective case series. SUBJECTS: Phakic eyes with RT that were successfully treated with laser photocoagulation or cryotherapy and subsequently underwent cataract surgery. METHOD: A retrospective review of phakic eyes treated for RTs between April 1, 2012 and May 31, 2023 was performed. Exclusions included prior vitreoretinal surgery before cataract removal and follow-up of less than 6 months post-cataract surgery. MAIN OUTCOME MEASURES: The incidence of RTs and RDs after cataract surgery, along with visual and anatomic outcomes. RESULTS: Of 12,109 phakic eyes treated for RTs, 1039 (8.6%) eyes underwent cataract surgery. After exclusions, 713 eyes of 660 patients were studied. The mean (standard deviation, SD) follow-up period post-cataract surgery was 34.8 (24.6) months with a median of 239 and 246 days to a new RT or RD development. The overall incidence for diagnosis of post-cataract surgery RT and RD was 7.3% (52/713) (2.9% and 4.3%, respectively), with a one-year incidence of 5.6% (2.2% and 3.4%, respectively). Multivariable regression analysis identified a higher risk of RT/RD among younger individuals (odds ratio [OR] 1.034; 95% confidence interval [CI] 1.004-1.065, P=0.028), males (OR 2.058; 95% CI 1.110-3.816, P=0.022), and those with shorter interval between laser treatment and cataract surgery (OR 1.001; 95% CI 1.001-1.001, P=0.011). Single surgery anatomic success for the RD repair was achieved in 25 eyes (80.6%) at 3 months, with a 100% final reattachment rate. The median final logMAR visual acuity was 0.10 (20/25) for RT, showing no significant change from post-cataract surgery, and 0.18 (20/30) for RD, a significant worsening from after cataract surgery. CONCLUSION: One year post-cataract surgery, the rate of diagnosed RT and RD in patients with previously treated RTs was relatively high, occurring in nearly 1 in 18 eyes. Higher risk was noted among younger individuals, males, and patients with a shorter interval between initial treatment for RT and cataract surgery. RD repair achieved good anatomical results, but vision declined.

Insulin Signaling Differentially Regulates the Trafficking of Insulin and Amyloid Beta Peptides at the Blood-Brain Barrier.

The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-ß (Aß) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aß levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aß and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aß peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aß42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aß42, [125I]iodo-Aß40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aß42, and [125I]iodo-Aß40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aß and insulin trafficking receptors (LRP-1 and IR-ß). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aß peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aß accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.

Predictors for infection severity for open tibial fractures: major trauma centre perspective.

INTRODUCTION: Open diaphyseal tibial fractures are the most common long-bone fractures and require a rapid approach to prevent devastating complications. Current literature reports the outcomes of open tibial fractures. However, there is no robust, up-to-date research on the predictive indicators of infection severity in a large open tibial fracture patient cohort. This study investigated the predictive factors of superficial infections and osteomyelitis in open tibial fractures. MATERIALS AND METHODS: A retrospective analysis of the tibial fracture database was carried out from 2014 to 2020. Criteria for inclusion was any tibial fracture including tibial plateau, shaft, pilon or ankle, with an open wound at the fracture site. Exclusion criteria included patients with a follow-up period of less than 12 months and who are deceased. A total of 235 patients were included in our study, of which 154 (65.6%), 42 (17.9%), and 39 (16.6%) developed no infection, superficial infection, or osteomyelitis, respectively. Patient demographics, injury characteristics, fracture characteristics, infection status and management details were collected for all patients. RESULTS: On multivariate modelling, patients with BMI > 30 (OR = 2.078, 95%CI [1.145-6.317], p = 0.025), Gustilo-Anderson (GA) type III (OR = 6.120, 95%CI [1.995-18.767], p = 0.001), longer time to soft tissue cover (p = 0.006) were more likely to develop a superficial infection, and patients with wound contamination (OR = 3.152, 95%CI [1.079-9.207], p = 0.036), GA-3 (OR = 3.387,95%CI [1.103-10.405], p = 0.026), longer to soft tissue cover (p = 0.007) were more likely to develop osteomyelitis. Univariate analysis also determined that risk factors for superficial infection were: BMI > 35 (OR = 6.107, 95%CI [2.283-16.332], p = 0.003) and wound contamination (OR = 2.249, 95%CI [1.015-5.135], p = 0.047); whilst currently smoking (OR = 2.298, 95%CI [1.087-4.856], p = 0.025), polytrauma (OR = 3.212, 95%CI [1.556-6.629], p = 0.001), longer time to definitive fixation (p = 0.023) were for osteomyelitis. However, none of these reached significance in multivariate analysis. CONCLUSION: Higher GA classification is a significant risk factor for developing superficial infection and osteomyelitis, with a stronger association with osteomyelitis, especially GA 3C fractures. Predictors for superficial infection included BMI and time to soft tissue closure. Time to definitive fixation, time to soft tissue closure, and wound contamination were associated with osteomyelitis.

Variation on a technique for the intra-muscular insertion of nerve endings to minimise neuropathic and residual pain in lower limb amputees: a retrospective cohort study.

INTRODUCTION: A major cause of morbidity in lower limb amputees is phantom limb pain (PLP) and residual limb pain (RLP). This study aimed to determine whether a variation of the surgical technique of inserting nerve endings into adjacent muscle bellies at the time of lower limb amputation can decrease the incidence and severity of PLP and RLP. METHODS: Data were retrospectively collected from January 2015 to January 2021, including eight patients that underwent nerve insertion (NI) and 36 that received standard treatment. Primary outcomes included the 11-point Numerical Rating Scale (NRS) for pain severity, and Patient-Reported Outcomes Measurement Information System (PROMIS) pain intensity, behaviour, and interference. Secondary outcome included Neuro-QoL Lower Extremity Function assessing mobility. Cumulative scores were transformed to standardised t scores. RESULTS: Across all primary and secondary outcomes, NI patients had lower PLP and RLP. Mean 'worst pain' score was 3.5 out of 10 for PLP in the NI cohort, compared to 4.89 in the control cohort (p = 0.298), and 2.6 out of 10 for RLP in the NI cohort, compared to 4.44 in the control cohort (p = 0.035). Mean 'best pain' and 'current pain' scores were also superior in the NI cohort for PLP (p = 0.003, p = 0.022), and RLP (p = 0.018, p = 0.134). Mean PROMIS t scores were lower for the NI cohort for RLP (40.1 vs 49.4 for pain intensity; p = 0.014, 44.4 vs 48.2 for pain interference; p = 0.085, 42.5 vs 49.9 for pain behaviour; p = 0.025). Mean PROMIS t scores were also lower for the NI cohort for PLP (42.5 vs 52.7 for pain intensity; p = 0.018); 45.0 vs 51.5 for pain interference; p = 0.015, 46.3 vs 51.1 for pain behaviour; p = 0.569). Mean Neuro-QoL t score was lower in NI cohort (45.4 vs 41.9; p = 0.03). CONCLUSION: Surgical insertion of nerve endings into adjacent muscle bellies during lower limb amputation is a simple yet effective way of minimising PLP and RLP, improving patients' subsequent quality of life. Additional comparisons with targeted muscle reinnervation should be performed to determine the optimal treatment option.

Ankle fusion with tibiotalocalcaneal retrograde nail for fragility ankle fractures: outcomes at a major trauma centre.

PURPOSE: Fragility ankles fractures in the geriatric population are challenging to manage, due to fracture instability, soft tissue compromise, and patient co-morbidities. Traditional management options include open reduction internal fixation, or conservative treatment, both of which are fraught with high complication rates. We aimed to present functional outcomes of elderly patients with fragility ankle fractures treated with retrograde ankle fusion nails. METHODS: A retrospective observational study was performed on patients who underwent intramedullary nailing with a tibiotalocalcaneal nail. Twenty patients met the inclusion criteria of being over sixty and having multiple co-morbidities. Patient demographics, AO/OTA fracture classification, intra-operative and post-operative complications, time to mobilisation and union, AOFAS and Olerud-Molander scores, and patient mobility were recorded. RESULTS: There were seven males and thirteen females, with a mean age of 77.82 years old, five of whom are type 2 diabetics. Thirteen patients returned to their pre-operative mobility state, and the average Charlson Co-morbidity Index (CCI) was 5.05. Patients with a low CCI are more likely to return to pre-operative mobility status (p = 0.16; OR = 4.00). All patients achieved radiographical union, taking on average between 92.5 days and 144.6 days. The mean post-operative AOFAS and Olerud-Molander scores were 53.0 and 50.9, respectively. There were four cases of superficial infection, four cases of broken or loose distal locking screws. There were no deep infections, periprosthetic fractures, nail breakages, or non-unions. CONCLUSION: Tibiotalocalcaneal nailing is an effective and safe option for managing unstable ankle fractures in the elderly. This technique leads to lower complication rates and earlier mobilisation than traditional fixation methods.

A retrospective analysis of the definitive management of open talus fractures at a major trauma centre, comparing ORIF to FUSION: cohort study and audit of BOAST 4 guidelines.

PURPOSE: Open talus fractures are notoriously difficult to manage, and they are commonly associated with a high level of complications including non-union, avascular necrosis and infection. Currently, the management of such injuries is based upon BOAST 4 guidelines although there is no suggested definitive management, and thus, definitive management is based upon surgeon preference. The key principles of open talus fracture management which do not vary between surgeons are early debridement, orthoplastic wound care, anatomic reduction and definitive fixation whenever possible. However, there is much debate over whether the talus should be preserved or removed after open talus fracture/dislocation and proceeded to tibiocalcaneal fusion. METHODS: A review of electronic hospital records for open talus fractures from 2014 to 2021 returned fourteen patients with fifteen open talus fractures. Seven cases were initially managed with ORIF, and five cases were definitively managed with FUSION, while the others were managed with alternative methods. We collected patient's age, gender, surgical complications, surgical risk factors and post-treatment functional ability and pain and compliance with BOAST guidelines. The average follow-up of the cohort was 4 years and one month. EQ-5D-5L and FAAM-ADL/Sports score was used as a patient reported outcome measure. Data were analysed using the software PRISM. RESULTS: Comparison between FUSION and ORIF groups showed no statistically significant difference in EQ-5D-5L score (P = 0.13), FAAM-ADL (P = 0.20), FAAM-Sport (P = 0.34), infection rate (P = 0.55), surgical times (P = 0.91) and time to weight bearing (P = 0.39), despite a higher proportion of polytrauma and Hawkins III and IV fractures in the FUSION group. CONCLUSION: FUSION is typically used as second line to ORIF or failed ORIF. However, there is a lack of studies that directly compared outcome in open talus fracture patients definitively managed with FUSION or ORIF. Our results demonstrate for the first time that FUSION may not be inferior to ORIF in terms of patient functional outcome, infection rate and quality of life, in the management of patients with open talus fracture patients. Of note, as open talus fractures have increased risks of complications such as osteonecrosis and non-union, FUSION should be considered as a viable option to mitigate these potential complications in these patients.

Management of post-traumatic femoral defects with a monorail external fixator over an intramedullary nail.

PURPOSE: The management of limb-length discrepancy secondary to traumatic femoral bone loss poses a unique challenge for surgeons. The Ilizarov technique is popular, but is associated with long external fixator time and many complications. This retrospective study assessed outcomes of post-traumatic femoral defects managed by monorail external fixation over an intramedullary nail. METHODS: Eight patients were included from October 2015 to May 2019 with post-traumatic femoral defects that underwent treatment with monorail fixator-assisted intramedullary nailing. Primary outcome was time to bone union and bone results according to ASAMI classification. Secondary outcomes were lengthening index, consolidation time and index, external fixator index (EFI), time to partial weight bearing(PWB) and full weight bearing (FWB), and complications. Patient reported outcome measures including EQ-5D-5L, SF-36, Oxford knee scores (OKS), and Oxford hip scores (OHS) were recorded after recovery. RESULTS: Mean follow-up time was 227 weeks. Average bone defect size was 9.69 cm. Average consolidation time and index were 11.35 months and 1.24 months/cm, respectively. Mean lengthening and external fixator index were 20.2 days/cm and 23.88 days/cm, respectively. On average, patients achieved FWB and bone union 56.25 weeks and 68.83 weeks after bone transport initiation, respectively. Two patients had docking site non-union, five patients had pin site infections, and two patients had osteomyelitis. EQ-5D-5L and EQ-VAS scores were compared to UK population norms (p = 0.104, p = 0.238, respectively). Average OKS was 32.17 and OHS was 34.00. CONCLUSION: Monorail external fixation over an intramedullary nail is an effective option for post-traumatic femoral defects, reducing external fixator time and returning patients' quality of life to a level comparable with the normal population.

Perioperative use of intra-articular steroids during the COVID-19 pandemic.

PURPOSE: There are growing concerns with the widely used glucocorticoids during the Coronavirus disease-19 (COVID-19) pandemic due to the associated immunosuppressive effects, which may increase the risk of COVID-19 infection and worsen COVID-19 patient outcome. Heavily affecting orthopaedics, the pandemic led to delay and cancellation of almost all surgical cases, and procedures including perioperative intra-articular corticosteroid injections (ICIs) saw similar decreases. However, the benefits of ICI treatments during the pandemic may outweigh these potential risks, and their continued use may be warranted. METHODS: A literature search was conducted, and all relevant articles including original articles and reviews were identified and considered in full for inclusion, and analysed with expert opinion. Epidemiological statistics and medical guidelines were consulted from relevant authorities. RESULTS: ICIs allow a targeted approach on the affected joint and are effective in reducing pain while improving functional outcome and patient quality-of-life. ICIs delay the requirement for surgery, accommodating for the increased healthcare burden during the pandemic, while reducing postoperative hospital stay, bringing significant financial benefits. However, ICIs can exert systemic effects and suppress the immune system. ICIs may increase the risk of COVID-19 infection and reduce the efficacy of COVID-19 vaccinations, leading to important public health implications. CONCLUSION: Perioperative ICI treatments may bring significant, multifaceted benefits during the pandemic. However, ICIs increase the risk of infection, and perioperative COVID-19 is associated with mortality. The use of ICIs during the COVID-19 pandemic should therefore be considered carefully on an individual patient basis, weighing the associated risks and benefits.

High-Density Lipoprotein Mimetic Peptide 4F Efficiently Crosses the Blood-Brain Barrier and Modulates Amyloid-ß Distribution between Brain and Plasma.

Treatments to elevate high-density lipoprotein (HDL) levels in plasma have decreased cerebrovascular amyloid -ß (Aß) deposition and mitigated cognitive decline in Alzheimer disease (AD) transgenic mice. Since the major protein component of HDL particles, apolipoprotein A-I (ApoA-I), has very low permeability at the blood-brain barrier (BBB), we investigated 4F, an 18-amino-acid ApoA-I/HDL mimetic peptide, as a therapeutic alternative. Specifically, we examined the BBB permeability of 4F and its effects on [125I]Aß trafficking from brain to blood and from blood to brain. After systemic injection in mice, the BBB permeability of [125I]4F, estimated as the permeability-surface area (PS) product, ranged between 2 and 5 × 10-6 ml/g per second in various brain regions. The PS products of [125I]4F were ∼1000-fold higher compared with those determined for [125I]ApoA-I. Moreover, systemic infusion with 4F increased the brain efflux of intracerebrally injected [125I]Aß42. Conversely, 4F infusion decreased the brain influx of systemically injected [125I]Aß42. Interestingly, 4F did not significantly alter the brain influx of [125I]Aß40. To corroborate the in vivo findings, we evaluated the effects of 4F on [125I]Aß42 transcytosis across polarized human BBB endothelial cell (hCMEC/D3) monolayers. Treatment with 4F increased the abluminal-to-luminal flux and decreased the luminal-to-abluminal flux of [125I]Aß42 across the hCMEC/D3 monolayers. Additionally, 4F decreased the endothelial accumulation of fluorescein-labeled Aß42 in the hCMEC/D3 monolayers. These findings provide a mechanistic interpretation for the reductions in brain Aß burden reported in AD mice after oral 4F administration, which represents a novel strategy for treating AD and cerebral amyloid angiopathy. SIGNIFICANCE STATEMENT: The brain permeability of the ApoA-I mimetic peptide 4F was estimated to be ∼1000-fold greater than ApoA-I after systemic injection of radiolabeled peptide/protein in mice. Further, 4F treatment increased the brain efflux of amyloid -ß and also decreased its brain influx, as evaluated in mice and in blood-brain barrier cell monolayers. Thus, 4F represents a potential therapeutic strategy to mitigate brain amyloid accumulation in cerebral amyloid angiopathy and Alzheimer disease.

Effect of Tungsten Oxide Nanostructures on Sensitivity and Selectivity of Pollution Gases.

We report on the different surface structures of tungsten oxides which have been synthesized using a simple post-annealing-free hot-filament CVD technique, including 0D nanoparticles (NPs), 1D nanorods (NRs), and 2D nanosheet assemblies of 3D hierarchical nanoflowers (NFs). The surface morphologies, crystalline structures, and material compositions have been characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and Raman spectroscopy, respectively. The sensor performances based on the synthesized samples of various surface morphologies have been investigated, as well as the influences of operating temperature and applied bias. The sensing property depends closely on the surface morphology, and the 3D hierarchical nanoflowers-based gas sensor offers the best sensitivity and fastest response time to NH3 and CH3 gases when operated at room temperature.

Apolipoprotein A-I Crosses the Blood-Brain Barrier through Clathrin-Independent and Cholesterol-Mediated Endocytosis.

Recent studies suggest that apolipoprotein A-I (ApoA-I), the major protein constituent of high-density lipoprotein particles, plays a critical role in preserving cerebrovascular integrity and reducing Alzheimer's risk. ApoA-I present in brain is thought to be primarily derived from the peripheral circulation. Although plasma-to-brain delivery of ApoA-I is claimed to be handled by the blood-cerebrospinal fluid barrier (BCSFB), a contribution by the blood-brain barrier (BBB), which serves as a major portal for protein delivery to brain, cannot be ruled out. In this study, we assessed the permeability-surface area product (PS) of radioiodinated ApoA-I (125I-ApoA-I) in various brain regions of wild-type rats after an intravenous bolus injection. The PS value at the cortex, caudate putamen, hippocampus, thalamus, brain stem, and cerebellum was found to be 0.39, 0.28, 0.28, 0.36, 0.69, and 0.76 (ml/g per second × 10-6), respectively. Solutes delivered into brain via the BCSFB are expected to show greater accumulation in the thalamus due to its periventricular location. The modest permeability for 125I-ApoA-I into the thalamus relative to other regions suggests that BCSFB transport accounts for only a portion of total brain uptake and thus BBB transport cannot be ruled out. In addition, we show that Alexa Flour 647-labeled ApoA-I (AF647-ApoA-I) undergoes clathrin-independent and cholesterol-mediated endocytosis in transformed human cerebral microvascular endothelial cells (hCMEC/D3). Further, Z-series confocal images of the hCMEC/D3 monolayers and Western blot detection of intact ApoA-I on the abluminal side demonstrated AF647-ApoA-I transcytosis across the endothelium. These findings implicate the BBB as a significant portal for ApoA-I delivery into brain.

A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

Switch-on fluorescent/FRET probes to study human histidine triad nucleotide binding protein 1 (hHint1), a novel target for opioid tolerance and neuropathic pain.

Histidine Triad Nucleotide Binding Protein 1 (Hint1) has emerged to be an important post-synaptic protein associated with a variety of central nervous system disorders such as pain, addiction, and schizophrenia. Recently, inhibition of histidine nucleotide binding protein 1 (Hint1) with a small nucleoside inhibitor has shown promise as a new therapeutic strategy for the treatment of neuropathic pain. Herein, we describe the first rationally designed small molecule switch-on probes with dual fluorescence and FRET properties to study Hint1. Two non-natural fluorescent nucleosides with a fluorescent lifetime of 20 and 25 ns were each coupled through a linker to the indole ring, i.e. probes 7 and 8. Both probes were found to be water soluble and quenched intramolecularly via photoinduced electron transfer (PET) resulting in minimal background fluorescence. Upon incubating with Hint1, compound 7 and 8 exhibited a 40- and 16-fold increase in the fluorescence intensity compared to the control. Compounds 7 and 8 bind Hint1 with a dissociation constant of 0.121 ± 0.02 and 2.2 ± 0.36 µM, respectively. We demonstrate that probe 8 exhibits a switch-on FRET property with an active site tryptophan residue (W123). We show the utility of probes in performing quantitative ligand displacement studies, as well as in selective detection of Hint1 in the cell lysates. These probes should be useful for studying the dynamics of the active site, as well as for the development of fluorescence lifetime based high throughput screening assay to identify novel inhibitors for Hint1 in future.

Safety of undiluted intracameral moxifloxacin without postoperative topical antibiotics in cataract surgery.

The objective of this study is to evaluate the safety of undiluted 0.5 % intracameral moxifloxacin for postoperative endophthalmitis prophylaxis in cataract surgery patients without the use of additional postoperative topical antibiotics. All phacoemulsification cataract surgeries performed by a single surgeon (B.A.) at the John A. Moran Eye Center from June 2012 to May 2015 were reviewed retrospectively. From June 2012 to April 2014, patients were given topical 0.5 % moxifloxacin postoperatively. From May 2014 to May 2015, all patients were given moxifloxacin intracamerally with no antibiotics postoperatively. The follow-up period was 1 month after surgery. Preoperative visual acuity and postoperative visual acuity, corneal edema, and anterior chamber reaction were recorded and compared between the two groups. 384 cataract surgeries were performed during the study period. None of the 384 eyes in the study developed endophthalmitis. Of those 384 eyes, 222 were included in the study for analysis based on the inclusion and exclusion criteria. 131 were part of the topical antibiotic group and 91 were part of the intracameral group. The differences in uncorrected visual acuity at 1 day postoperatively (p = 0.595) and best corrected visual acuity at 1 month postoperatively (p = 0.099) were not statistically significant. Differences in corneal edema (p = 0.370) and anterior chamber reaction (p = 0.069) at 1 day postoperatively and corneal edema (p = 0.512) and anterior chamber reaction (p = 0.512) at 1 month postoperatively were also not statistically significant. Undiluted 0.5 % moxifloxacin can be safely injected intracamerally following cataract surgery without additional postoperative antibiotic prophylaxis to prevent endophthalmitis without adverse effects on patient outcomes.

The accuracy and diagnostic value of gram staining joint aspirates in suspected joint infections.

BACKGROUND: Septic arthritis is a debilitating condition with prolonged treatment and adverse outcomes. A gram stain is often performed from the joint aspirate sample, followed by a definitive culture. In our study, we assessed the accuracy of gram staining for suspected septic arthritis and explored factors associated with positive culture growth and false negatives in the gram stain. METHODS: We retrospectively reviewed joint aspirates performed from 2015-2021 at a major trauma centre. Aspirates not cultured for septic arthritis were excluded. Data collected included aspirate site, gram stain and culture result delay, patient demographics, orthopaedic/rheumatological history, and comorbidities. Outcomes measured were gram stain sensitivity and specificity. Factors influencing positive cultures and false negative gram stain results were analysed using logistic regression. RESULTS: Of 408 joint aspirates meeting the criteria, 37 did not undergo initial gram staining. Gram stain sensitivity was 30.4%, specificity was 97.6%. The delay from aspirate to definitive gram stain and culture results was 1.1 and 5.4 days, respectively Logistic regression identified that prosthetic joint(p = 0.007), past joint infections(p = 0.006), arthritis(p < 0.001), hypertension(p = 0.007), diabetes(p = 0.019) were positively associated with positive cultures. Past joint infections(p = 0.004) were positively associated with false negative gram stain results. Patients on antibiotics during the aspirate had a higher risk of false negative gram stain results (OR = 5.538, 95%CI, 2.802-10.948; p < 0.001). CONCLUSIONS: In conclusion, the initial gram stain has limited sensitivity and caution should be exercised when interpreting negative results. Vigilance is crucial when the highlighted comorbidities or antibiotic use are present, to assess patients with potential joint infections.

The evolution of poller screws.

Compared to other techniques, poller screws with intramedullary nailing are technically simple, practical, and reproducible for the fixation of metaphyseal fractures. In addition, poller screws do not require special instruments or hardware and are minimally invasive. This review takes a historical perspective to evaluate poller screws holistically. A non-systematic search on PubMed was performed using 'Poller screw' or 'Blocking screw' to find early use of poller blocking screws. Relevant references from these primary studies were then followed up. In 1999, Krettek et al. first coined the term poller screws after the small metal bollards that block and direct traffic. Poller screws were introduced as an adjunct to aid the union of metaphyseal long bone fractures during intramedullary nailing. However, as more evidence was published, the true effectiveness of poller screws was not appreciated, leading to split opinions. Through our research, we have built upon our understanding of poller screws, and we present a novel classification of poller screws over the years while exploring our novel technique and what we believe to be the fourth generation of poller screws. Currently, there is a paucity of research focussing on poller screws. However, studying the original evidence regarding poller screws through the most recent articles has demonstrated a confusion of research in this field. Therefore, we suggest a more organised approach to classify the use of poller screws.

Prospective Evaluation of the Clinical Benefits of a Novel Tattoo-less Workflow for Nonspine Bone Stereotactic Body Radiation Therapy: Integrating Surface-Guidance With Triggered Imaging Reduces Treatment Time and Eliminates the Need for Tattoos.

PURPOSE: Oligometastatic disease has expanded the indications for nonspine bone stereotactic body radiation therapy (NSB SBRT). We investigated whether optical surface monitoring systems (OSMS) could enable tattoo-less setup and substitute for 2-dimensional/3-dimensional or cone beam computed tomography (CBCT)-based mid-imaging in NSB SBRT. METHODS AND MATERIALS: OSMS was incorporated in parallel with an existing workflow using pretreatment CBCT and 2-dimensional/3-dimensional kV/kV mid-imaging beginning November 2019. The ability of OSMS to detect out-of-tolerance (>2 mm/>2°) and commanded couch shifts was analyzed. A workflow incorporating OSMS reference captures, CBCT for pretreatment verification, and OSMS/triggered imaging (TI) for intrafraction monitoring was developed for rib/sternum SBRT beginning November 2021 and all NSB SBRT beginning February 2022. Treatment time and CBCT-related radiation dose between the OSMS and the non-OSMS intrafraction monitoring group was analyzed pre- and post-OSMS/TI workflow adoption. All fractions were analyzed through statistical process control with use of an XmR chart of treatment time per quarter from February 2019 to February 2023. Special cause rules were based on Institute for Healthcare Improvement criteria. RESULTS: From February 2019 to February 2023, 1993 NSB SBRT fractions were delivered, including 234 rib, 109 sternum, 214 ilium, and 682 multisite. Over 20 commanded shifts, OSMS could detect 2-mm shifts to within 0.4 mm 67% of the time and 0.8 mm 95% of the time. All NSB SBRT sites showed significant reductions in treatment time, including the greatest improvement in rib total treatment (21.6-13.4 minutes; P = 1.16 × 10-17) and beam time (7.9-3.2 minutes; P = 7.32 × 10-27). Significant reductions in CBCT-related radiation were also observed for several NSB sites. These process improvements were associated with OSMS adoption. CONCLUSIONS: Adoption of a novel NSB SBRT workflow incorporating OSMS/TI for bone intrafraction motion monitoring reduced treatment time and CBCT-related radiation exposure while also allowing for more continuous intrafraction motion monitoring for NSB SBRT. OSMS/TI enabled the transition to a tattoo-less workflow.

Sensitivity of activatable reactive oxygen species probes by fluorescence spectroelectrochemistry.

We have developed a new analytical method of evaluating activatable fluorescent probes for ROS detection using integrated fluorescence spectroelectrochemistry. The Tafel formalism was applied to describe the process of the probes' oxidation under electrochemical conditions and identify a novel parameter defined as the threshold oxidation potential. This potential can serve as an approximation to the equilibrium potential and can be utilized for determining the sensitivity of a probe to oxidation. Based upon the measured values of threshold potentials, the order of sensitivity towards oxidation among several commonly used probes was determined to be the following (from highest to lowest): 2,7-dihydrodichlorofluorescein > dihydroethidium > dihydrorhodamine 123 > dihydrorhodamine 6G. The presented approach opens up a new direction in synthesizing and screening novel ROS probes with a well-defined sensitivity for in vitro and in vivo applications.

Identification of age- and immune-related gene signatures for clinical outcome prediction in lung adenocarcinoma.

BACKGROUND: The understanding of the factors causing decreased overall survival (OS) in older patients compared to younger patients in lung adenocarcinoma (LUAD) remains. METHODS: Gene expression profiles of LUAD were obtained from publicly available databases by Kaplan-Meier analysis was performed to determine whether age was associated with patient OS. The immune cell composition in the tumor microenvironment (TME) was evaluated using CIBERSORT. The fraction of stromal and immune cells in tumor samples were also using assessed using multiple tools including ESTIMATE, EPIC, and TIMER. Differentially expressed genes (DEGs) from the RNA-Seq data that were associated with age and immune cell composition were identified using the R package DEGseq. A 22-gene signature composed of DEGs associated with age and immune cell composition that predicted OS were constructed using Least Absolute Shrinkage and Selection Operator (LASSO). RESULTS: In The Cancer Genome Atlas (TCGA)-LUAD dataset, we found that younger patients (≤70) had a significant better OS compared to older patients (>70). In addition, older patients had significantly higher expression of immune checkpoint proteins including inhibitory T cell receptors and their ligands. Moreover, analyses using multiple bioinformatics tools showed increased immune infiltration, including CD4+ T cells, in older patients compared to younger patients. We identified a panel of genes differentially expressed between patients >70 years compared to those ≤70 years, as well as between patients with high or low immune scores and selected 84 common genes to construct a prognostic gene signature. A risk score calculated based on 22 genes selected by LASSO predicted 1, 3, and 5-year OS, with an area under the curve (AUC) of 0.72, 0.72, 0.69, receptively, in TCGA-LUAD dataset and an independent validation dataset available from the European Genome-phenome Archive (EGA). CONCLUSION: Our results demonstrate that age contributes to OS of LUAD patients atleast in part through its association with immune infiltration in the TME.