CYCP2;1 integrates genetic and nutritional information to promote meristem cell division in Arabidopsis.

In higher plants, cell cycle activation in the meristems at germination is essential for the initiation of post-embryonic development. We previously identified the signaling pathways of homeobox transcription factor STIMPY and metabolic sugars as two interacting branches of the regulatory network that is responsible for activating meristematic tissue proliferation in Arabidopsis. In this study, we found that CYCP2;1 is both a direct target of STIMPY transcriptional activation and an early responder to sugar signals. Genetic and molecular studies show that CYCP2;1 physically interacts with three of the five mitotic CDKs in Arabidopsis, and is required for the G2 to M transition during meristem activation. Taken together, our results suggest that CYCP2;1 acts as a permissive control of cell cycle progression during seedling establishment by directly linking genetic control and nutritional cues with the activity of the core cell cycle machinery.

Adapting Contingency Management for Hospitalized Patients with Stimulant Use Disorder.

BACKGROUND: Hospitals struggle to engage patients with stimulant use disorders, and little is known about how to adapt evidence-based behavioral interventions, such as contingency management (CM), for hospital settings. Our study is the first step in informing the design of a hospital CM intervention. METHODS: We performed a qualitative study at a quaternary referral academic medical center in Portland, Oregon. We conducted semistructured qualitative interviews with CM experts, hospital staff, and hospitalized patients, eliciting input about hospital CM adaptations, anticipated challenges, and potential opportunities. We performed a reflexive thematic analysis at a semantic level and shared results for respondent validation. RESULTS: We interviewed 8 CM experts (researchers and clinicians), 5 hospital staff, and 8 patients. Participants felt CM could benefit hospitalized patients by supporting patient substance use disorder and physical health goals, especially by addressing the boredom, sadness, and loneliness of hospitalization. Participants emphasized that in-person interactions could improve patient-staff relationships by using "super positive" experiences to improve rapport. For successful hospital CM, participants emphasized CM core concepts and potential hospital adaptations, including identifying hospital-specific high-yield target behaviors, ensuring staff training, and using CM to support the hospital discharge transition. Participants also encouraged considering novel mobile app interventions, which may offer more flexibility in the hospital, recommending that such interventions include an in-person CM facilitator. CONCLUSIONS: Contingency management has potential to support hospitalized patients and improve patient and staff experience. Our findings can inform CM interventions for hospital systems seeking to expand access to CM and stimulant use disorder treatment.

Challenges of Diagnosing and Managing Designer Benzodiazepine Dependence and Withdrawal: A Case Report.

BACKGROUND: Over the last 10 years, an increasing number of unregulated novel psychoactive substances, including "designer benzodiazepines" (DBZDs), have emerged on the recreational drug market. Despite the rapidly increasing usage of DBZDs, there is a significant lack of information regarding clinical management. Here we present a case illustrating the difficulties of diagnosing and managing DBZD related sedative-hypnotic use disorder. CASE PRESENTATION: Our patient is a 30-year-old man with severe opioid and sedative-hypnotic use disorders. He had a 10-year history of using heroin, clonazolam, and alprazolam. He stopped using heroin when on methadone maintenance therapy but continued using clonazolam and nonprescribed alprazolam. His opioid treatment program discontinued methadone due to benzodiazepine intoxication, and he returned to heroin use. He then presented for residential withdrawal management where he underwent successful buprenorphine induction and benzodiazepine withdrawal management. During a 3-month period of benzodiazepine abstinence, he struggled with ongoing cravings and post-acute withdrawal syndrome, ultimately leading to return to DBZD use. DISCUSSION: Despite the increasing prevalence of DBZD use, the usage of DBZDs is likely under-recognized because these compounds are generally not included on standard in-office urine drug immunoassay tests. Initial studies suggest that DBZDs have high potencies, shorter half-lives, are more addictive, and can result in more severe withdrawal symptoms compared to known benzodiazepines. However, there remains a lack of information about the pharmacokinetics and pharmacodynamics of DBZDs, making clinical management for DBZD related sedative-hypnotic use disorders challenging to treat.

Benzodiazepines and Related Sedatives.

Benzodiazepine and related sedative use has been increasing. There has been a growing number of unregulated novel psychoactive substances, including designer benzodiazepines. Benzodiazepines have neurobiological and pharmacologic properties that result in a high potential for misuse and physical dependence. Options for discontinuing long-term benzodiazepine use include an outpatient benzodiazepine taper or inpatient withdrawal management at a hospital or detoxification facility. The quality of evidence on medications for benzodiazepine discontinuation is overall low, whereas cognitive behavioral therapy has shown the most benefit in terms of behavioral treatments. Benzodiazepines may also have significant adverse effects, increasing the risk of overdose and death.

Dual checkpoint targeting of B7-H3 and PD-1 with enoblituzumab and pembrolizumab in advanced solid tumors: interim results from a multicenter phase I/II trial.

BACKGROUND: Availability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable interest in developing combination therapies to improve response rates and outcomes. B7-H3 (CD276) is a member of the B7 family of cell surface molecules and provides an alternative immune checkpoint molecule to therapeutically target alone or in combination with programmed cell death-1 (PD-1)-targeted therapies. Enoblituzumab, an investigational anti-B7-H3 humanized monoclonal antibody, incorporates an immunoglobulin G1 fragment crystallizable (Fc) domain that enhances Fcγ receptor-mediated antibody-dependent cellular cytotoxicity. Coordinated engagement of innate and adaptive immunity by targeting distinct members of the B7 family (B7-H3 and PD-1) is hypothesized to provide greater antitumor activity than either agent alone. METHODS: In this phase I/II study, patients received intravenous enoblituzumab (3-15 mg/kg) weekly plus intravenous pembrolizumab (2 mg/kg) every 3 weeks during dose-escalation and cohort expansion. Expansion cohorts included non-small cell lung cancer (NSCLC; checkpoint inhibitor [CPI]-naïve and post-CPI, programmed death-ligand 1 [PD-L1] <1%), head and neck squamous cell carcinoma (HNSCC; CPI-naïve), urothelial cancer (post-CPI), and melanoma (post-CPI). Disease was assessed using Response Evaluation Criteria in Solid Tumors version 1.1 after 6 weeks and every 9 weeks thereafter. Safety and pharmacokinetic data were provided for all enrolled patients; efficacy data focused on HNSCC and NSCLC cohorts. RESULTS: Overall, 133 patients were enrolled and received ≥1 dose of study treatment. The maximum tolerated dose of enoblituzumab with pembrolizumab at 2 mg/kg was not reached. Intravenous enoblituzumab (15 mg/kg) every 3 weeks plus pembrolizumab (2 mg/kg) every 3 weeks was recommended for phase II evaluation. Treatment-related adverse events occurred in 116 patients (87.2%) and were grade ≥3 in 28.6%. One treatment-related death occurred (pneumonitis). Objective responses occurred in 6 of 18 (33.3% [95% CI 13.3 to 59.0]) patients with CPI-naïve HNSCC and in 5 of 14 (35.7% [95% CI 12.8 to 64.9]) patients with CPI-naïve NSCLC. CONCLUSIONS: Checkpoint targeting with enoblituzumab and pembrolizumab demonstrated acceptable safety and antitumor activity in patients with CPI-naïve HNSCC and NSCLC. TRIAL REGISTRATION NUMBER: NCT02475213.

Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

Validation of a modification to Performance-Tested Method 070601: Reveal Listeria Test for detection of Listeria spp. in selected foods and selected environmental samples.

A modification to Performance-Tested Method (PTM) 070601, Reveal Listeria Test (Reveal), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 27-30 h at 30 degrees C and environmental samples for 24-48 h at 30 degrees C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there was a statistically significant difference in performance between the Reveal and reference culture [U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS)] methods for only a single food in one trial (pasteurized crab meat) at the 27 h enrichment time point, with more positive results obtained with the FDA/BAM reference method. No foods showed statistically significant differences in method performance at the 30 h time point. Independent laboratory testing of 3 foods again produced a statistically significant difference in results for crab meat at the 27 h time point; otherwise results of the Reveal and reference methods were statistically equivalent. Overall, considering both internal and independent laboratory trials, sensitivity of the Reveal method relative to the reference culture procedures in testing of foods was 85.9% at 27 h and 97.1% at 30 h. Results from 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the Reveal method was more productive than the reference USDA-FSIS culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the Reveal method at the 24 h time point. Overall, sensitivity of the Reveal method at 24 h relative to that of the USDA-FSIS method was 153%. The Reveal method exhibited extremely high specificity, with only a single false-positive result in all trials combined for overall specificity of 99.5%.

Validation of a modification to Performance-Tested Method 010403: microwell DNA hybridization assay for detection of Listeria spp. in selected foods and selected environmental surfaces.

A modification to Performance-Tested Method 010403, GeneQuence Listeria Test (DNAH method), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 27-30 h at 30 degrees C, and environmental samples for 24-48 h at 30 degrees C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there were statistically significant differences in method performance between the DNAH method and reference culture procedures for only 2 foods (pasteurized crab meat and lettuce) at the 27 h enrichment time point and for only a single food (pasteurized crab meat) in one trial at the 30 h enrichment time point. Independent laboratory testing with 3 foods showed statistical equivalence between the methods for all foods, and results support the findings of the internal trials. Overall, considering both internal and independent laboratory trials, sensitivity of the DNAH method relative to the reference culture procedures was 90.5%. Results of testing 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the DNAH method was more productive than the reference U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the DNAH method at the 24 h time point. Overall, sensitivity of the DNAH method at 24 h relative to that of the USDA-FSIS method was 152%. The DNAH method exhibited extremely high specificity, with only 1% false-positive reactions overall.

Highly conserved influenza T cell epitopes induce broadly protective immunity.

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.