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Tumor Treating Fields (TTFields) therapy is an anti-cancer treatment modality that is delivered noninvasively to the tumor site via skin-placed arrays. The therapy is US Food and Drug Administration (FDA) approved and Conformité Européenne (CE) marked for adults with newly diagnosed and recurrent glioblastoma (GBM) (grade 4 glioma in the European Union). To date, there are limited data on the safety and efficacy of TTFields therapy in patients with implanted cardiac pacemakers. Herein, we report a case of a 79-year-old male patient with GBM receiving TTFields therapy with a prior medical history of cardiac events necessitating a cardiac pacemaker. The patient presented to the emergency department in May 2021 with newly onset left-sided weakness along with seizures. Based on an initial evaluation and results of the initial computed tomography (CT) scans (May 2021), the patient was clinically diagnosed with a high-grade glioma which was later confirmed as IDH wildtype following a biopsy. He was treated with radiotherapy (40 Gy in 15 fractions), followed by adjuvant temozolomide (TMZ) (75 mg/m2). TTFields therapy was initiated alongside maintenance TMZ (150 mg/m2). Average TTFields therapy usage was 67% throughout the duration of treatment. Follow-up CT scans (February and May of 2022) indicated stable disease. CT scans in August 2022 showed an increase in size of a mass with heterogeneous contrast enhancement and the patient subsequently passed away in October 2022. The patient's last cardiac tests demonstrated that the pacemaker was operational with adequate cardiac function. This report suggests that TTFields therapy concomitant with an implanted electronic device may be safe in patients with GBM.

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During its cell cycle, the bacterium Caulobacter crescentus switches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the transition from the motile to the sessile lifestyle. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and promotes the motile lifestyle: HmrA (holdfast and motility regulator A). Genetic screens and genomic localization led to the identification of additional genes that form a putative phosphorelay pathway with HmrA. We postulate that the Hmr pathway acts as a rheostat to control the proportion of cells harboring a flagellum or a holdfast in the population. Further genetic analysis suggests that the Hmr pathway impacts c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Our results also indicate that the Hmr pathway is involved in the regulation of motile to sessile lifestyle transition as a function of various environmental factors: biofilm formation is repressed when excess copper is present and derepressed under non-optimal temperatures. Finally, we provide evidence that the Hmr pathway regulates motility and adhesion without modulating the transcription of the holdfast synthesis regulator HfiA. IMPORTANCE: Complex communities attached to a surface, or biofilms, represent the major lifestyle of bacteria in the environment. Such a sessile state enables the inhabitants to be more resistant to adverse environmental conditions. Thus, having a deeper understanding of the underlying mechanisms that regulate the transition between the motile and the sessile states could help design strategies to improve biofilms when they are beneficial or impede them when they are detrimental. For Caulobacter crescentus motile cells, the transition to the sessile lifestyle is irreversible, and this decision is regulated at several levels. In this work, we describe a putative phosphorelay that promotes the motile lifestyle and inhibits biofilm formation, providing new insights into the control of adhesin production that leads to the formation of biofilms.

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Barley grain sources with variable kernel sizes makes adequate and consistent processing of kernels challenging. This study evaluated how the severity of processing for reconstituted high moisture (65% on DM basis) barley (RHB) affects ensiling characteristics and in vitro ruminal fermentation. Three independent sources of light (<630 g/500 mL) and heavy (>670 g/500 mL) barley were blended to create 4 sources of variable kernel sized barley (646 g/500 mL). Reconstituted barley was rolled through a roller gap width of 1.40 (RHBF), 1.86 (RHBM), or 2.31 mm (RHBC) and ensiled for 1 or 5 mo with dry rolled barley (DRB; roller gap width 1.86 mm) used as a control. The 1-mo RHB and the DRB were further evaluated using the artificial rumen technique (RUSITEC) to investigate the effects of severity of processing for RHB on ruminal fermentation, and gas, methane, and microbial protein production. Using a randomized complete block design (n = 4), 16 fermenters from 2 RUSITEC apparatuses were used to assess the 4 sources and 4 processing treatments. The addition of water increased kernel width before rolling and resulted in increased kernel length, width, and thickness for RHB relative to dry rolled barley. Increasing processing severity for RHB linearly increased kernel width. The percentage of fine particles (<1.18 mm) was greater for DRB than RHBF, but did not differ by processing severity for RHB. Dry matter, organic matter, and starch disappearance were not different between DRB and RHBF, but linearly increased with increasing processing severity for RHB. Fermenter pH tended to be less for DRB relative to RHBF. In conclusion, the reduction in fine particles with the addition of water for RHB may prevent a decline in fermenter pH and when processed to achieve the same PI using a smaller roller gap width, yielded similar DM and OM disappearance suggesting a lesser risk for low ruminal pH without compromising digestibility.

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INTRODUCTION: While piecemeal endoscopic mucosal resection (EMR) for T1a oesophageal adenocarcinoma is acceptable, enbloc-R0 excision is advocated for T1b disease as it may offer a potential cure and mitigate recurrence. Thus, distinguishing between T1a and T1b disease is imperative under current treatment paradigms. We sought to ascertain whether expert Barrett's endoscopists were able to make this distinction based on optical evaluation. METHODS: Sixty sets of endoscopic images of histologically confirmed high grade dysplasia (HGD), T1a and T1b disease (n=20 for each) were compiled from consecutive patients at a single institution. Each set contained four images, and were standardized to include an overview, a close-up in high-definition white light, a near-focus magnification image, and a narrow-band image. Experts were invited to predict histology for each set. RESULTS: 19 experts from 8 countries (Australia, USA, Italy, Netherlands, Germany, Canada, Belgium, and Portugal) participated. The majority had been practicing for >20 years, with a median annual case volume for Barrett's EMR of 50 (IQR 18-75), and Barrett's ESD of 25 (IQR 10-45). Oesophageal adenocarcinoma (T1a/b) could be distinguished from HGD, with a pooled sensitivity of 89.1% (95% CI:84.7-93.4. When predicting T-stage for T1b adenocarcinoma cases, pooled sensitivity was 43.8% (95% CI:29.9-57.7). Fleiss' kappa was 0.421 (95% CI:0.399-0.442, P<0.001), indicating fair-to-moderate agreement. CONCLUSIONS: Expert Barrett's endoscopists can reliably differentiate T1a/T1b oesophageal adenocarcinoma from HGD. Although there is fair-to-moderate agreement for T-staging, T1b disease cannot be reliably distinguished from T1a disease. This may have implications on clinical decision making and selection of endoscopic treatment methods.

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Importance: Functional outcomes after repair of rhegmatogenous retinal detachments (RRDs) are highly dependent on baseline visual acuity and foveal status. Adverse social determinants of health (SDOH) can present barriers to timely presentation for repair and limit vision outcomes. Objective: To evaluate the association between neighborhood-level SDOH with baseline severity (visual acuity and fovea status) of RRD. Design, Setting, and Participants: This was a retrospective cohort study that included adult patients 18 years and older who underwent primary repair of uncomplicated RRD at the Wilmer Eye Institute from January 2008 to December 2018. Study data were analyzed from December 2023 to April 2024. Exposures: The census block group of patient home addresses were matched to multiple neighborhood-level SDOH including the Area Deprivation Index (ADI), per capita income, percentage of renters, percentage of rent burden, percentage of people using a food assistance program, percentage of uninsured individuals, mode of transportation to work, distance to the nearest transit stop, total road density, National Walkability Index, Index of Medical Underservice score, and aggregate cost of medical care. Main Outcomes and Measures: Odds of presenting with vision worse than 20/40 or fovea-involving RRD using multivariable logistic regression adjusting for age, sex, race and ethnicity, and insurance. Results: A total of 700 patients (mean [SD] age, 57.9 [12.4] years; 432 male [61.7%]) were included. Every decile increase in ADI, indicating more socioeconomic disadvantage, was associated with an increased odds of presenting with worse visual acuity and fovea-involving RRD (odds ratio [OR], 1.14; 95% CI, 1.04-1.24; P = .004 and OR, 1.13; 95% CI, 1.04-1.22; P = .005, respectively). Each $1000 increase in per capita income was associated with lower odds of presenting with worse vision (OR, 0.99; 95% CI, 0.98-0.99; P = .001). Every 1% increase in percentage of workers who drove to work was associated with an increased odds of presenting with worse vision and fovea-involving RRD (OR, 1.02; 95% CI, 1.01-1.03; P = .005 and OR, 1.01; 95% CI, 1.00-1.03; P = .04, respectively). Conclusions and Relevance: Results of this cohort study suggest that patients with a residence in neighborhoods with more socioeconomic deprivation or a higher percentage of workers who drove to work were more likely to present with more severe RRD even after accounting for multiple individual-level characteristics. These findings support consideration of public policy changes to address the barriers faced by patients residing in certain neighborhoods who seek prompt surgical intervention for RRD to reduce health disparities in RRD outcomes.

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BACKGROUND: The reported incidence of posttraumatic knee osteoarthritis (PTOA) after primary anterior cruciate ligament reconstruction (ACLR) varies considerably. Further, there are gaps in identifying which patients are at risk for PTOA after ACLR and whether there are modifiable factors. PURPOSE: To (1) determine the incidence of PTOA in a primary ACLR cohort and (2) identify patient and perioperative factors associated with the development of PTOA after primary ACLR. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Data from the Kaiser Permanente ACLR Registry were used to conduct a cohort study. Patients who had undergone primary ACLR without a previous diagnosis of osteoarthritis were identified (2009-2020). The crude incidence of PTOA was calculated using the Aalen-Johansen estimator with a multistate model. The association of patient and operative factors with the development of PTOA after primary ACLR was modeled as a time to event using multistate Cox proportional hazards regression. Models stratified by age (<22 and ≥22 years) were also conducted because of the effect modification of age. RESULTS: The study sample included 41,976 cases of primary ACLR. The incidence of PTOA was 1.7%, 5.1%, and 13.6% at 2, 5, and 10 year follow-ups, respectively. Risk factors for PTOA that were consistently identified in the overall cohort and age-stratified groups included a body mass index ≥30 versus <30 and an allograft or quadriceps tendon autograft versus a hamstring tendon autograft. Patients presenting with knee pain after ACLR were further identified when considering postoperative factors. Other risk factors for PTOA in the overall cohort included age ≥22 versus <22 years, bone-patellar tendon-bone autograft versus hamstring tendon autograft, hypertension, cartilage injury, meniscal injury, revision after primary ACLR with concomitant meniscal/cartilage surgery, multiligament injury, other activity at the time of injury compared with sport, and tibial tunnel drilling technique rather than the anteromedial portal. CONCLUSION: Knee pain after ACLR may be an early sign of PTOA. Surgeons should consider the adverse associations of a higher body mass index and an allograft or quadriceps tendon autograft with the development of PTOA, as these were factors identified with a higher risk, regardless of a patient's age at the time of primary ACLR.

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The objectives were to determine the effects of dietary crude protein (CP) content and corn grain processing on whole-body urea kinetics and the functional roles of urea transporter-B (UT-B) and aquaporins (AQP) in serosal-to-mucosal urea flux (Jsm-urea) in ovine ruminal epithelia. Thirty-two Rideau-Arcott ram lambs were blocked by bodyweight into groups of 4 and then randomly allocated within blocks to 1 of 4 diets (n = 8) in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn grain processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). Whole-body urea kinetics and N balance were determined using 4-d continuous intrajugular infusions of [15N15N]-urea with concurrent collections of urine and feces with four blocks of lambs (n = 4). After 23 d on diets, lambs were killed to collect ruminal epithelia for mounting in Ussing chambers to determine Jsm-urea and the measurement of mRNA abundance of UT-B and AQP. Serosal and mucosal additions of phloretin and NiCl2 were used to inhibit UT-B- and AQP-mediated urea transport, respectively. Lambs fed HP had a greater (P < 0.01) N intake (29.4 vs. 19.1 g/d) than those fed LP; however, retained N (g/d or % of N intake) was not different. As a % of N intake, lambs fed SFC tended (P = 0.09) to have a lower N excretion (72.2 vs. 83.5%) and a greater N retention (27.8 vs. 16.6%) compared to those fed WSC. Endogenous urea-N production (UER) was greater in lambs fed HP compared to those fed LP (29.9 vs. 20.6 g/d; P = 0.02), whereas urea-N secreted into the gut (GER; g/d) and urea-N used for anabolic purposes (UUA; g/d) were similar. Lambs fed LP tended (P = 0.05) to have greater GER:UER (0.78 vs. 0.66) and UUA:GER (0.23 vs. 0.13) ratios, and a greater Jsm-urea (144.7 vs. 116.1 nmol/[cm2 × h]; P = 0.07) compared to those fed HP. Lambs fed SFC tended to have a lower NiCl2-insensitive Jsm-urea (117.4 vs. 178.4 nmol/[cm2 × h]; P = 0.09) and had a lower phloretin-insensitive Jsm-urea (87.1 vs. 143.1 nmol/[cm2 × h]; P = 0.02) compared to those fed WSC. The mRNA abundance of UT-B (0.89 vs. 1.07; P = 0.08) and AQP-3 (0.90 vs. 1.05; P = 0.07) tended to be lower in lambs fed SFC compared to those fed WSC. Overall, reducing CP content tended to increase the GER:UER ratio with no changes in the expression or function of UT-B and AQP. Although corn grain processing had no effects on GER, feeding SFC increased the portion of urea secretion into the rumen that was mediated via UT-B and AQP.

In ruminants, urea produced in the liver as a nitrogenous waste can be secreted into the rumen where it can be used by rumen microorganisms as a source of nitrogen (N) for their growth. Therefore, urea secretion into the rumen is nutritionally important for ruminants particularly when dietary N intake is deficient. Urea secretion into the rumen occurs via transporter proteins in rumen tissue referred to as urea transporters (UT-B) and aquaporins (AQP). The purpose of this research was to investigate the effects of dietary crude protein (CP) content and corn grain processing on urea secretion into the rumen and the function of UT-B and AQP. Thirty-two Rideau-Arcott lambs were assigned to 1 of 4 diets in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). When compared to feeding HP, feeding LP tended to increase urea secretion into the rumen, but there were no corresponding changes in UT-B and AQP function. Corn processing did not influence urea secretion into the rumen; however, the portion of urea secretion that was facilitated via UT-B and AQP was greater in lambs fed SFC compared to those fed WSC.

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Computed tomography is commonly used to evaluate the reptile coelom. One challenge in the acquisition of contrast-enhanced CT studies in reptiles is the difficulty in placing intravenous catheters due to the poor conspicuity of peripheral vessels. To overcome these limitations, intraosseous catheterization of the long bones (humerus, femur, and tibia) can be used. The purpose of this prospective study was to assess the feasibility and diagnostic value of contrast-enhanced CT of the coelom using a femoral intraosseous route for contrast administration. Twelve healthy juvenile green iguanas were enrolled. In 9 of 12 iguanas, vascular phases were obtained, providing strong, homogeneous enhancement of the arterial and venous tree concurrently. In these nine cases, Phase #1, acquired 60 s after beginning the injection of contrast medium, provided maximal contrast between the hepatic parenchyma and hepatic vasculature. In the remaining three cases, injection failure resulted from intracortical placement of the catheter tip. Histological examination of the liver, kidneys, and femur was performed to determine the pathomorphological correlates of the CT findings; in a third of patients, some degree of contrast-induced renal toxicity was documented, which in mammalians shows potential for reversibility. Four iguanas had iatrogenic structural damage to the femoral growth plates attributed to intraosseous catheter placement. A femoral intraosseous route can be used in green iguanas for iodinated contrast medium administration for CT angiography. For the greatest contrast between coelomic organs and vascular structures, at least one postcontrast acquisition 60 s after initiation of contrast administration is recommended when using this technique.

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The development of electrophilic ligands that rapidly modify specific lysine residues remains a major challenge. Salicylaldehyde-based inhibitors have been reported to form stable imine adducts with the catalytic lysine of protein kinases. However, the targeted lysine in these examples is buried in a hydrophobic environment. A key unanswered question is whether this strategy can be applied to a lysine on the surface of a protein, where rapid hydrolysis of the resulting salicylaldimine is more likely. Here, we describe a series of aminomethyl-substituted salicylaldehydes that target a fully solvated lysine on the surface of the ATPase domain of Hsp90. By systematically varying the orientation of the salicylaldehyde, we discovered ligands with long residence times, the best of which engages Hsp90 in a quasi-irreversible manner. Crystallographic analysis revealed a daisy-chain network of intramolecular hydrogen bonds in which the salicylaldimine is locked into position by the adjacent piperidine linker. This study highlights the potential of aminomethyl salicylaldehydes to generate conformationally stabilized, hydrolysis-resistant imines, even when the targeted lysine is far from the ligand binding site and is exposed to bulk solvent.

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Basking sharks (Cetorhinus maximus) seasonally aggregate in coastal surface waters of the North Atlantic, providing opportunities for visual observation. While putative courtship displays have been observed, actual copulation has not been documented. Here we examine video collected by an unmanned aerial vehicle ("drone") of novel behavioral interactions between basking sharks in Cape Cod Bay, Massachusetts in May 2021. The behaviors, including close following and tight concentric circling, are consistent with pre-copulatory behavior observed in other shark species. These observations provide new insights into the pre-copulatory behavior of basking sharks.

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METHODS: This qualitative interview study was part of a larger study that interviewed19 participants recruited among patients with cancer registered with Samoa Cancer Society, or patients recently discharged from the main tertiary hospital in Samoa. Interview transcripts were reviewed, and the research team identified the key palliative-care-related themes. RESULTS: Analysis led to the generation of the following key themes: living with pain; resource constraints; support from family and faith; and interactions with healthcare professionals. Most participants were experiencing unresolved pain as a result of poor health knowledge, lack of access to medications and practical issues, such as financial limitations and lack of transport. Participants also reported a lack of resources, including financial constraints, as a significant barrier to effective healthcare. Sources of support for patients with cancer were most commonly strong family relationships and religious faith. Although it was difficult for the participants to access care from healthcare professionals, they found that honest communication about their condition was comforting. However, they were frustrated if they felt communication was unsatisfactory. CONCLUSION: This study provides much needed evidence on the issues that affect the quality of life patients with cancer receiving palliative care and their families in Samoa. It also highlights the intersectionality of these issues and how this compounds the patient experience. Practical recommendations for improving palliative care lie in a few key areas, including a need for improved access to opioids and support for family caregivers. However, from a systemic perspective, the way forward should lie in harnessing the strengths of Samoan culture, including the strong sense of family and the role of religion, to provide support and care for palliative patients.

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BACKGROUND: Recently, radio-frequency ablation has been used to modulate slow-wave activity in the porcine stomach. Gastric ablation is, however, still in its infancy compared to its history in the cardiac field, and electrophysiological studies have been restricted to temperature-controlled, non-irrigated ablation. Power-controlled, irrigated ablation may improve lesion formation at lower catheter-tip temperatures that produce the desired localized conduction block. METHODS AND RESULTS: Power-controlled, irrigated radio-frequency ablation was performed on the gastric serosal surface of female weaner pigs (n = 5) in vivo. Three combinations of power (10-15 W) and irrigation settings (2-5 mL min-1) were investigated. A total of 12 linear lesions were created (n = 4 for each combination). Slow waves were recorded before and after ablation using high-resolution electrical mapping. KEY RESULTS: Irrigation maintained catheter-tip temperature below 50°C. Ablation induced a complete conduction block in 8/12 cases (4/4 for 10 W at 2 mL min-1, 1/4 for 10 W at 5 mL min-1, 3/4 for 15 W at 5 mL min-1). Blocks were characterized by a decrease in signal amplitude at the lesion site, along with changes in slow-wave propagation patterns, where slow waves terminated at and/or rotated around the edge of the lesion. CONCLUSIONS AND INFERENCES: Power-controlled, irrigated ablation can successfully modulate gastric slow-wave activity at a reduced catheter-tip temperature compared to temperature-controlled, non-irrigated ablation. Reducing the irrigation rate is more effective than increasing power for blocking slow-wave activity. These benefits suggest that irrigated ablation is a suitable option for further translation into a clinical intervention for gastric electrophysiology disorders.

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Gastroenteropancreatic high-grade (HG) neuroendocrine carcinoma (GEP-NEC) is an aggressive malignancy with limited treatment options and increasing incidence in the United States. Due to the rarity of the cancer and heterogeneity of the primary tumor location, data on GEP-NEC oncogenesis and its interaction with the host immune system are limited. A greater understanding of GEP-NEC and its tumor microenvironment (TME) would benefit efforts to develop more effective targeted therapies and rationally adapt immunotherapy to this disease. In this study, we profiled the expression of 770 unique genes using 21 biopsy samples from patients with GEP-NEC using the NanoString nCounter PanCancer IO 360 platform. Our results show several trends evident within the GEP-NEC TME. Greater expression of genes indicative of immune cell infiltration was present within the TME of patients <60 years of age and in patients with greater overall survival (OS). Tumors from patients with non-pancreatic NEC had diminished MHCII expression compared to pancreatic NEC, suggesting more prominent adaptive immune responses in the pancreatic GEP-NEC subtype. Patients with a >6 months OS had tumors with elevated NK cell gene signatures compared to patients with poor survival. Further, the analysis revealed numerous differentially expressed genes based on patient age, tumor location, response to treatment, and OS, which warrant future validation for assessing the relationship with clinical outcomes in patients.

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Pulsed-field ablation (PFA) is an emerging ablative technology that has been used successfully to eliminate cardiac arrhythmias. As a nonthermal technique, it has significant benefits over traditional radiofrequency ablation with improved target tissue specificity and reduced risk of adverse events during cardiac applications. We investigated whether PFA is safe for use in the stomach and whether it could modulate gastric slow waves. Female weaner pigs were fasted overnight before anesthesia was induced using tiletamine hydrochloride (50 mg·mL-1) and zolazepam hydrochloride (50 mg·mL-1) and maintained with propofol (Diprivan 2%, 0.2-0.4 mg·kg-1·min-1). Pulsed-field ablation was performed on their gastric serosa in vivo. Adjacent point lesions (n = 2-4) were used to create a linear injury using bipolar pulsed-field ablation consisting of 40 pulses (10 Hz frequency, 0.1 ms pulse width, 1,000 V amplitude). High-resolution electrical mapping defined baseline and postablation gastric slow-wave patterns. A validated five-point scale was used to evaluate tissue damage in hematoxylin and eosin-stained images. Results indicated that PFA successfully induced complete conduction blocks in all cases, with lesions through the entire thickness of the gastric muscle layers. Consistent postablation slow-wave patterns emerged immediately following ablation and persisted over the study period. Pulsed-field ablation induces rapid conduction blocks as a tool to modulate slow-wave patterns, indicating it may be suitable as an alternative to radiofrequency ablation.NEW & NOTEWORTHY Results show that pulsed-field ablation can serve as a gastric slow-wave intervention by preventing slow-wave propagation across the lesion site. Stable conduction blocks were established immediately following energy delivery, faster than previous examples of radiofrequency gastric ablation. Pulsed-field ablation may be an alternative for gastric slow-wave intervention, and further functional and posthealing studies are now warranted.

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(R,S)-Ketamine (ketamine) is a dissociative anesthetic that also possesses analgesic and antidepressant activity. Undesirable dissociative side effects and misuse potential limit expanded use of ketamine in several mental health disorders despite promising clinical activity and intensifying medical need. (2R,6R)-Hydroxynorketamine (RR-HNK) is a metabolite of ketamine that lacks anesthetic and dissociative activity but maintains antidepressant and analgesic activity in multiple preclinical models. To enable future assessments in selected human indications, we report the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of RR-HNK in a Phase 1 study in healthy volunteers (NCT04711005). A six-level single-ascending dose (SAD) (0.1-4 mg/kg) and a two-level multiple ascending dose (MAD) (1 and 2 mg/kg) study was performed using a 40-minute IV administration emulating the common practice for ketamine administration for depression. Safety assessments showed RR-HNK possessed a minimal adverse event profile and no serious adverse events at all doses examined. Evaluations of dissociation and sedation demonstrated that RR-HNK did not possess anesthetic or dissociative characteristics in the doses examined. RR-HNK PK parameters were measured in both the SAD and MAD studies and exhibited dose-proportional increases in exposure. Quantitative electroencephalography (EEG) measurements collected as a PD parameter based on preclinical findings and ketamine's established effect on gamma-power oscillations demonstrated increases of gamma power in some participants at the lower/mid-range doses examined. Cerebrospinal fluid examination confirmed RR-HNK exposure within the central nervous system (CNS). Collectively, these data demonstrate RR-HNK is well tolerated with an acceptable PK profile and promising PD outcomes to support the progression into Phase 2.

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Type 2C protein phosphatases (PP2Cs) are emerging as important regulators of plant immune responses, although little is known about how they might impact nucleotide-binding, leucine-rich repeat (NLR)-triggered immunity (NTI). We discovered that expression of the PP2C immunity-associated candidate 14 gene (Pic14) is induced upon activation of the Pto/Prf-mediated NTI response in tomato. Pto/Prf recognizes the effector AvrPto translocated into plant cells by the pathogen Pseudomonas syringae pv. tomato (Pst) and activate a MAPK cascade and other responses which together confer resistance to bacterial speck disease. Pic14 encodes a PP2C with an N-terminal kinase-interacting motif (KIM) and a C-terminal phosphatase domain. Upon inoculation with Pst-AvrPto, Pto/Prf-expressing tomato plants with loss-of-function mutations in Pic14 developed less speck disease, specifically in older leaves, compared to wild-type plants. Transient expression of Pic14 in leaves of Nicotiana benthamiana and tomato inhibited cell death typically induced by Pto/Prf and the MAPK cascade members M3Kα and Mkk2. The cell death-suppressing activity of Pic14 was dependent on the KIM and the catalytic phosphatase domain. Pic14 inhibited M3Kα- and Mkk2-mediated activation of immunity-associated MAPKs and Pic14 was shown to be an active phosphatase that physically interacts with and dephosphorylates Mkk2 in a KIM-dependent manner. Together, our results reveal Pic14 as an important negative regulator of Pto/Prf-triggered immunity by interacting with and dephosphorylating Mkk2.

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Type 2C protein phosphatases (PP2Cs) constitute a large family in most plant species but relatively few of them have been implicated in immunity. To identify and characterize PP2C phosphatases that affect tomato (Solanum lycopersicum) immunity, we used CRISPR/Cas9 to generate loss-of-function mutations in 11 PP2C-encoding genes whose expression is altered in response to immune elicitors or pathogens. We report that two closely related PP2C phosphatases, Pic3 (PP2C immunity-associated candidate 3) and Pic12, are involved in regulating resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Loss-of-function mutations in Pic3 led to enhanced resistance to Pst in older but not younger leaves, whereas such mutations in Pic12 resulted in enhanced resistance in both older and younger leaves. Overexpression of Pic3 and Pic12 proteins in leaves of Nicotiana benthamiana inhibited resistance to Pst, and this effect was dependent on Pic3/12 phosphatase activity and an N-terminal palmitoylation motif associated with localization to the cell periphery. Pic3, but not Pic12, had a slight negative effect on flagellin-associated reactive oxygen species generation, although their involvement in the response to Pst appeared independent of flagellin. RNA-sequencing analysis of Rio Grande (RG)-PtoR wild-type plants and two independent RG-pic3 mutants revealed that the enhanced disease resistance in RG-pic3 older leaves is associated with increased transcript abundance of multiple defense related genes. RG-pic3/RG-pic12 double mutant plants exhibited stronger disease resistance than RG-pic3 or RG-pic12 single mutants. Together, our results reveal that Pic3 and Pic12 negatively regulate tomato immunity in an additive manner through flagellin-independent pathways.

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Assays that detect viral infections play a significant role in limiting the spread of diseases such as SARS-CoV-2. Here, we present Rolosense, a virus sensing platform that leverages the motion of 5 µm DNA-based motors on RNA fuel chips to transduce the presence of viruses. Motors and chips are modified with aptamers, which are designed for multivalent binding to viral targets and lead to stalling of motion. Therefore, the motors perform a "mechanical test" of the viral target and stall in the presence of whole virions, which represents a unique mechanism of transduction distinct from conventional assays. Rolosense can detect SARS-CoV-2 spiked in artificial saliva and exhaled breath condensate with a sensitivity of 103 copies/mL and discriminates among other respiratory viruses. The assay is modular and amenable to multiplexing, as demonstrated by our one-pot detection of influenza A and SARS-CoV-2. As a proof of concept, we show that readout can be achieved using a smartphone camera with a microscopic attachment in as little as 15 min without amplification reactions. Taken together, these results show that mechanical detection using Rolosense can be broadly applied to any viral target and has the potential to enable rapid, low-cost point-of-care screening of circulating viruses.

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The objective of this study was to evaluate effects of increasing the inclusion of dry-rolled hybrid rye (HR) as a replacement for dry-rolled barley grain (DRB) on feed intake, ruminal fermentation, and the site and extent of nutrient digestion for finishing cattle. Eight ruminally and duodenally cannulated Hereford-cross heifers were used in a replicated 4 × 4 Latin square design with 21-d periods including 15 d of dietary adaptation and 6 d of data and sample collection. Dietary treatments included a control diet with 10.00% grass hay, 85.21% DRB, 4.51% of a vitamin and mineral supplement, and 0.28% of urea on a dry matter (DM) basis. Hybrid rye grain replaced 33%, 67%, or 100% of the DRB. Feed ingredients, feed refusals, ruminal pH, ruminal fluid, duodenal digesta, and fecal samples were collected from days 18 to 21 in each period. Data were analyzed using the Proc Glimmix procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC) to evaluate the linear, quadratic, and cubic effects of increasing HR inclusion. Increasing HR inclusion as a substitute for DRB linearly decreased (P < 0.01) DM intake, linearly decreased mean ruminal pH (P < 0.01), and increased the duration (P < 0.01) and area (P = 0.02) that ruminal pH was < 5.5. There were no effects of HR inclusion on total short chain fatty acid and lactic acid concentrations in ruminal fluid. Likewise, the molar proportions of acetate and butyrate were not affected by HR inclusion. Propionate was cubically affected by HR inclusion (P = 0.02). Ruminal ash-free neutral detergent fiber (aNDFom) digestibility linearly increased (P = 0.03) with increasing HR, but there was no effect on ruminal starch digestibility averaging 71.1% (SEM = 3.611). Increasing HR inclusion linearly increased intestinal DM digestibility (% of flow to the duodenum; P = 0.03), tended to linearly increase intestinal digestibility of organic matter (P = 0.08), and tended to quadratically affect intestinal digestibility of aNDFom (P = 0.07). Increasing hybrid rye linearly increased apparent total tract DM, organic matter, crude protein, aNDFom, and starch digestibility (P ≤ 0.05). In addition, increasing HR inclusion linearly increased GE digestibility (P < 0.01) and the DE concentration (P < 0.01). Increasing the inclusion rate of HR grain as a substitute for DRB in finishing diets decreased DMI and increased risk for low ruminal pH, which may be influenced by greater digestible energy concentration arising from greater DM, OM, aNDFom, and starch digestibility.

Rye grain is not a common cereal grain used for finishing cattle due to risk for ergot contamination and concerns with palatability; however, the development of varieties with low ergot risk may increase its use. In this study, dry matter intake, ruminal fermentation, and the site and extent of nutrient digestion were evaluated when dry-rolled hybrid rye (HR) replaced dry-rolled barley (DRB) in diets for finishing beef cattle. Increasing the inclusion of HR as a substitute for DRB linearly decreased dry matter intake while linearly increasing the duration that ruminal pH was <5.5. Ruminal digestibility of ash-free neutral detergent fiber (aNDFom) linearly increased with increasing HR inclusion, while ruminal starch digestibility was not affected. Total tract digestibility of dry matter, organic matter, crude protein, aNDFom, and starch, along with the digestible energy concentration, linearly increased as HR inclusion increased as a substitute for DRB. These results suggest that incorporating HR as a replacement for DRB has the potential to improve the ruminal digestibility of aNDFom and total tract digestibility for most chemical constituents but may reduce dry matter intake while increasing risk for low ruminal pH.

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