Versatile nanobody-based approach to image, track and reconstitute functional Neurexin-1 in vivo.

Neurexins are key adhesion proteins that coordinate extracellular and intracellular synaptic components. Nonetheless, the low abundance of these multidomain proteins has complicated any localization and structure-function studies. Here we combine an ALFA tag (AT)/nanobody (NbALFA) tool with classic genetics, cell biology and electrophysiology to examine the distribution and function of the Drosophila Nrx-1 in vivo. We generate full-length and ΔPDZ ALFA-tagged Nrx-1 variants and find that the PDZ binding motif is key to Nrx-1 surface expression. A PDZ binding motif provided in trans, via genetically encoded cytosolic NbALFA-PDZ chimera, fully restores the synaptic localization and function of NrxΔPDZ-AT. Using cytosolic NbALFA-mScarlet intrabody, we achieve compartment-specific detection of endogenous Nrx-1, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles. Our findings illustrate the versatility of the ALFA system and pave the way towards dissecting functional domains of complex proteins in vivo.

Mapping Milwaukee's Blueprint for Peace: Evaluating the Geospatial Reach of a Cure Violence Implementation, 414LIFE.

BACKGROUND: Cure Violence interruption programs are evidence-based interventions aimed at reducing the transmission of gun violence and its related injuries. Assessing the implementation of these programs can include the metric of "reach." This study evaluated one such program - 414LIFE - in Milwaukee, Wisconsin. The evaluation reconceptualized "reach" as a metric for reaching the individuals and neighborhoods at greatest risk for gun violence. METHODS: 414LIFE's reach was analyzed descriptively and geospatially through its program evaluation dataset from May 2019 through September 2020 using a cross-sectional design. Program referral criteria includes patients who sustained a gunshot wound, are less than 36 years old, and a resident of, or injured in, the city of Milwaukee. A choropleth map visualized location of participants' residence, which justified a global Moran's I, and then a local Moran's I calculation to identify statistically significant clustering of referrals. RESULTS: In the first 1.5 years of the program's partnership with the local level I trauma center and affiliated academic medical institution, 398 patients were referred. Three hundred referrals (75.4%) met program criteria; 53.8% were Black men. Statistically significant clusters were identified and mapped. Half of the top 10 neighborhoods with referrals were the city's identified priority neighborhoods. CONCLUSIONS: 414LIFE successfully reaches its intended population and geographic locations. Geospatial reach should be considered routinely in program evaluations of Cure Violence programs to track growth and reach over time.

Predictive Factors for the Application of Pelvic Binders in the Prehospital Setting.

OBJECTIVES: Early pelvic binder placement in the field stabilizes pelvic fractures and tamponades potential hemorrhage within the pelvis. Despite known risk factors for pelvic fracture, it remains challenging to quickly triage and correctly apply a pelvic binder. We aim to develop a prediction model that exclusively uses prehospital criteria to inform the decision to place a pelvic binder. METHODS: The trauma registry was used to identify all trauma patients admitted to an urban Level I trauma center between January 2013 and December 2017. Variables collected included patient demographics, mechanism of injury, prehospital vital signs, and the presence of a pelvic fracture. Participants were randomly assigned to a training group (70%) or a validation group (30%). Univariate analyses were used to identify significant predictors for use in multivariate predictive models. RESULTS: A total of 8,480 (65% male; median age 49; median ISS 9) and 3,676 (65% male; median age 48; median ISS 9) trauma patients were randomly assigned to the training and validation groups, respectively. Univariate analysis showed significant likelihood of pelvic fracture associated with female sex, hemodynamic instability (initial systolic blood pressure < 90 mmHg), blunt injury type, specific mechanisms of injury (motor vehicle collision, motorcycle collision, pedestrian struck by motor vehicle, crushing injury, and riding an animal), impact location, and position in vehicle. Multivariate models adjusting for blunt type injury, hemodynamic instability, impact location, and position in vehicle showed that presence of two or more of these risk factors is significantly associated with presence of pelvic fracture. CONCLUSION: Establishing select prehospital criteria for the empiric application of pelvic binders for patients in the field with blunt injuries, hemodynamic instability, frontal or side motor vehicle collision impact, and non-front seat passenger may improve outcomes among patients with pelvic fractures.

Immune microenvironment of basal cell carcinoma and tumor regression following combined PD-1/LAG-3 blockade.

Systemic treatment options for patients with locally advanced or metastatic basal cell carcinoma (BCC) are limited, particularly when tumors are refractory to anti-programmed cell death protein-1 (PD-1). A better understanding of immune checkpoint expression within the BCC tumor microenvironment may inform combinatorial treatment strategies to optimize response rates. CD3, PD-1, programmed death ligand-1 (PD-L1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin domain and mucin domain 3 (TIM-3)+ cell densities within the tumor microenvironment of 34 archival, histologically aggressive BCCs were assessed. Tumor infiltrating lymphocyte (TIL) expression of PD-1, PD-L1, and LAG-3, and to a lesser degree TIM-3, correlated with increasing CD3+ T-cell densities (Pearson's r=0.89, 0.72, 0.87, and 0.63, respectively). 100% of BCCs (34/34) demonstrated LAG-3 and PD-1 expression in >1% TIL; and the correlation between PD-1 and LAG-3 densities was high (Pearson's r=0.89). LAG-3 was expressed at ~50% of the level of PD-1. Additionally, we present a patient with locally-advanced BCC who experienced stable disease during and after 45 weeks of first-line anti-PD-1 (nivolumab), followed by a partial response after the addition of anti-LAG-3 (relatlimab). Longitudinal biopsies throughout the treatment course showed a graduated increase in LAG-3 expression after anti-PD-1 therapy, lending support for coordinated immunosuppression and suggesting LAG-3 as a co-target for combination therapy to augment the clinical impact of anti-PD-(L)1.

Arterial revascularization in patients with hand pain dialyzing with upper arm Arteriovenous (AV) fistulas: A single center experience.

BACKGROUND: Distal hand ischemia syndrome (DHIS) is a well reported adverse outcome in patients with upper arm AV access. 25%-40% of these patients have been reported to be due to primary arterial disease complicated with significant arterial calcification. The effectiveness of revascularization of the distal arterial circulation on symptom resolution has not been reported yet. METHODS: Retrospective single center analysis of patients evaluated for hand/forearm pain in patients with upper arm AV access who had arterial revascularization between 01/2016 and 12/2020 were included for the analysis. Fifty-one patients met inclusion criteria. Stenotic lesions greater than 70% in the subclavian, axillary, brachial, radial, or ulnar artery were treated with balloon angioplasty. Institutional approval was obtained to review charts. OUTCOMES: Successful revascularization, improvement in pain in 48 h, 1 month, and 3 months. RESULTS: Seventy six percent of patients had an upper arm Arteriovenous Fistula (AVF) and 24% patients had an upper arm Arteriovenous Graft (AVG). Mean access flow was 1210 (556) ml/min. 55% of patients had radial or ulnar arterial stenosis, 45% had brachial/axillary or subclavian artery stenosis. 45% patients had lesions in both radial and ulnar arteries, 88% of patients were successfully revascularized. 76% (18) of patients had improvement in symptoms within 48 h and 68% remained symptom free in 3 months. Mean DHIS stage was 3.1 before intervention and improved to 1.1 post intervention (p < 0.001). Patient satisfaction with their AV access improved from 34% to 72% (p < 0.01). Multiple regression analysis did not reveal statistically significant correlations between time on dialysis vintage and other chronic medical conditions on post procedure symptom improvement. CONCLUSIONS: DHIS with occlusive arterial disease can be successfully revascularized to improve symptoms. Complete evaluation of the inflow arterial segment and optimal endovascular revascularization could decrease the need for access revision procedures or access abandonment.

Vancomycin dosing in high-intensity continuous renal replacement therapy: A retrospective cohort study.

INTRODUCTION: An inverse relationship exists between vancomycin serum concentrations and the intensity of continuous renal replacement therapy (CRRT), reflected through the dialysate flow rate (DFR). There remains a lack of evidence to guide initial vancomycin dosing in the setting of high-intensity CRRT (i.e., DFR >30 mL/kg/h). Additionally, recommendations for pharmacokinetic monitoring of vancomycin have transitioned from a trough-based to area under the curve (AUC)-based dosing strategy to optimize efficacy and safety. Therefore, an improved understanding of the impact of CRRT intensity on AUC/MIC (minimum inhibitory concentration) has the potential to enhance vancomycin dosing in this patient population. OBJECTIVES: The goal of this study is to evaluate current vancomycin dosing strategies and achievement of pharmacokinetic targets in patients on high-intensity CRRT. METHODS: This was a single-center, retrospective cohort study of adult critically ill patients admitted to Houston Methodist Hospital between May 2019 and October 2021 and received vancomycin therapy while on high-intensity CRRT. High-intensity CRRT was defined by a DFR that was both ≥3 L/h and >30 mL/kg/h. Depending on the initial vancomycin dosing strategy, patients were stratified into either the traditional (15 mg/kg/day) or enhanced (≥15 mg/kg/day) dosing group. The primary outcome was the percent of patients who attained steady-state AUC24 /MIC ≥400 mg*h/L at the first obtained vancomycin level in the enhanced group compared with the traditional group. RESULTS: A total of 125 patients were included in the final analysis, 56 in the traditional and 69 in the enhanced dosing group. The primary end point occurred in 74% and 54% of patients in the enhanced and traditional dosing groups, respectively (p = 0.029). Therapeutic vancomycin trough levels (10-20 µg/mL) were more commonly achieved in the enhanced dosing group compared with the traditional dosing group (66.7% vs. 45%, p = 0.013). As DFR rose, increasingly higher doses of vancomycin, up to 27 mg/kg/day, were required to achieve the therapeutic targets. CONCLUSION: This is the first study to evaluate the influence of variable CRRT intensities on vancomycin AUC/MIC. Our findings suggest that vancomycin doses of ≥15 mg/kg/day are needed to achieve early therapeutic targets in patients on high-intensity CRRT.

Jumping performance in tree squirrels: Insights into primate evolution.

Morphological traits suggesting powerful jumping abilities are characteristic of early crown primate fossils. Because tree squirrels lack certain 'primatelike' grasping features but frequently travel on the narrow terminal branches of trees, they make a viable extant model for an early stage of primate evolution. Here, we explore biomechanical determinants of jumping performance in the arboreal Eastern gray squirrel (Sciurus carolinensis, n = 3) as a greater understanding of the biomechanical strategies that squirrels use to modulate jumping performance could inform theories of selection for increased jumping ability during early primate evolution. We assessed vertical jumping performance by using instrumented force platforms upon which were mounted launching supports of various sizes, allowing us to test the influence of substrate diameter on jumping kinetics and performance. We used standard ergometric methods to quantify jumping parameters (e.g., takeoff velocity, total displacement, peak mechanical power) from force platform data during push-off. We found that tree squirrels display divergent mechanical strategies according to the type of substrate, prioritizing force production on flat ground versus center of mass displacement on narrower poles. As jumping represents a significant part of the locomotor behavior of most primates, we suggest that jumping from small arboreal substrates may have acted as a potential driver of the selection for elongated hindlimb segments in primates, allowing the center of mass to be accelerated over a longer distance-and thereby reducing the need for high substrate reaction forces.

Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/µL and a time to result of under 30 minutes.

Survival outcomes for patients with colorectal cancer with synchronous liver only metastasis.

BACKGROUND: Colorectal cancer with synchronous liver-only metastasis is managed with a multimodal approach, however, optimal sequencing of modalities remains unclear. METHODS: A retrospective review of all consecutive rectal or colon cancer cases with synchronous liver-only metastasis was conducted from the South Australian Colorectal Cancer Registry from 2006 to 2021. This study aimed to investigate how order and type of treatment modality affects overall survival. RESULTS: Data of over 5000 cases were analysed (n = 5244), 1420 cases had liver-only metastasis. There were a greater number of colon than rectal primaries (N = 1056 versus 364). Colonic resection was the preferred initial treatment for the colon cohort (60%). In the rectal cohort, 30% had upfront resection followed by 27% that had chemo-radiotherapy as 1st line therapy. For the colon cohort, there was an improved 5-year survival with surgical resection as initial treatment compared to chemotherapy (25% versus 9%, P < 0.001). In the rectal cohort, chemo-radiotherapy as the initial treatment was associated with an improved 5-year survival compared to surgery or chemotherapy (40% versus 26% versus 19%, P = 0.0015). Patients who were able to have liver resection had improved survival, with 50% surviving over 5 years compared to 12 months in the non-resected group (P < 0.001). Primary rectal KRAS wildtype patients who underwent liver resection and received Cetuximab had significantly worse outcomes compared to KRAS wildtype patients who did not (P = 0.0007). CONCLUSIONS: Where surgery is possible, resection of liver metastasis and primary tumour improved overall survival. Further research is required on the use of targeted treatments in patients undergoing liver resection.

Designing a decision aid for cancer prevention: a qualitative study.

OBJECTIVES: Australian guidelines recommend people aged 50-70 years old consider taking low-dose aspirin to reduce their risk of colorectal cancer. The aim was to design sex-specific decision aids (DAs) with clinician and consumer input, including expected frequency trees (EFTs) to communicate the risks and benefits of taking aspirin. METHODS: Semi-structured interviews were conducted with clinicians. Focus groups were conducted with consumers. The interview schedules covered ease of comprehension, design, potential effects on decision-making, and approaches to implementation of the DAs. Thematic analysis was employed; independent coding by 2 researchers was inductive. Themes were developed through consensus between authors. RESULTS: Sixty-four clinicians were interviewed over 6 months in 2019. Twelve consumers aged 50-70 years participated in two focus groups in February and March 2020. The clinicians agreed that the EFTs would be helpful to facilitate a discussion with patients but suggested including an additional estimate of the effects of aspirin on all-cause mortality. The consumers felt favourable about the DAs and suggested changes to the design and wording to ease comprehension. CONCLUSION: DAs were designed to communicate the risks and benefits of low-dose aspirin for disease prevention. The DAs are currently being trialled in general practice to determine their impact on informed decision-making and aspirin uptake.

Aspirin can help to prevent bowel cancer up by to 25% and the chances of dying from it by up to 33%. Australian guidelines recommend that people aged 50­70 years old to consider taking low-dose aspirin to reduce their risk of bowel cancer. To encourage GPs and their patients to discuss the guidelines, we designed a brochure called a decision aid with the help of clinicians and people in the community of Victoria, Australia. The decision aid covered the benefits and risks of taking aspirin. Clinicians participated in interviews and provided feedback on the statistics presented in a chart called an expected frequency tree. People in the community participated in group discussions and improved the design and comprehension of the decision aid. The clinicians and people who participated in this study do not fully represent the diversity of the Australian population, as they were mostly white and highly educated. We are now testing if the decision aid is effective for supporting a discussion between patients and general practitioners, helping their patients make an informed decision about taking aspirin, and whether it encourages them to take aspirin daily after being shown the decision aid in general practice.

Successful aortic valve replacement by overexpanding a SAPIEN transcatheter heart valve for a large aortic annulus (>900†mm2): a case report.

Background: There are limited commercial valve sizes available for transcatheter aortic valve implantation (TAVI). This makes it challenging or even prohibitive to perform TAVI on large aortic annuli. Case summary: A 78-year-old male with known low-flow, low-gradient severe aortic stenosis presented with progressive dyspnoea, chest pressure, and decompensated heart failure. Successful off-label TAVI was performed for tricuspid aortic valve stenosis with an aortic annulus of >900 mm2. An Edwards S3 29 mm valve was overexpanded with an extra 7 mL of volume at valve deployment. No complications occurred, and only trivial paravalvular leak was seen following implantation. The patient died from a non-cardiovascular cause 8 months following the procedure. Discussion: Patients that require aortic valve replacement with prohibitive surgical risk and have very large aortic valve annuli pose significant technical challenges. This case shows the feasibility of TAVI by overexpanding an Edwards S3 valve.

Improving Quality Metrics with a Day-only Skin Abscess Protocol: Experience from Australia.

BACKGROUND: Skin abscesses are a common emergency presentation often requiring incision and drainage; however, issues with theatre access lead to delays in management and high costs. The long-term impact in a tertiary centre of a standardised day-only protocol is unknown. The aim was to evaluate the impact of day-only skin abscess protocol (DOSAP) for emergency surgery of skin abscesses in a tertiary institution in Australia and to provide a blueprint for other institutions. METHODS: A retrospective cohort study analysed several time periods: Period A (July 2014 to 2015, n = 201) pre-DOSAP implementation, Period B (July 2016 to 2017, n = 259) post-DOSAP, and Period C (July 2018 to 2022, n = 1,625) prospectively analysed four 12-month periods to assess long-term utilisation of DOSAP. Primary outcomes were length of stay and delay to theatre. Secondary outcome measures included theatre start time, representation rates and total costs. Statistical analysis using nonparametric methods was used to analyse the data. RESULTS: There was a significant decrease in ward length of stay (1.25 days vs. 0.65 days, P < 0.0001), delay to theatre (0.81 days vs. 0.44 days, P < 0.0001) and theatre start time before 10AM (44 cases vs. 96 cases, P < 0.0001) after implementation of DOSAP. There was a significant decrease in median cost of admission of $711.74 after accounting for inflation. Period C reported 1,006 abscess presentations successfully managed by DOSAP over the four-year period. CONCLUSION: Our study demonstrates the successful implementation of DOSAP in an Australian tertiary centre. The ongoing utilisation of the protocol demonstrates the ease of applicability.

Synergistic and Antagonistic Effects of the Co-Pyrolysis of Plastics and Corn Stover to Produce Char and Activated Carbon.

The physicochemical properties of char and activated carbon produced from the co-pyrolysis of corn stover (CS) and plastics, polystyrene (PS) and polyethylene terephthalate (PET), were studied. Non-isothermal gas analysis of the volatiles was conducted using an online mass spectrometer to correlate the thermal degradation of gaseous byproducts to the formation of pores in the char materials. The findings determined that the addition of PS or PET promotes the formation of the solid char product with either higher than average pore sizes or surface areas compared to control samples. The addition of PET to corn stover increases the surface area of the char formed. The char formed from a CS:PET mass ratio of 1:1 produced char with a surface area of 423.8 ± 24.8 m2/g at 500 °C and a duration of 2 h. The surface area of the chars formed from CS and PET decreased as the amount of PET decreased, showing a tendency for PET to increase the surface area of the char materials synergistically. The addition of PS to corn stover promoted the formation of chars with, on average, larger pore sizes than the control char samples. The chars were chemically activated with potassium hydroxide, and the activated carbon that formed had lower surface areas but comparable surface functional groups to the control samples. Vanillin adsorption testing showed that activated carbon from corn stover performed the best at removing 95% of the vanillin after 2 h. In contrast, the activated carbon from the chars produced from the co-pyrolysis of corn stover and polystyrene or corn stover and polyethylene terephthalate removed 45% and 46% of vanillin after 2 h, respectively. The findings suggest that plastics have a synergistic relationship in producing char precursors with improved porosity but antagonistically affect the activated carbon adsorbent properties.

Harnessing synthetic biology to enhance ocean health.

Ocean health is faltering, its capability for regeneration and renewal being eroded by a steady pulse of anthropomorphic impacts. Plastic waste has infiltrated all ocean biomes, climate change threatens coral reefs with extinction, and eutrophication has unleashed vast algal blooms. In the face of these challenges, synthetic biology approaches may hold untapped solutions to mitigate adverse effects, repair ecosystems, and put us on a path towards sustainable stewardship of our planet. Leveraging synthetic biology tools would enable innovative engineering approaches to augment the natural adaptive capacity of ocean biological systems to cope with the swiftness of human-induced change. Here, we present a framework for developing synthetic biology solutions for the challenges of plastic pollution, coral bleaching, and harmful algal blooms.

Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 µM/min ± 1.76 µM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.

End-to-end design of wearable sensors.

Wearable devices provide an alternative pathway to clinical diagnostics by exploiting various physical, chemical and biological sensors to mine physiological (biophysical and/or biochemical) information in real time (preferably, continuously) and in a non-invasive or minimally invasive manner. These sensors can be worn in the form of glasses, jewellery, face masks, wristwatches, fitness bands, tattoo-like devices, bandages or other patches, and textiles. Wearables such as smartwatches have already proved their capability for the early detection and monitoring of the progression and treatment of various diseases, such as COVID-19 and Parkinson disease, through biophysical signals. Next-generation wearable sensors that enable the multimodal and/or multiplexed measurement of physical parameters and biochemical markers in real time and continuously could be a transformative technology for diagnostics, allowing for high-resolution and time-resolved historical recording of the health status of an individual. In this Review, we examine the building blocks of such wearable sensors, including the substrate materials, sensing mechanisms, power modules and decision-making units, by reflecting on the recent developments in the materials, engineering and data science of these components. Finally, we synthesize current trends in the field to provide predictions for the future trajectory of wearable sensors.

Missed diagnosis of cirrhosis in the inpatient setting.

Cirrhosis accounts for a large number of deaths in the United States and worldwide, leading to an increasing burden on the healthcare system. Cirrhosis is, however, a progressive disease with different potential complications related to liver dysfunction and portal hypertension. Often, patients may present with complications of cirrhosis without having been diagnosed previously. It is pertinent that clinicians recognize these signs to place patients on an appropriate course of management to help delay or avoid further disease progression while avoiding deleterious outcomes and unnecessary utilization. We will discuss the epidemiology of liver disease, cirrhosis, and its complications (hepatic encephalopathy, ascites, and varices). In this study, we will discuss the rationale and impact of missing these diagnoses on the healthcare system and patient.