Personalized, parcel-guided non-invasive transcranial magnetic stimulation for the treatment of post-concussive syndrome: safety and proof of concept.

OBJECTIVE: To determine the safety and proof of concept of a parcel-guided, repetitive Transcranial Magnetic Stimulation (rTMS) in patients who develop a heterogeneous array of symptoms, known collectively as post-concussive syndrome (PCS), following traumatic brain injury (TBI). METHODS: We performed a retrospective review of off-label, individualized, parcel-guided rTMS in 19 patients from December 2020 to May 2023. Patients had at least one instance of mild, moderate, or severe TBI and developed symptoms not present prior to injury. rTMS targets were identified based on machine learning connectomic software using functional connectivity anomaly matrices compared to healthy controls. EuroQol (EQ-5D), as a measurement of quality of life, and additional questionnaires dependent on individual's symptoms were submitted prior to, after, and during follow-up from rTMS. RESULTS: Nineteen patients showed improvement in EQ-5D and Rivermead Post Concussion Symptoms Questionnaires - 3 after treatment and follow-up. For nine patients who developed depression, five (55%) attained response and remission based on the Beck Depression Inventory after treatment. Eight of ten patients with anxiety had a clinically significant reduction in Generalized Anxiety Disorder-7 scores during follow-up. CONCLUSION: Parcel-guided rTMS is safe and may be effective in reducing PCS symptoms following TBI and should incite further controlled studies.

Taxonomic and genomic attributes of oligotrophic soil bacteria.

Not all bacteria are fast growers. In soil as in other environments, bacteria exist along a continuum-from copiotrophs that can grow rapidly under resource-rich conditions to oligotrophs that are adapted to life in the "slow lane." However, the field of microbiology is built almost exclusively on the study of copiotrophs due, in part, to the ease of studying them in vitro. To begin understanding the attributes of soil oligotrophs, we analyzed three independent datasets that represent contrasts in organic carbon availability. These datasets included 185 samples collected from soil profiles across the USA, 950 paired bulk soil and rhizosphere samples collected across Europe, and soils from a microcosm experiment where carbon availability was manipulated directly. Using a combination of marker gene sequencing and targeted genomic analyses, we identified specific oligotrophic taxa that were consistently more abundant in carbon-limited environments (subsurface, bulk, unamended soils) compared to the corresponding carbon-rich environment (surface, rhizosphere, glucose-amended soils), including members of the Dormibacterota and Chloroflexi phyla. In general, putative soil oligotrophs had smaller genomes, slower maximum potential growth rates, and were under-represented in culture collections. The genomes of oligotrophs were more likely to be enriched in pathways that allow oligotrophs to metabolize a range of energy sources and store carbon, while genes associated with energy-intensive functions like chemotaxis and motility were under-represented. However, few genomic attributes were shared, highlighting that oligotrophs likely use a range of different metabolic strategies and regulatory pathways to thrive in resource-limited soils.

F@#$ pain! A mini-review of the hypoalgesic effects of swearing.

Swearing, or the use of taboo language, has been repeatedly shown to induce hypoalgesia. While reliable hypoalgesic effects have been observed across studies, the mechanisms by which swearing influences pain and the optimal dosage of swearing remain poorly understood. Plausible mechanistic rationale for swearing's impact on pain include sympathetic response, emotion, humor, distraction, aggression, state disinhibition, psychological flow, risky behavior, and self-confidence. It remains unknown how the intensity of the swear word, speech volume, frequency, or timing influences pain modulation. While the majority of evidence demonstrates the efficacy of swearing at attenuating acute pain responses, these studies have utilized healthy populations with controlled experiments in laboratory settings. Comparatively, less is known about how laboratory findings translate practically/clinically to diverse populations, various dosages, and different pain chronicities. A greater understanding of mechanistic underpinnings and practical implications are necessary to feasibly implement swearing as a therapeutic modality to combat pain. The purpose of the following mini-review is to provide an overview of the current evidence on swearing for the reduction of pain, speculate on plausible underlying mechanisms, and discuss the potential for optimization of swearing for real-world translation. Lastly, identifying knowledge gaps to aid in directing future research will be discussed.

The clinical impact of estimating low-density lipoprotein cholesterol (LDL-C) using different equations in the general population.

BACKGROUND: Low-density lipoprotein cholesterol (LDL-C) is associated with atherosclerotic cardiovascular disease (ASCVD). Friedewald, Sampson, and Martin-Hopkins equations are used to calculate LDL-C. This study compares the impact of switching between these equations in a large geographically defined population. MATERIALS AND METHODS: Data for individuals who had a lipid panel ordered clinically between 2010 and 2019 were included. Comparisons were made across groups using the two-sample t-test or chi-square test as appropriate. Discordances between LDL measures based on clinically actionable thresholds were summarized using contingency tables. RESULTS: The cohort included 198,166 patients (mean age 54 years, 54% female). The equations perform similarly at the lower range of triglycerides but began to diverge at a triglyceride level of 125 mg/dL. However, at triglycerides of 175 mg/dL and higher, the Martin-Hopkins equation estimated higher LDL-C values than the Samson equation. This discordance was further exasperated at triglyceride values of 400 to 800 mg/dL. When comparing the Sampson and Friedewald equations, at triglycerides are below 175 mg/dL, 9% of patients were discordant at the 70 mg/dL cutpoint, whereas 42.4% were discordant when triglycerides are between 175 and 400 mg/dL. Discordance was observed at the clinically actionable LDL-C cutpoint of 190 mg/dL with the Friedewald equation estimating lower LDL-C than the other equations. In a high-risk subgroup (ASCVD risk score > 20%), 16.3% of patients were discordant at the clinical cutpoint of LDL-C < 70 mg/dL between the Sampson and Friedewald equations. CONCLUSIONS: Discordance at clinically significant LDL-C cutpoints in both the general population and high-risk subgroups were observed across the three equations. These results show that using different methods of LDL-C calculation or switching between different methods could have clinical implications for many patients.

Elevated Mood Induced by Subthalamic Nucleus Deep Brain Stimulation: A Video-Recorded Case Report.

Background: Deep brain stimulation (DBS) can be an effective therapy to control motor signs in patients with Parkinson's disease (PD). However, subthalamic nucleus (STN) DBS can induce undesirable psychiatric adverse effects, including elevated mood. Case report: We reported a video case of a 73-year-old male implanted with bilateral STN DBS who experienced stimulation-induced elevated mood. A correlation between mood changes and enhanced activation of the ventromedial region in the left STN was observed. Discussion: This video case report illustrates STN DBS-induced elevated mood and enhances early symptom recognition for patients and diagnostic awareness for professionals.

Identifying Person-Specific Drivers of Depression in Adolescents: Protocol for a Smartphone-Based Ecological Momentary Assessment and Passive Sensing Study.

BACKGROUND: Adolescence is marked by an increasing risk of depression and is an optimal window for prevention and early intervention. Personalizing interventions may be one way to maximize therapeutic benefit, especially given the marked heterogeneity in depressive presentations. However, empirical evidence that can guide personalized intervention for youth is lacking. Identifying person-specific symptom drivers during adolescence could improve outcomes by accounting for both developmental and individual differences. OBJECTIVE: This study leverages adolescents' everyday smartphone use to investigate person-specific drivers of depression and validate smartphone-based mobile sensing data against established ambulatory methods. We describe the methods of this study and provide an update on its status. After data collection is completed, we will address three specific aims: (1) identify idiographic drivers of dynamic variability in depressive symptoms, (2) test the validity of mobile sensing against ecological momentary assessment (EMA) and actigraphy for identifying these drivers, and (3) explore adolescent baseline characteristics as predictors of these drivers. METHODS: A total of 50 adolescents with elevated symptoms of depression will participate in 28 days of (1) smartphone-based EMA assessing depressive symptoms, processes, affect, and sleep; (2) mobile sensing of mobility, physical activity, sleep, natural language use in typed interpersonal communication, screen-on time, and call frequency and duration using the Effortless Assessment of Risk States smartphone app; and (3) wrist actigraphy of physical activity and sleep. Adolescents and caregivers will complete developmental and clinical measures at baseline, as well as user feedback interviews at follow-up. Idiographic, within-subject networks of EMA symptoms will be modeled to identify each adolescent's person-specific drivers of depression. Correlations among EMA, mobile sensor, and actigraph measures of sleep, physical, and social activity will be used to assess the validity of mobile sensing for identifying person-specific drivers. Data-driven analyses of mobile sensor variables predicting core depressive symptoms (self-reported mood and anhedonia) will also be used to assess the validity of mobile sensing for identifying drivers. Finally, between-subject baseline characteristics will be explored as predictors of person-specific drivers. RESULTS: As of October 2023, 84 families were screened as eligible, of whom 70% (n=59) provided informed consent and 46% (n=39) met all inclusion criteria after completing baseline assessment. Of the 39 included families, 85% (n=33) completed the 28-day smartphone and actigraph data collection period and follow-up study visit. CONCLUSIONS: This study leverages depressed adolescents' everyday smartphone use to identify person-specific drivers of adolescent depression and to assess the validity of mobile sensing for identifying these drivers. The findings are expected to offer novel insights into the structure and dynamics of depressive symptomatology during a sensitive period of development and to inform future development of a scalable, low-burden smartphone-based tool that can guide personalized treatment decisions for depressed adolescents. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/43931.

Exome Sequencing Identifies Carriers of the Autosomal Dominant Cancer Predisposition Disorders Beyond Current Practice Guideline Recommendations.

PURPOSE: The autosomal dominant cancer predisposition disorders hereditary breast and ovarian cancer (HBOC) and Lynch syndrome (LS) are genetic conditions for which early identification and intervention have a positive effect on the individual and public health. The goals of this study were to determine whether germline genetic screening using exome sequencing could be used to efficiently identify carriers of HBOC and LS. METHODS: Participants were recruited from three geographically and racially diverse sites in the United States (Rochester, MN; Phoenix, AZ; Jacksonville, FL). Participants underwent Exome+ sequencing (Helix Inc, San Mateo, CA) and return of results for specific genetic findings: HBOC (BRCA1 and BRCA1) and LS (MLH1, MSH2, MSH6, PMS2, and EPCAM). Chart review was performed to collect demographics and personal and family cancer history. RESULTS: To date, 44,306 participants have enrolled in Tapestry. Annotation and interpretation of all variants in genes for HBOC and LS resulted in the identification of 550 carriers (prevalence, 1.24%), which included 387 with HBOC (27.2% BRCA1, 42.8% BRCA2) and 163 with LS (12.3% MSH6, 8.8% PMS2, 4.5% MLH1, 3.8% MSH2, and 0.2% EPCAM). More than half of these participants (52.1%) were newly diagnosed carriers with HBOC and LS. In all, 39.2% of HBOC/LS carriers did not satisfy National Comprehensive Cancer Network (NCCN) criteria for genetic evaluation. NCCN criteria were less commonly met in underrepresented minority populations versus self-reported White race (51.5% v 37.5%, P = .028). CONCLUSION: Our results emphasize the need for wider utilization of germline genetic sequencing for enhanced screening and detection of individuals who have LS and HBOC cancer predisposition syndromes.

FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model.

Fibroblast activation protein (FAP) has attracted considerable attention as a possible target for the radiotherapy of solid tumors. Unfortunately, initial efforts to treat solid tumors with FAP-targeted radionuclides have yielded only modest clinical responses, suggesting that further improvements in the molecular design of FAP-targeted radiopharmaceutical therapies (RPT) are warranted. In this study, we report several advances on the previously described FAP6 radioligand that increase tumor retention and accelerate healthy tissue clearance. Seven FAP6 derivatives with different linkers or albumin binders were synthesized, radiolabeled, and investigated for their effects on binding and cellular uptake. The radioligands were then characterized in 4T1 tumor-bearing Balb/c mice using both single-photon emission computed tomography (SPECT) and ex vivo biodistribution analyses to identify the conjugate with the best tumor retention and tumor-to-healthy organ ratios. The results reveal an optimized FAP6 radioligand that exhibits efficacy and safety properties that potentially justify its translation into the clinic.

Transcriptomic convergence despite genomic divergence drive field cancerization in synchronous squamous tumors.

Introduction: Field cancerization is suggested to arise from imbalanced differentiation in individual basal progenitor cells leading to clonal expansion of mutant cells that eventually replace the epithelium, although without evidence. Methods: We performed deep sequencing analyses to characterize the genomic and transcriptomic landscapes of field change in two patients with synchronous aerodigestive tract tumors. Results: Our data support the emergence of numerous genetic alterations in cancer-associated genes but refutes the hypothesis that founder mutation(s) underpin this phenomenon. Mutational signature analysis identified defective homologous recombination as a common underlying mutational process unique to synchronous tumors. Discussion: Our analyses suggest a common etiologic factor defined by mutational signatures and/or transcriptomic convergence, which could provide a therapeutic opportunity.

Mobility-Assisted Pseudo-MS3 Sequencing of Protein Ions.

The sequencing of intact proteins within a mass spectrometer has many benefits but is frequently limited by the fact that tandem mass spectrometry (MS/MS) techniques often generate poor sequence coverages when applied to protein ions. To overcome this limitation, exotic MS/MS techniques that rely on lasers and radical chemistry have been developed. These techniques generate high sequence coverages, but they require specialized instrumentation, create products through multiple dissociation mechanisms, and often require long acquisition times. Recently, we demonstrated that protein ions can be dissociated in a trapped ion mobility spectrometry (TIMS) device prior to mobility separation in a commercial timsTOF. All generated product ions were distributed throughout the mobility dimension, and this separation enabled deconvolution of complex tandem mass spectra and could enable facile pseudo-MS3 interrogation of generated product ions with the downstream quadrupole and collision cell. A second activation step improves sequence coverage because the most labile bonds have been depleted during the first dissociation and subsequent dissociation events are more evenly distributed throughout the product ion backbone. In this work, we explore the potential of this mobility-assisted pseudo-MS3 (MAP) method on a commercial timsTOF and timsTOF Pro 2. We demonstrate that while MAP only generates 92% of the sequence coverage of the most effective MS/MS technique, it accomplished this feat in 1.5 min and could be facilely integrated with liquid chromatographic separations.

The path forward for protein footprinting, covalent labeling, and mass spectrometry-based protein conformational analyses.

Mass spectrometry-based approaches to assess protein conformation have become widely utilized due to their sensitivity, low sample requirements, and broad applicability to proteins regardless of size and environment. Their wide applicability and sensitivity also make these techniques suitable for the analysis of complex mixtures of proteins, and thus, they have been applied at the cell and even the simple organism levels. These works are impressive, but they predominately employ "bottom-up" workflows and require proteolytic digestion prior to analysis. Once digested, it is not possible to distinguish the proteoform from which any single peptide is derived and therefore, one cannot associate distal-in primary structure-concurrent post-translational modifications (PTMs) or covalent labels, as they would be found on separate peptides. Thus, analyses via bottom-up proteomics report the average PTM status and higher-order structure (HOS) of all existing proteoforms. Second, these works predominately employ promiscuous reagents to probe protein HOS. While this does lead to improved conformational resolution, the formation of many products can divide the signal associated with low-copy number proteins below signal-to-noise thresholds and complicate the bioinformatic analysis of these already challenging systems. In this perspective, I further detail these limitations and discuss the positives and negatives of top-down proteomics as an alternative.

Does the aerosol box accommodate the double-lumen tube intubation?

The aerosol box was widely used to shield healthcare providers from exposure to COVID-19 during single-lumen intubation procedures. However, it has not previously been evaluated for its use in double-lumen tube intubations. This report presents the case of a 25-year-old COVID-19-positive male with a fever who required an emergency thoracotomy for a mediastinal abscess. During the rapid-sequence induction of general anesthesia, an attempt to use the aerosol box for double-lumen tube intubation was made. The attempt faced unique challenges due to the aerosol box's restrictive dimensions and the double-lumen tube's physical characteristics, such as length and flexibility, resulting in an unsuccessful first attempt. Consequently, the aerosol box was removed, and a successful intubation was achieved without it. Postoperatively, the patient remained intubated, was transferred to the intensive care unit, and was extubated on the second postoperative day, followed by intensive care unit discharge. This experience suggests that the standard aerosol box size (50 cm wide, 40 cm deep, and 50 cm tall) may not be suitable for double-lumen tube intubations. This highlights the importance of assessing the feasibility of each aerosol box before its clinical use in such procedures.

Spectroscopic Manifestations Of (Anti)Aromaticity In Oxidized And Reduced Porphyrin And Norcorrole.

Aromaticity and antiaromaticity are foundational principes in organic chemistry, regularly invoked to explain stability, structure, and magnetic and electronic properties. There are ongoing challenges in assigning molecules as aromatic or antiaromatic using optical spectroscopy. Here we report spectroelectrochemical and computational analyses of porphyrin (18π neutral, aromatic) and norcorrole (16π neutral, antiaromatic), and their oxidized (16π porphyrin dication) and reduced (norcorrole 18π dianion) forms. Our results show that while the visible spectra are characteristic of (anti)aromaticity consistent with Hückel's rules, the IR spectra are much less informative, owing to the relative rigidity of norcorrole. The results have implications for the assignment of (anti)aromaticity in both ground-state and time-resolved excited-state spectra.

Discernible interindividual patterns of global efficiency decline during theoretical brain surgery.

The concept of functional localization within the brain and the associated risk of resecting these areas during removal of infiltrating tumors, such as diffuse gliomas, are well established in neurosurgery. Global efficiency (GE) is a graph theory concept that can be used to simulate connectome disruption following tumor resection. Structural connectivity graphs were created from diffusion tractography obtained from the brains of 80 healthy adults. These graphs were then used to simulate parcellation resection in every gross anatomical region of the cerebrum by identifying every possible combination of adjacent nodes in a graph and then measuring the drop in GE following nodal deletion. Progressive removal of brain parcellations led to patterns of GE decline that were reasonably predictable but had inter-subject differences. Additionally, as expected, there were deletion of some nodes that were worse than others. However, in each lobe examined in every subject, some deletion combinations were worse for GE than removing a greater number of nodes in a different region of the brain. Among certain patients, patterns of common nodes which exhibited worst GE upon removal were identified as "connectotypes". Given some evidence in the literature linking GE to certain aspects of neuro-cognitive abilities, investigating these connectotypes could potentially mitigate the impact of brain surgery on cognition.

Management of bone disease with concurrent chimeric antigen receptor T-cell therapy for multiple myeloma.

In the intricate landscape of multiple myeloma, a hematologic malignancy of plasma cells, bone disease presents a pivotal and often debilitating complication. The emergence of Chimeric Antigen Receptor T-cell (CAR-T) therapy has marked a pivotal shift in the therapeutic landscape, offering novel avenues for the management of MM, particularly for those with relapsed or refractory disease. This innovative treatment modality not only targets malignant cells with precision but also influences the bone microenvironment, presenting both challenges and opportunities in patient care. In this comprehensive review, we aim to examine the multifaceted aspects of bone disease in patients with multiple myeloma and concurrent CAR-T therapy, highlighting its clinical ramifications and the latest advancements in diagnostic modalities and therapeutic interventions. The article aims to synthesize current understanding of the interplay between myeloma cells, CAR-T cells, and the bone microenvironment in the context of current treatment strategies in this challenging and unique patient population.

Integrated platform for multiscale molecular imaging and phenotyping of the human brain.

Understanding cellular architectures and their connectivity is essential for interrogating system function and dysfunction. However, we lack technologies for mapping the multiscale details of individual cells and their connectivity in the human organ-scale system. We developed a platform that simultaneously extracts spatial, molecular, morphological, and connectivity information of individual cells from the same human brain. The platform includes three core elements: a vibrating microtome for ultraprecision slicing of large-scale tissues without losing cellular connectivity (MEGAtome), a polymer hydrogel-based tissue processing technology for multiplexed multiscale imaging of human organ-scale tissues (mELAST), and a computational pipeline for reconstructing three-dimensional connectivity across multiple brain slabs (UNSLICE). We applied this platform for analyzing human Alzheimer's disease pathology at multiple scales and demonstrating scalable neural connectivity mapping in the human brain.

Postoperative opioid consumption patterns diverge between propensity matched patients undergoing traumatic and elective cervical spine fusion.

BACKGROUND CONTEXT: Prolonged opioid therapy following spine surgery is an ongoing postoperative concern. While prior studies have investigated postoperative opioid use patterns in the elective cervical surgery patient population, to our knowledge, opioid use patterns in patients undergoing surgery for traumatic cervical spine injuries have not been elucidated. PURPOSE: The purpose of this study was to compare opioid use and prescription patterns in the postoperative pain management of patients undergoing traumatic and elective cervical spine fusion surgery. STUDY DESIGN: Retrospective cohort study. PATIENT SAMPLE: Adult patients with traumatic cervical injuries who underwent primary anterior cervical discectomy and fusion (ACDF) or posterior cervical decompression and fusion (PCDF) during their initial hospital admission. The propensity matched, control group consisted of adult elective cervical fusion patients who underwent primary ACDF or PCDF. OUTCOME MEASURES: Demographic data, surgical characteristics, spinal disease diagnosis, location of cervical injury, procedure type, operative levels fused, and Prescription Drug Monitoring Program (PDMP) data. PDMP data included the number of opioid prescriptions filled, preoperative opioid use, postoperative opioid use, and use of perioperative benzodiazepines, muscle relaxants, or gabapentin. Opioid consumption data was collected in morphine milligram equivalents (MME) and standardized per day. METHODS: A 1:1 propensity match was performed to match traumatic injury patients undergoing cervical fusion surgery with elective cervical fusion patients. Traumatic injury patients were matched based on age, sex, CCI, procedure type, and cervical levels fused. Pre- and postoperative opioid, benzodiazepine, muscle relaxant, and gabapentin use were assessed for the traumatic injury and elective patients. T- or Mann-Whitney U tests were used to compare continuous data and Chi-Squared or Fisher's Exact were used to compare categorical data. Multivariate stepwise regression using MME per day 0 - 30 days following surgery as the dependent outcome was performed to further evaluate associations with postoperative opioid use. RESULTS: A total of 48 patients underwent fusion surgery for a traumatic cervical spine injury and 48 elective cervical fusion with complete PDMP data were assessed. Elective patients were found to fill more prescriptions (3.19 vs. 0.65, p=.023) and take more morphine milligram equivalents (MME) per day (0.60 vs. 0.04, p=.014) within 1 year prior to surgery in comparison to traumatic patients. Elective patients were also more likely to use opioids (29.2% vs. 10.4%, p=.040) and take more MMEs per day (0.70 vs. 0.05, p=.004) within 30 days prior to surgery. Within 30 days postoperatively, elective patients used opioids more frequently (89.6% vs. 52.1%, p<.001) and took more MMEs per day (3.73 vs. 1.71, p<.001) than traumatic injury patients. Multivariate stepwise regression demonstrated preoperative opioid use (Estimate: 1.87, p=.013) to be correlated with higher postoperative MME per day within 30 days of surgery. Surgery after traumatic injury was correlated with lower postoperative MME use per day within 30 days of surgery (Estimate: -1.63 p=.022). CONCLUSION: Cervical fusion patients with a history of traumatic spine injury consume fewer opioids in the early postoperative period in comparison to elective cervical fusion patients, however both cohorts consumed a similar amount after the initial 30-day postoperative period. Preoperative opioid use was also a risk factor for higher consumption in the short-term postoperative period. These results may aid physicians in further understanding patients' postoperative care needs based on presenting injury characteristics and highlights the need for enhanced follow-up care for traumatic cervical spine injury patients after fusion surgery.

Cannabis, Tobacco Use, and COVID-19 Outcomes.

Importance: It is unclear whether cannabis use is associated with adverse health outcomes in patients with COVID-19 when accounting for known risk factors, including tobacco use. Objective: To examine whether cannabis and tobacco use are associated with adverse health outcomes from COVID-19 in the context of other known risk factors. Design, Setting, and Participants: This retrospective cohort study used electronic health record data from February 1, 2020, to January 31, 2022. This study included patients who were identified as having COVID-19 during at least 1 medical visit at a large academic medical center in the Midwest US. Exposures: Current cannabis use and tobacco smoking, as documented in the medical encounter. Main Outcomes and Measures: Health outcomes of hospitalization, intensive care unit (ICU) admission, and all-cause mortality following COVID-19 infection. The association between substance use (cannabis and tobacco) and these COVID-19 outcomes was assessed using multivariable modeling. Results: A total of 72 501 patients with COVID-19 were included (mean [SD] age, 48.9 [19.3] years; 43 315 [59.7%] female; 9710 [13.4%] had current smoking; 17 654 [24.4%] had former smoking; and 7060 [9.7%] had current use of cannabis). Current tobacco smoking was significantly associated with increased risk of hospitalization (odds ratio [OR], 1.72; 95% CI, 1.62-1.82; P < .001), ICU admission (OR, 1.22; 95% CI, 1.10-1.34; P < .001), and all-cause mortality (OR, 1.37, 95% CI, 1.20-1.57; P < .001) after adjusting for other factors. Cannabis use was significantly associated with increased risk of hospitalization (OR, 1.80; 95% CI, 1.68-1.93; P < .001) and ICU admission (OR, 1.27; 95% CI, 1.14-1.41; P < .001) but not with all-cause mortality (OR, 0.97; 95% CI, 0.82-1.14, P = .69) after adjusting for tobacco smoking, vaccination, comorbidity, diagnosis date, and demographic factors. Conclusions and Relevance: The findings of this cohort study suggest that cannabis use may be an independent risk factor for COVID-19-related complications, even after considering cigarette smoking, vaccination status, comorbidities, and other risk factors.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.