Assessing effects of Cannabis on various neuropathologies: A systematic review.

Natural bioactives possess a wide range of chemical structures that can exert a plethora of pharmacological and toxicological actions, resulting in neuroprotection or neurotoxicity. These pharmacodynamic properties can positively or negatively impact human and animal global healthcare. Remarkably, Ayurvedic botanical Cannabis has been used worldwide by different ethnicities and religions for spiritual, commercial, recreational, nutraceutical, cosmeceutical, and medicinal purposes for centuries. Cannabis-based congeners have been approved by the United States of America's (USA) Food & Drug Administration (FDA) and other global law agencies for various therapeutic purposes. Surprisingly, the strict laws associated with possessing cannabis products have been mitigated in multiple states in the USA and across the globe for recreational use. This has consequently led to a radical escalation of exposure to cannabis-related substances of abuse. However, there is a lacuna in the literature on the acute and chronic effects of Cannabis and its congeners on various neuropathologies. Moreover, in the post-COVID era, there has been a drastic increase in the incidence and prevalence of numerous neuropathologies, leading to increased morbidity and mortality. There is an impending necessity for a safe, economically viable, multipotent, natural bioactive to prevent and treat various neuropathologies. The ayurvedic herb, Cannabis is one of the oldest botanicals known to humans and has been widely used. However, the comprehensive effect of Cannabis on various neuropathologies is not well established. Hence, this review presents effects of Cannabis on various neuropathologies.

Unlocking the future of patient Education: ChatGPT vs. LexiComp® as sources of patient education materials.

BACKGROUND: ChatGPT is a conversational artificial intelligence technology that has shown application in various facets of healthcare. With the increased use of AI, it is imperative to assess the accuracy and comprehensibility of AI platforms. OBJECTIVE: This pilot project aimed to assess the understandability, readability, and accuracy of ChatGPT as a source of medication-related patient education as compared with an evidence-based medicine tertiary reference resource, LexiComp®. METHODS: Patient education materials (PEMs) were obtained from ChatGPT and LexiComp® for 8 common medications (albuterol, apixaban, atorvastatin, hydrocodone/acetaminophen, insulin glargine, levofloxacin, omeprazole, and sacubitril/valsartan). PEMs were extracted, blinded, and assessed by 2 investigators independently. The primary outcome was a comparison of the Patient Education Materials Assessment Tool-printable (PEMAT-P). Secondary outcomes included Flesch reading ease, Flesch Kincaid grade level, percent passive sentences, word count, and accuracy. A 7-item accuracy checklist for each medication was generated by expert consensus among pharmacist investigators, with LexiComp® PEMs serving as the control. PEMAT-P interrater reliability was determined via intraclass correlation coefficient (ICC). Flesch reading ease, Flesch Kincaid grade level, percent passive sentences, and word count were calculated by Microsoft® Word®. Continuous data were assessed using the Student's t-test via SPSS (version 20.0). RESULTS: No difference was found in the PEMAT-P understandability score of PEMs produced by ChatGPT versus LexiComp® [77.9% (11.0) vs. 72.5% (2.4), P=0.193]. Reading level was higher with ChatGPT [8.6 (1.2) vs. 5.6 (0.3), P < 0.001). ChatGPT PEMs had a lower percentage of passive sentences and lower word count. The average accuracy score of ChatGPT PEMs was 4.25/7 (61%), with scores ranging from 29% to 86%. CONCLUSION: Despite comparable PEMAT-P scores, ChatGPT PEMs did not meet grade level targets. Lower word count and passive text with ChatGPT PEMs could benefit patients, but the variable accuracy scores prevent routine use of ChatGPT to produce medication-related PEMs at this time.

A case study to engage students in the research design and ethics of high-throughput metagenomics.

Case studies present students with an opportunity to learn and apply course content through problem solving and critical thinking. Supported by the High-throughput Discovery Science & Inquiry-based Case Studies for Today's Students (HITS) Research Coordination Network, our interdisciplinary team designed, implemented, and assessed two case study modules entitled "You Are What You Eat." Collectively, the case study modules present students with an opportunity to engage in experimental research design and the ethical considerations regarding microbiome research and society. In this manuscript, we provide instructors with tools for adopting or adapting the research design and/or the ethics modules. To date, the case has been implemented using two modalities (remote and in-person) in three courses (Microbiology, Physiology, and Neuroscience), engaging over 200 undergraduate students. Our assessment data demonstrate gains in content knowledge and students' perception of learning following case study implementation. Furthermore, when reflecting on our experiences and student feedback, we identified ways in which the case study could be modified for different settings. In this way, we hope that the "You Are What You Eat" case study modules can be implemented widely by instructors to promote problem solving and critical thinking in the traditional classroom or laboratory setting when discussing next-generation sequencing and/or metagenomics research.

A survey on awareness, knowledge and preferences toward genetic testing among the United States general public.

Aim: To understand awareness, knowledge and preferences regarding genetic testing among the USA general public. Methods: A cross-sectional online survey using a Qualtrics Panel. Results: Among 1600 respondents, 545 (34%) were White, 411 (26%) Black, 412 (26%) Hispanic or Latin(x) and 232 (15%) Asian. Most had heard of ancestry testing (87%) and genetic health risk testing (69%), but a third thought inherited genes were only a little or not at all responsible for obesity (36%) and mental health (33%). The majority preferred pre-emptive pharmacogenetic testing (n = 74%) compared with reactive testing. Statistically significant differences between racial/ethnic groups and rural-urban respondents were observed. Conclusion: Most preferred pre-emptive pharmacogenetic testing; however, about one-quarter preferred reactive testing. Preferences should be discussed during patient-clinician interactions.

What is this study about? This study presents a large online survey among the USA general public to understand their awareness, knowledge and preferences about genetic testing and how this may vary by racial/ethnic group and rural/urban status.What were the results? Most survey respondents had heard of ancestry testing (87%) and genetic health risk testing (69%). However, over a third of respondents thought that inherited genes may be only a little or not at all responsible for obesity (36%) and mental health (33%). When asked about preferences for pre-emptive compared with reactive pharmacogenetic testing, the majority preferred pre-emptive testing (n = 74%). Statistically significant differences between racial/ethnic groups as well as rural-urban respondents were seen.What do the results mean? The US general public may have a different understanding of genetic testing for different diseases, and have different preferences when it comes to the timing of testing. Appropriate educational content targeting the link between genetics and specific diseases should be prepared, and preferences for pre-emptive or reactive testing should be discussed during visits with healthcare providers.

The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies.

As the kynurenine pathway's links to inflammation, the immune system, and neurological disorders became more apparent, it attracted more and more attention. It is the main pathway through which the liver breaks down Tryptophan and the initial step in the creation of nicotinamide adenine dinucleotide (NAD+) in mammals. Immune system activation and the buildup of potentially neurotoxic substances can result from the dysregulation or overactivation of this pathway. Therefore, it is not shocking that kynurenines have been linked to neurological conditions (Depression, Parkinson's, Alzheimer's, Huntington's Disease, Schizophrenia, and cognitive deficits) in relation to inflammation. Nevertheless, preclinical research has demonstrated that kynurenines are essential components of the behavioral analogs of depression and schizophrenia-like cognitive deficits in addition to mediators associated with neurological pathologies due to their neuromodulatory qualities. Neurodegenerative diseases have been extensively associated with neuroactive metabolites of the kynurenine pathway (KP) of tryptophan breakdown. In addition to being a necessary amino acid for protein synthesis, Tryptophan is also transformed into the important neurotransmitters tryptamine and serotonin in higher eukaryotes. In this article, a summary of the KP, its function in neurodegeneration, and the approaches being used currently to target the route therapeutically are discussed.

A Monovalent Fab Affinity-Capture and Elution Bridging Immunoassay Overcomes Rheumatoid Factor Interference while Accurately Detecting Antidrug Antibodies.

BACKGROUND: Rheumatoid factor (RF) consists of autoantibodies that bind the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) and present in sera of rheumatoid arthritis (RA) patients. Immunoassays to detect antidrug antibodies (ADA) in RA patient samples may experience interference due to RF binding and crosslinking Fc regions of the capture and detection antibody reagents. To overcome this interference, a novel Fab affinity-capture and elution (ACE)-bridging immunoassay (Fab ACE-Bridge) was developed with monovalent-recombinant Fab to avoid RF interference. METHODS: ACE and ACE-Bridge assays were developed to detect ADA against a therapeutic monoclonal antibody using samples from healthy donors, psoriasis patients, and RA patients. The performance of these assays was compared to a novel Fab ACE-Bridge assay, in which monoclonal antibody was replaced with monovalent Fab. RESULTS: High screening signals in the ACE and ACE-Bridge assays were detected in RA patient samples but not in samples from healthy donors or psoriasis patients. The high screening signals in RA samples did not inhibit to the expected extent in the confirmatory assay, a consistent feature of false-positive screening results. Further investigation revealed RF as the interferent affecting assay performance. Modification of the ACE-Bridge assay by using monovalent Fab eliminated RF interference while allowing for sensitive and drug-tolerant detection of authentic ADA. CONCLUSIONS: RF interfered significantly in traditional ACE and ACE-Bridge assays. Implementation of a novel monovalent Fab ACE-Bridge assay overcame RF interference. The use of monovalent Fab is recommended for immunogenicity assays when assessing ADA in RA patient samples.

Current published evidence on barriers and proposed strategies for genetic testing implementation in health care settings: A scoping review.

BACKGROUND: The slow uptake of genetic testing in routine clinical practice warrants the attention of researchers and practitioners to find effective strategies to facilitate implementation. OBJECTIVES: This study aimed to identify the barriers to and strategies for pharmacogenetic testing implementation in a health care setting from published literature. METHODS: A scoping review was conducted in August 2021 with an expanded literature search using Ovid MEDLINE, Web of Science, International Pharmaceutical Abstract, and Google Scholar to identify studies reporting implementation of pharmacogenetic testing in a health care setting, from a health care system's perspective. Articles were screened using DistillerSR and findings were organized using the 5 major domains of Consolidated Framework for Implementation Research (CFIR). RESULTS: A total of 3536 unique articles were retrieved from the above sources, with only 253 articles retained after title and abstract screening. Upon screening the full texts, 57 articles (representing 46 unique practice sites) were found matching the inclusion criteria. We found that most reported barriers and their associated strategies to the implementation of pharmacogenetic testing surrounded 2 CFIR domains: intervention characteristics and inner settings. Factors relating to cost and reimbursement were described as major barriers in the intervention characteristics. In the same domain, another major barrier was the lack of utility studies to provide evidence for genetic testing uptake. Technical hurdles, such as integrating genetic information to medical records, were identified as an inner settings barrier. Collaborations and lessons from early implementers could be useful strategies to overcome majority of the barriers across different health care settings. Strategies proposed by the included implementation studies to overcome these barriers are summarized and can be used as guidance in future. CONCLUSION: Barriers and strategies identified in this scoping review can provide implementation guidance for practice sites that are interested in implementing genetic testing.

Myrica cerifera, a Medicinal Plant of the Lumbee Tribe, has Antibacterial and Nematicidal Properties.

Currently threatening the world of medicine is a growing number of antibiotic-resistant diseases. More specifically, bacteria and nematodes have gained resistance to many of the world's leading antibiotics and nematicides, respectively, making infections more difficult to treat. Subsequently, these parasitic organisms are able to continue damaging crops and other living organisms like humans without strong interference. To help people and the environment, the development of new and novel antibiotics is vital. Previous research suggests that phytochemicals are a potential solution that will not only help inhibit bacterial growth but also reduce nematode survival. We hypothesized that Myrica cerifera, a plant often used by the Lumbee tribe to treat illness, possesses antibacterial and nematicidal properties. To answer our hypothesis, we began by collecting plant specimens to extract material for biological assays and to subsequently isolate and elucidate the structures of active components. The extract was evaluated for antibacterial properties with an agar diffusion assay and then nematicidal properties using Caenorhabditis elegans. M. cerifera extract was added onto an agar lawn at various doses, and the nematodes' lifespans were scored. The findings of this study show that extracts of this plant, more commonly referred to as 'wax myrtle', do significantly decrease the lifespan of C. elegans and increase the zone of inhibition for Staphylococcus epidermidis and Staphylococcus aureus. In addition, two compounds were isolated and characterized through chemical extraction, chromatographic separation, and spectroscopic analysis. These compounds could potentially be used to treat bacterial and nematode infections.

Hypoxia Inducible Factor-1α binds and activates γ-secretase for Aß production under hypoxia and cerebral hypoperfusion.

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer's disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aß). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aß production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O2. Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aß production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aß production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment.

HspB1 Overexpression Improves Life Span and Stress Resistance in an Invertebrate Model.

To explore the role of the small heat shock protein beta 1 (HspB1, also known as Hsp25 in rodents and Hsp27 in humans) in longevity, we created a Caenorhabiditis elegans model with a high level of ubiquitous expression of the naked mole-rat HspB1 protein. The worms showed increased life span under multiple conditions and also increased resistance to heat stress. RNAi experiments suggest that HspB1-induced life extension is dependent on the transcription factors skn-1 (Nrf2) and hsf-1 (Hsf1). RNAseq from HspB1 worms showed an enrichment in several skn-1 target genes, including collagen proteins and lysosomal genes. Expression of HspB1 also improved functional outcomes regulated by SKN-1, specifically oxidative stress resistance and pharyngeal integrity. This work is the first to link a small heat shock protein with collagen function, suggesting a novel role for HspB1 as a hub between canonical heat response signaling and SKN-1 transcription.

Predementia Brain Changes in Progranulin Mutation: A Systematic Review of Neuroimaging Evidence.

BACKGROUND: Mutations in the progranulin (GRN) gene are a major cause of familial frontotemporal dementia. They result in a loss of progranulin levels and in GRN-related brain degenerative changes that unfold over years if not decades. The aim of our review was to summarize the evidence on emerging functional and structural brain abnormalities in carriers of GRN mutations. SUMMARY: We performed a systematic search for studies that used at least one modality (structural MRI, fMRI, fluorodeoxyglucose positron emission tomography, diffusion tensor imaging) to compare mutation carriers to non-carrier controls. Our search produced 13 studies published between 2008 and 2017, the majority cross-sectional, with carrier sample sizes ranging from 5 to 65. Key Messages: The aggregate findings suggest that (1) measurable brain changes are detectable in at least some mutation carriers 20-25 years prior to disease onset; (2) functional/metabolic changes progress more consistently over time than structural changes; (3) the topographic pattern is anterior to posterior, not always asymmetric, and maps onto known functional networks.

Decreased Prefrontal Activation during Matrix Reasoning in Predementia Progranulin Mutation Carriers.

We tested the potential of task-based functional neuroimaging as a biomarker of emerging prefrontal brain changes in progranulin (GRN) mutations carriers. Five GRN mutation carriers free of frontotemporal dementia (FTD) and 11 non-carriers from families with FTD-GRN underwent functional MRI while solving matrix-reasoning problems. Mutation carriers displayed slower responses for more difficult problems and lower lateral prefrontal activation across all problems. Overall task-evoked posterior ventrolateral prefrontal activation predicted mutation status with 100% sensitivity and 91% specificity. Volumetric differences did not account for activation differences. Prefrontal activation may have utility as a biomarker in GRN mutation.

Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Sprouty2 regulates endochondral bone formation by modulation of RTK and BMP signaling.

Skeletal development is regulated by the coordinated activity of signaling molecules that are both produced locally by cartilage and bone cells and also circulate systemically. During embryonic development and postnatal bone remodeling, receptor tyrosine kinase (RTK) superfamily members play critical roles in the proliferation, survival, and differentiation of chondrocytes, osteoblasts, osteoclasts, and other bone cells. Recently, several molecules that regulate RTK signaling have been identified, including the four members of the Sprouty (Spry) family (Spry1-4). We report that Spry2 plays an important role in regulation of endochondral bone formation. Mice in which the Spry2 gene has been deleted have defective chondrogenesis and endochondral bone formation, with a postnatal decrease in skeletal size and trabecular bone mass. In these constitutive Spry2 mutants, both chondrocytes and osteoblasts undergo increased cell proliferation and impaired terminal differentiation. Tissue-specific Spry2 deletion by either osteoblast- (Col1-Cre) or chondrocyte- (Col2-Cre) specific drivers led to decreased relative bone mass, demonstrating the critical role of Spry2 in both cell types. Molecular analyses of signaling pathways in Spry2(-/-) mice revealed an unexpected upregulation of BMP signaling and decrease in RTK signaling. These results identify Spry2 as a critical regulator of endochondral bone formation that modulates signaling in both osteoblast and chondrocyte lineages.

Wnt signaling interacts with bmp and edn1 to regulate dorsal-ventral patterning and growth of the craniofacial skeleton.

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton.

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton.

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.

Expression and characterization of penicillin-binding proteins in Burkholderia cenocepacia.

Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His(6)-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind beta-lactams, indicating these putative PBPs have penicillin-binding activity.