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Clostridioides difficile is a Gram-positive anaerobe that can cause a spectrum of disorders that range in severity from mild diarrhoea to fulminant colitis and/or death. The bacterium produces up to three toxins, which are considered the major virulence factors in C. difficile infection. These toxins promote inflammation, tissue damage and diarrhoea. In this Review, we highlight recent biochemical and structural advances in our understanding of the mechanisms that govern host-toxin interactions. Understanding how C. difficile toxins affect the host forms a foundation for developing novel strategies for treatment and prevention of C. difficile infection.

Objectives To describe clinicopathologic characteristics and survival outcomes of endometrial adenocarcinomas stratified by mismatch repair (MMR) status. Methods Single-institution, retrospective study of all women with endometrioid adenocarcinomas treated from January 2012 through December 2017. Patients were categorized into one of three groups based on MMR testing: intact MMR expression (MMR+), probable MMR mutation (MMR–), or MLH1 hypermethylation (hMLH1+). Demographics, pathologic characteristics, recurrence rates, and survival differences were analyzed. Results In total, 316 women were included in the analysis: 235 (74.4%) patients in the MMR+ group, 10 (3.1%) in the MMR– group, and 71 (22.5%) in the hMLH1+ group. Patients with hMLH1+ were significantly older, exhibited higher-grade histology and presence of lymphovascular space invasion, and were more likely to have received adjuvant treatment. The early stage hMLH1+ patients were more likely to recur (15.3% hMLH1+ vs 2.3% MMR+ vs 12.5% MMR–, P Conclusions Women with hMLH1+ endometrial cancer have worse prognostic features and recur more frequently, even in patients traditionally considered low risk for recurrence.

Introduction. Drug checking services for harm reduction and overdose prevention have been implemented in many jurisdictions as a public health intervention in response to the opioid overdose crisis. This study demonstrates the first on-site use of paper spray mass spectrometry for quantitative drug checking to address the limitations of current on-site drug testing technologies. Methods. Paper spray mass spectrometry was used to provide on-site drug checking services at a supervised consumption site in the Downtown Eastside of Vancouver, British Columbia, Canada during a 2-day pilot test in August 2019. The method included the targeted quantitative measurement of 49 drugs and an untargeted full scan to assist in identifying unknown/unexpected components. Results. During the pilot, 113 samples were submitted for analysis, with 88 (78%) containing the client expected substance. Fentanyl was detected in 45 of 59 expected fentanyl samples, and in 50 (44%) samples overall at a median concentration of 3.6% (w/w%). The synthetic precursor of fentanyl, 4-anilino-N-phenethyl-piperidine (4-ANPP), was found in 74.0% of all fentanyl samples at a median concentration of 2.2%, suggesting widespread poor manufacturing practices. Etizolam was detected in 10 submitted samples anticipated to be fentanyl at a median concentration of 2.5%. No clients submitting these samples expected etizolam or a benzodiazepine in their sample. In three instances, it was co-measured with fentanyl, and in seven cases it was detected alone. Discussion and Conclusions. The quantitative capabilities and low detection limits demonstrated by paper spray mass spectrometry offer distinct benefits over existing on-site drug checking methods and harm reduction services. This work was supported by the Natural Science and Engineering Research Council of Canada Discovery Grant Program funding (RGPIN-2016-05380), Vancouver Island University and the Health Canada Drug Checking Technology Challenge. This article was originally published as: Borden, S.A., Saatchi, A., Vandergrift, G.W., Palaty, J., Lysyshyn, M., & Gill, C.G. (2021). A new quantitative drug checking technology for harm reduction: Pilot study in Vancouver, Canada using paper spray mass spectrometry. Drug and Alcohol Review, 1-8. https://doi.org/10.1111/dar.13370 This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. https://viurrspace.ca/bitstream/handle/10613/24515/Gill2021.pdf?sequence=3

Electroadhesion, i.e., adhesion induced by an electric field, occurs between non-sticky cationic and anionic hydrogels. Here, we demonstrate electroadhesion between cationic gels and animal (bovine) tissues. When gel and tissue are placed under an electric field (DC, 10 V) for 20 s, the pair strongly adhere, and the adhesion persists indefinitely thereafter. Applying the DC field with reversed polarity eliminates the adhesion. Electroadhesion works with the aorta, cornea, lung, and cartilage. We demonstrate the use of electroadhesion to seal cuts or tears in tissues or model anionic gels. Electroadhered gel-patches provide a robust seal over openings in bovine aorta, and a gel sleeve is able to rejoin pieces of a severed gel tube. These studies raise the possibility of using electroadhesion in surgery while obviating the need for sutures. Advantages include the ability to achieve adhesion on-command, and moreover the ability to reverse this adhesion in case of error., The authors demonstrate strong adhesion of cationic hydrogels to bovine tissues under a DC electric field. Such electroadhesion can be reversed by switching the polarity of the field. This approach could enable simpler surgeries, where sutures are not needed.

Peripheral nerve blocks are commonly used in total knee arthroplasty (TKA). Liposomal bupivacaine is an extended-release anesthetic medication that maintains efficacy upwards of 72 hours. This study compared single-shot liposomal bupivacaine (LB) with the standard single-shot bupivacaine (SB) in a preoperative adductor canal block in TKA patients. A double-blind randomized, controlled trial at a single institution was performed in patients undergoing TKA. A standard preoperative single-shot adductor canal nerve injection was performed in 31 patients using 266 mg of liposomal bupivacaine (20 mL), whereas 32 patients received a standard formulation of 0.5% bupivacaine hydrogen chloride (20 mL). The primary outcome measure was postoperative gait velocity. Secondary outcomes included knee range of motion, pain scores, patient satisfaction, knee extension strength, opioid consumption, length of stay, and adverse events. There were no differences in baseline measures between groups. Improved pain ratings with activity ( P =.009) were noted on postoperative day 1 with LB (mean, 4.4; SD, 2.0) compared with SB (mean, 5.9; SD, 2.6). Fewer opioids were used with LB compared with SB on postoperative day 1 (mean, 51.2 vs 66.1; P =.020) and on postoperative day 2 (mean, 39.5 vs 54.8; P =.016). No statistically significant differences in gait velocity, knee range of motion, knee extension strength, or patient satisfaction occurred. Peripheral nerve blockade with a single-shot adductor canal injection demonstrated improved pain scores with activity and diminished postoperative narcotic use when using LB compared with SB in TKA patients. There may be early postoperative advantages with LB as a single-shot injection in adductor canal blockade for patients undergoing TKA. [ Orthopedics . 2021;44(4):249–255.]

Background This study sought to define duplex ultrasound (DUS) velocity criteria predicting ≥70% stenosis in superior mesenteric artery (SMA) stents by correlating in-stent peak systolic velocity (PSV) with computed tomographic angiography (CTA) measurements of percent stenosis. Methods A retrospective review of 109 patients undergoing SMA stenting between 2003 and 2018 was conducted at a single institution. Thirty-seven surveillance duplex ultrasound studies were found to have a CTA performed within 30 days of study completion. Bare metal (n = 20) and covered stents (n = 17) were included. Velocities were paired to in-stent restenosis (ISR) measured by mean vessel diameter reduction on SMA centerline reconstructions from CTA. Receiver operating characteristic (ROC) curves was generated and logistic regression models for ≥70% ISR probability were used to define velocity criteria in the stented SMA. Results At a PSV of 300 cm/sec, the sensitivity is 100% and specificity 80% for a ≥70% in-stent SMA stenosis. At a PSV of 400 cm/sec, the sensitivity and positive predictive value (PPV) is 63% and the specificity and negative predictive value (NPV) is 90%. A PSV of 450 cm/sec was consistent with the highest specificity (100%) and PPV (100%) but lower sensitivity (50%) and NPV (87.9%). One patient with a PSV of 441 cm/sec on surveillance DUS died from complications of acute-on-chronic mesenteric ischemia. Conclusions A PSV of 400 cm/sec on mesenteric DUS can predict ≥70% ISR with high sensitivity and should be considered as a diagnostic threshold for SMA in-stent restenosis.

Background The impact of workplace discrimination has gained recognition. Nearly two-thirds of all medical residency applicants reported being asked inappropriate or potentially illegal interview questions. The use of such questions during neurosurgery residency interviews has not yet been studied. Objective To evaluate the prevalence of inappropriate or potentially illegal questions in residency interviews and the impact on applicants' rank lists. Methods All 2018 to 2019 United States neurosurgery resident applicants were anonymously surveyed. The survey included 46 questions focused on demographics; if they were asked questions regarding rank list, age, gender, marital status, family planning, religion, sexual orientation, or disability and whether such questions affected their rank list formation. Results Of 265 surveyed United States applicants, 133 (50%) responded. Most respondents were male (78%), 24% were married, and 10% had children. During the formal interview, 94% were asked at least 1 inappropriate or potentially illegal question. About 78% reported being asked about marital status, 29% were asked about intent to have children. About 46% reported being counseled on their personal life, 30% were asked about their ethnic background, and 15% were asked about their religion. A total of 2 candidates reported questions about mental illness/disability, and 2 candidates reported being asked about sexual orientation. About 45% of applicants that were asked at least 1 of these questions ranked those programs lower. Conclusion Nearly all (94%) neurosurgical residency applicants reported being asked at least 1 inappropriate or potentially illegal question during interviews. Our results indicate that inappropriate questions negatively affected program rankings.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Platinum-based chemotherapy is commonly associated with toxic sensory neuropathies, but also, although rarely, with Guillain-Barré syndrome (GBS). We describe five patients who developed GBS while receiving platinum-based chemotherapy for a solid tumor and report the five cases published so far. Most patients had received cumulative platinum doses below known neurotoxic levels, and all of them had an optimal outcome after platinum discontinuation, associated in most cases with administration of intravenous immunoglobulin. Clinical presentation, electroneuromyography, and cerebrospinal fluid analysis help clinicians to differentiate GBS from toxic neuropathy. Platinum compounds are the only chemotherapeutic agents used for solid tumors that have been associated to GBS. Thus, we propose that GBS may constitute a non–dose-dependent side effect of platinum drugs and that awareness needs to be raised among oncologists on this rare but potentially life-threatening complication of platinum chemotherapy. Implications for Practice: Many patients on platinum-based chemotherapy for solid tumors develop sensory neuropathy, a common dose-dependent side effect. The authors propose that Guillain-Barré syndrome may constitute an immune-mediated, non-dose-related side effect of platinum-based chemotherapy. Prompt diagnosis of Guillain-Barré syndrome and distinction from classical toxic neuropathy are crucial for optimal treatment. Platinum discontinuation, associated if needed to intravenous immunoglobulin administration, radically changes the course of the disease and minimizes neurological sequelae.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, has been implicated in the pathogenesis of many cancers, with involvement in cell-cycle regulation, apoptosis, and immune response. Small-molecule GSK-3β inhibitors are currently undergoing clinical investigation. Tumor sequencing has revealed genomic alterations in GSK-3β, yet an assessment of the genomic landscape in malignancies is lacking. This study assessed >100,000 tumors from two databases to analyze GSK-3β alterations. GSK-3β expression and immune cell infiltrate data were analyzed across cancer types, and programmed death-ligand 1 (PD-L1) expression was compared between GSK-3β–mutated and wild-type tumors. GSK-3β was mutated at a rate of 1%. The majority of mutated residues were in the kinase domain, with frequent mutations occurring in a GSK-3β substrate binding pocket. Uterine endometrioid carcinoma was the most commonly mutated (4%) tumor, and copy-number variations were most commonly observed in squamous histologies. Significant differences across cancer types for GSK-3β–mutated tumors were observed for B cells (P = 0.018), monocytes (P = 0.002), dendritic cells (P = 0.005), neutrophils (P = 0.0003), and endothelial cells (P = 0.014). GSK-3β mRNA expression was highest in melanoma. The frequency of PD-L1 expression was higher among GSK-3β–mutated tumors compared with wild type in colorectal cancer (P = 0.03), endometrial cancer (P = 0.05), melanoma (P = 0.02), ovarian carcinoma (P = 0.0001), and uterine sarcoma (P = 0.002). Overall, GSK-3β molecular alterations were detected in approximately 1% of solid tumors, tumors with GSK-3β mutations displayed a microenvironment with increased infiltration of B cells, and GSK-3β mutations were associated with increased PD-L1 expression in selected histologies. These results advance the understanding of GSK-3β complex signaling network interfacing with key pathways involved in carcinogenesis and immune response.

Pediatric patients with complex colorectal and genitourinary conditions often require coordinated multidisciplinary care; however, this coordinated care can be hard to structure and deliver. The purpose of this paper is to review the development and implementation of a multidisciplinary colorectal and pelvic health program, one year after the program's initiation.This is an observational retrospective 1-year study (10/1/2017 to 9/30/2018). In fiscal year (FY) 2018, a multidisciplinary colorectal and pelvic health program was initiated. The program development incorporated bimonthly team meetings, educational conferences, and initiation of three clinics: a complex colorectal and genitourinary reconstruction clinic, a bowel management clinic, and a colonic motility clinic. Conditions treated included complex anorectal and cloacal malformations, Hirschsprung disease, and idiopathic constipation. The fiscal year was selected to provide comparative administrative data after program implementation.During the study period, 121 patients underwent comprehensive collaborative evaluation of which 58 (47%) were new to the institution compared to 12 (19%) new patients in the previous year (p 0.001). In FY 2018, there were 130 procedures performed and 512 collaborative visits with an average of 47 visits per month. This was a 3.4-fold increase in visits compared to FY2017 (171 visits). Of the new patients, 60% (35/58), traveled a median of 181 miles, representing 33 statewide counties, and 4 states compared to a median of 93 miles in the previous fiscal year (p = 0.004).The development of a colorectal and pelvic health program is feasible and requires a collaborative approach, necessitating multiple service lines within an institution. Program creation and implementation can result in rapid institutional clinical growth by filling a local and regional need through coordinated multidisciplinary care.IV.

Muscle tissue from three-spotted tilapia (Oreochromis andersonii) (n = 11), sharp tooth catfish (Clarias gariepinus) (n = 14) and tigerfish (Hydrocynus vittatus) (n = 12) were sampled from a market...

Objective To review key arguments supporting and criticizing syringe services programs (SSPs). Data sources Peer-reviewed literature and publicly available documents. Summary Pharmacy organizations—including the American Pharmacists Association and the American Society of Health-System Pharmacists—have stated that pharmacists should support programs that supply sterile injection materials to persons who use injection drugs. SSPs may include needle exchanges or other programs that meet these aims. Pharmacists should know that observational public health research demonstrates that SSPs can lower the transmission of blood-borne illnesses, improve linkage to care for substance use disorders, reduce health care expenditures, and reduce drug overdoses. Concerns about SSPs and increases in syringe litter or crime have not been borne out by research. Despite these findings and the positions of professional organizations, contemporary research suggests that pharmacists may be reluctant to support SSPs and other programs that would increase supply of sterile injection supplies to their communities. Conclusion The review of evidence in this commentary should help pharmacists better understand the evidence in favor of SSPs, the potential criticisms of SSPs, and the reasons that their profession is moving to support these programs.

Breast conservation surgery (BCS) aims to excise all cancerous tissue while minimizing the amount of healthy breast tissue removed. Up to 30% of patients undergoing BCS require a second operation for re-excision to obtain negative margins. Previous studies reported a lower re-excision rate with intraoperative use of the MarginProbe device (Dune Medical Devices). This device utilizes radiofrequency spectroscopy to detect differences between cancerous and normal tissue. From July 2009 to January 2010, our institution enrolled 46 patients electing for BCS in a prospective double-arm randomized controlled trial and had a significantly lower re-excision rate than that reported in the multicenter trial. Intraoperatively, after performing conventional lumpectomy with excision of any additional shavings deemed necessary based on palpation and visual inspection alone, patients were then randomized. In the device arm, the surgeon used the MarginProbe to interrogate the lumpectomy specimen, taking additional shavings from the cavity surfaces corresponding to the parts of the specimen read as positive by the device. In the control arm, only standard intraoperative assessments were performed. All specimens were evaluated by pathologists who were blinded to the study arm. In this population, 72% had invasive ductal carcinoma (IDC), 20% had ductal carcinoma in situ (DCIS), and 8% had invasive lobular carcinoma (ILC). Average age was 64 years old. The average size of the specimen was 5.6 cm, the average volume was 37.8 cm3 , and the average weight was 32.7 g. The mean size of DCIS was 1.4 cm. For invasive specimens, 32 were T1 and 7 were T2. Prior to randomization, 43 patients were thought to have positive or close margins and therefore underwent additional shavings. Twenty-three patients were randomized to the device arm and 23 to the control arm. In the device arm, 14 (60%) patients had IDC, 7 (30%) had DCIS, and 2 (8%) had ILC, vs the control arm where 19 (82%) patients had IDC, 2 (8%) had DCIS, and 2 (8%) had ILC. Eight (35%) patients in the control group vs 1 (4%) in the device group underwent re-excision for margin involvement (P < .05). The use of the MarginProbe device at our institution significantly improved the ability of our surgeons to obtain clear margins during initial BCS. Our results show a lower re-excision rate (4%) than those published in the multicenter trial (19.8%). We postulate that in the face of more patients having DCIS in our device group (30%), our surgeons responded by taking thicker shavings when the MarginProbe device reported margin involvement during the initial lumpectomy, resulting in greater success achieving clear final margins on the shaved tissue and a significantly lower re-excision rate than previously reported with the MarginProbe device.

Objective Overdose from opioids has reached epidemic proportions. Large healthcare systems can utilize existing technology to encourage responsible opioid prescribing practices. Our study measured the effects of using the electronic medical record (EMR) with direct clinician feedback to standardize opioid prescribing practices within a large healthcare system. Methods This retrospective multicenter study compared a 12 month pre- and post-intervention in 14 emergency departments after four interventions utilizing the EMR were implemented: (1) deleting clinician preference lists, (2) defaulting dose, frequency, and quantity, (3) standardizing formulary to encourage best practices, and (4) creating dashboards for clinician review with current opioid prescribing practices. Outlying clinicians received feedback through email and direct counseling. Total number of opioid prescriptions per 100 discharges pre- and post-intervention were recorded as primary outcome. Secondary outcomes included number of prescriptions per 100 discharges/clinician exceeding 3-day supply (defined as 12 tablets), number exceeding 30 morphine equivalent daily dose (MEDD)/day, and number of non-formulary prescriptions. Results There were >700,000 discharges during pre- and post-intervention periods. Percentage of total number opioid prescriptions per 100 discharges decreased from 14.4% to 7.4%, a 7.0% absolute reduction, (95% CI,6.9%–7.2%). There was a 5.9% to 0.7% reduction in prescriptions exceeding 3-days, (95% CI, 5.1%–5.3%), a 4.3% to 0.3% reduction in prescriptions exceeding 30 MEDD, (95% CI, 3.9%–4.0%), and a 0.3% to 0.1% reduction in non-formulary prescriptions, (95% CI, 0.2%–0.3%). Conclusions A multi modal approach using EMR interventions which provide real time data and direct feedback to clinicians can facilitate appropriate opioid prescribing.

No abstract available Keywords: CNS Tumors; Childhood Neuropsychiatric Disorders; Mood; Pediatrics; Psychosis.

We present a case of an 18-month-old, 8.69 kg, female, who presented with a coronary fistula between the left circumflex coronary artery and coronary sinus (CS) for remote computed tomography (CT) imaging and transcatheter closure. This is the fifth published case report to describe this congenital anomaly and the first to discuss general anesthesia (GA) and the hemodynamic management considerations for the anesthesiologist.

Background The rarity of perianesthetic catastrophic events creates a challenge in training support staff in the treatment of emergencies such as local anesthetic systemic toxicity (LAST). Simulation learning offers a unique venue in which to safely encounter rare events. Aims This study aimed to evaluate knowledge retention regarding LAST in perianesthetic nursing staff using high-fidelity simulation and a short didactic session. Design Nurses were recruited from perioperative locations to participate in a simulated scenario of LAST and engage in a short didactic session. Settings Simulation and education occurred in the high-fidelity simulation center at the University of Wisconsin Hospital. Participants/Subjects Thirteen nurses from the preoperative, post-anesthesia, and block nursing teams participated in the study. Methods Participants completed a pre-test before participating in the simulation, followed by a formal debrief and short lecture. They then completed post-tests and a short survey focused on self-efficacy and review of the simulation experience. Results Test scores, compared to the pre-test, improved significantly on the immediately-post, 1-month, and 3-month assessments. Participants felt more equipped to handle crisis scenarios. Conclusions Experiential learning often results in significant knowledge acquisition and retention. Participants in this study improved their test scores regarding LAST and increased their sense of self-efficacy and ability to handle crisis scenarios after taking part in a high-fidelity simulation.

Background Patient boarding in the emergency department (ED) is a significant issue leading to increased morbidity/mortality, longer lengths of stay, and higher hospital costs. We examined the impact of boarding patients on the ED waiting room. Additionally, we determined whether facility type, patient acuity, time of day, or hospital occupancy impacted waiting rooms in 18 EDs across a large healthcare system. Methods This was a retrospective multicenter study that included all ED encounters between January 1, 2018, and September 30, 2019. Encounters with missing Emergency Severity Index (ESI) level were excluded. ESI levels were defined as high (ESI 1,2), middle (ESI 3), and low (ESI 4,5). Spearman correlation coefficients measured the relationship between boarded patients and number of patients in ED waiting room. A multivariable mixed effects model identified drivers of this relationship. Results A total of 1,134,178 encounters were included. Spearman correlation coefficient was significant between number of patients in the ED waiting room and patient boarding (0.54). For every additional patient boarded/hour, the number of patients waiting/hour in the waiting room increased by 8% (95% confidence interval [CI] = 1.08–1.09). The number of patients waiting for a room/hour was 2.28 times higher for middle than for high acuity. The number of patients in waiting room slightly decreased as hospital occupancy increased (95% CI = 0.997–0.997). Conclusion Number of patients in ED waiting room are directly related to boarding times and hospital occupancy. ED waiting room times should be considered as not just an ED operational issue, but an aspect of hospital throughput.

Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, PLoS One 8, e73204 (2013); S. Kozar et al., Cell Stem Cell 13, 626–633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen Clostridioides difficile, we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

The field of youth development continues to evolve and grow, as many professionals (e.g., social workers, health care workers, public health workers, etc.) have begun to recognize the role youth development plays in their daily work. However, the challenge now is to properly identify the youth development-related work of these professionals, in order to appropriately support and resource the workforce to ensure their success with youth. The current article concludes this special issue on the Youth Development Workforce, with insights from key leaders in the field of youth development on the strengths and challenges that the field will face as it transforms. This article, in combination with the prior articles in this special issue, reflects the current efforts of the youth development workforce that acts as a foundation for the future of the field.

Significance Clostridioides difficile is a bacterial pathogen of global importance that is a major cause of hospital-acquired diarrhea. Antibiotic-mediated disruptions to the gut microbiota and associated metabolome promote C. difficile growth and infection through mechanisms that are poorly understood. Here, we show that intestinal bile acids, which are known to play a role in C. difficile germination and outgrowth, also directly bind and inhibit TcdB toxin, the primary virulence determinant of C. difficile . Bile acid binding induces a major conformational change in TcdB structure that prevents receptor binding and uptake into cells. In addition to suggesting a role for bile acids in protecting against C. difficile pathogenesis, these findings highlight an approach to block C. difficile virulence.

Clostridioides difficile is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of C. difficile infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT). Reported here is a 3.8-Å cryo–electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to β-barrel pore, although we also observe a single bound CDTa in the prepore and β-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.

Background Minimally-invasive methods to treat scars address a common pathway of altering collagen structure, leading to collagen remodeling. Objective In this study, we employed in situ redox chemistry to create focal pH gradients in skin, altering dermal collagen, in a process we refer to as electrochemical therapy (ECT). The effects of ECT to induce biochemical and structural changes in ex vivo porcine skin were examined. Methods During ECT, two platinum electrodes were inserted into fresh porcine skin, and following saline injection, an electrical potential was applied. pH mapping, high frequency ultrasonography, and two photon excitation microscopy and second harmonic generation (SHG) microscopy were used to evaluate treatment effects. Findings were correlated with histology. Results Following ECT, pH mapping depicted acid and base production at anode and cathode sites respectively, with increasing voltage and application time. Gas formation during ECT was observed with ultrasonography. Anode sites showed significant loss of SHG signal, while cathode sites showed disorganized collagen structure with fewer fibrils emitting an attainable signal. Histologically, collagen denaturation at both sites was confirmed. Conclusion We demonstrated the production of in situ acid and base in skin occurring via ECT. The effects chemically and precisely alter collagen structure through denaturation, giving insight on the potential of ECT as a simple, low-cost, and minimally-invasive means to remodel skin and treat scars.

BACKGROUND: A retrospective clinical case series study was conducted to evaluate the use of a novel, spherical bioactive glass bone graft (BioSphere Putty) as a graft material for cervical and lumbar interbody fusion. METHODS: Data was collected from a single surgeon using BioSphere Putty along with standardized hardware in anterior cervical decompression and fusion (ACDF), transforaminal lumbar interbody fusion (TLIF), and anterior lumbar interbody fusion (ALIF) surgical procedures. BioSphere Putty was used in combination with cancellous allograft (ACDF and ALIF) or in combination with autograft (TLIF). Clinical outcomes were assessed at 1- and 2-year using radiographic imaging and the visual analog pain scale (VAS). VAS scores at the 1- and 2-year follow-up periods were statistically compared to pre-operative scoring. Successful clinical outcomes were determined by a combination of the presence of a complete radiographic fusion and a decrease in VAS at 1-year and 1- and 2-year follow-up periods. RESULTS: The retrospective review of the patient data identified 248 cases that had either 1- or 1- & 2-year follow-up. This consisted of 115 ACDF procedures and 133 lumbar fusion procedures. Lumbar fusion cases were further sub-grouped with 103 patients undergoing TLIF procedures and 30 patients undergoing ALIF procedures. The global results for the series as a whole showed clinical outcomes comparative to other advanced biologic bone grafts. Radiographically all patients demonstrated fusion (100% fusion rate) and there were no clinical adverse events, infections, or graft-related complications in any of the patients in the series. One-year VAS scores were consistent with historical norms and demonstrated significant decreases in pre-operative pain for both ACDF patients (78% decrease) and lumbar patients (66% decrease TLIF/ALIF) (t test, P

OBJECTIVES: Integration of pharmacogenomics into clinical care is being studied in multiple disciplines. We hypothesized that understanding attitudes and perceptions of anesthesiologists, critical care, and pain medicine providers would uncover unique considerations for future implementation within perioperative care. METHODS: A survey (multiple choice and Likert-scale) was administered to providers within our Department of Anesthesia and Critical Care prior to initiation of a department-wide prospective pharmacogenomics implementation program. The survey addressed knowledge, perceptions, experiences, resources, and barriers. RESULTS: Of 153 providers contacted, 149 (97%) completed the survey. Almost all providers (92%) said that genetic results influence drug therapy, and few (22%) were skeptical about the usefulness of pharmacogenomics. Despite this enthusiasm, 87% said their awareness about pharmacogenomic information is lacking. Feeling well-informed about pharmacogenomics was directly related to years in practice/experience: only 38% of trainees reported being well-informed, compared to 46% of those with 1–10 years of experience, and nearly two-thirds with 11+ years (p

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives – 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryoelectron microscopy, and in the current study, we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effects of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative-stain electron microscopy and single-particle two-dimensional class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization, and cell vacuolation. Internalization of a nonoligomerizing mutant form of VacA by host cells was detected without a requirement for acid activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ("iDrugSnFRs") for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs, in combination with previously described nicotine and varenicline sensors, provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing β-cells within the pancreatic islets of Langerhans (insulitis), resulting in loss of glucose homeostasis. Early diagnosis during pre-symptomatic T1D would allow for therapeutic intervention prior to substantial loss of β-cell mass at T1D onset. There are limited methods to track the progression of insulitis and β-cell mass decline in pre-symptomatic T1D. During insulitis, the islet microvasculature increases permeability, such that sub-micron sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Sub-micron sized nanodroplet (ND) phasechange agents can be vaporized into micron-sized bubbles; serving as a circulating microbubble precursor. We tested if NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in NOD mice and NOD;Rag1ko controls, and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10w NOD mice following ND infusion and vaporization, but was absent in both the non-infiltrated kidney of NOD mice and pancreas of Rag1ko controls. High contrast elevation also correlated with rapid diabetes onset. In pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression to guide and assess preventative therapeutic interventions for T1D.SignificanceThere is a need for imaging methods to detect type1 diabetes (T1D) progression prior to clinical diagnosis. T1D is a chronic disease that results from autoreactive T cells infiltrating the islet of Langerhans and destroying insulin-producing β-cells. Overt disease takes years to present and is only diagnosed after significant β-cells loss. As such, the possibility of therapeutic intervention to preserve β-cell mass is hampered by an inability to follow pre-symptomatic T1D progression. There are immunotherapies that can delay T1D development. However identifying ‘at risk’ individuals, and tracking whether therapeutic interventions are impacting disease progression, prior to T1D onset, is lacking. A method to detect insulitis and β-cell mass decline would present an opportunity to guide therapeutic treatments to prevent T1D.

Clostridioides difficilecauses antibiotic-associated diseases in humans ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ∼20 to 30 % of the patients with a primaryC. difficileinfection (CDI). During CDI,C. difficileforms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown thatC. difficilespores interact with intestinal epithelial cells (IECs), which contributes to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence thatC. difficilespores interact with E-cadherin, contributing to spore-adherence and internalization into IECs.C. difficiletoxins TcdA/TcdB lead to adherens junctions opening and increase spore-adherence to IECs. Confocal micrographs demonstrate thatC. difficilespores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA/TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By ELISA, immunofluorescence, and immunogold labelling, we observed that E-cadherin binds toC. difficilespores, specifically to the hair-like projections of the spore, reducing spore-adherence to IECs. Overall, these results expand our knowledge of howC. difficilespores bind to IECs by providing evidence that E-cadherin acts as a spore-adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with “microbubble” ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study.

Regression analysis was used to evaluate teeth examination as a tool for estimating age in beef and dairy cattle at Neudamm farm, Namibia, in 2018. There was a significant difference in age (months) between the overall mean actual age (M=60, SD=4.57) and the mean estimated age (M=57, SD=4.52 months) in beef cattle; t(74) = 3.61, p