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1.
J Imaging ; 10(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38921608

RESUMO

Hyperspectral images include information from a wide range of spectral bands deemed valuable for computer vision applications in various domains such as agriculture, surveillance, and reconnaissance. Anomaly detection in hyperspectral images has proven to be a crucial component of change and abnormality identification, enabling improved decision-making across various applications. These abnormalities/anomalies can be detected using background estimation techniques that do not require the prior knowledge of outliers. However, each hyperspectral anomaly detection (HS-AD) algorithm models the background differently. These different assumptions may fail to consider all the background constraints in various scenarios. We have developed a new approach called Greedy Ensemble Anomaly Detection (GE-AD) to address this shortcoming. It includes a greedy search algorithm to systematically determine the suitable base models from HS-AD algorithms and hyperspectral unmixing for the first stage of a stacking ensemble and employs a supervised classifier in the second stage of a stacking ensemble. It helps researchers with limited knowledge of the suitability of the HS-AD algorithms for the application scenarios to select the best methods automatically. Our evaluation shows that the proposed method achieves a higher average F1-macro score with statistical significance compared to the other individual methods used in the ensemble. This is validated on multiple datasets, including the Airport-Beach-Urban (ABU) dataset, the San Diego dataset, the Salinas dataset, the Hydice Urban dataset, and the Arizona dataset. The evaluation using the airport scenes from the ABU dataset shows that GE-AD achieves a 14.97% higher average F1-macro score than our previous method (HUE-AD), at least 17.19% higher than the individual methods used in the ensemble, and at least 28.53% higher than the other state-of-the-art ensemble anomaly detection algorithms. As using the combination of greedy algorithm and stacking ensemble to automatically select suitable base models and associated weights have not been widely explored in hyperspectral anomaly detection, we believe that our work will expand the knowledge in this research area and contribute to the wider application of this approach.

2.
Prehosp Emerg Care ; : 1-5, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38781490

RESUMO

Sodium nitrite overdose leads to profound methemoglobinemia and may quickly progress to death. It is an increasingly common method of suicide and is often fatal. Methylene blue is an effective but time-sensitive antidote that has the potential to save lives when administered early. In this case report, we describe a fatal sodium nitrite overdose and the subsequent creation of a prehospital protocol for our large urban Emergency Medical Services system.

3.
Prehosp Emerg Care ; : 1-6, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38808969

RESUMO

OBJECTIVE: Agitation is a common prehospital problem and frequently presents without a clear etiology. Given the dynamic environment of the prehospital setting, there has historically been a varied approach to treating agitation with a heavy reliance on parenteral medications. Newer best practice guidelines recommend the incorporation of oral medications to treat patients experiencing agitation. Therefore, we evaluated the use of oral risperidone in a single system after a change in protocol occurred. METHODS: This was conducted as a retrospective chart review of an urban/suburban Emergency Medical Services system over the period of 8 months. The first day this medication was implemented throughout the service was included. Charts were included for selection if they included risperidone oral dissolving tablet (ODT) as a charted medication. The primary outcome was administration of additional medications to treat agitation. Exploratory outcome measures included acceptance of medication, documented injury to paramedics, documented injuries to patients, scene times, and adverse events that could possibly be linked to the medication. RESULTS: A total of 552 records were screened for inclusion. Risperidone was offered to 530 patients and accepted by 512 (96.6%). Of these 512 patients, the median age of included patients was 39 years old (IQR 29-52 years old) with a range of 18-89 years old. Rescue or additional medications for agitation were required in 9 (1.8%) cases. There were a total of 4 (0.8%) potential complications following administration of risperidone. There were no reported assaults with subsequent injuries to prehospital personnel or injuries sustained by patients reported in this study. CONCLUSIONS: Risperidone ODT was found to be a safe and effective medication to treat mild agitation in a large urban and suburban EMS system. The need for additional medications to treat agitation was rare, and there were no documented injuries to either patients or paramedics.

4.
ISME Commun ; 4(1): ycae057, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38812718

RESUMO

Microbial communities are diverse biological systems that include taxa from across multiple kingdoms of life. Notably, interactions between bacteria and fungi play a significant role in determining community structure. However, these statistical associations across kingdoms are more difficult to infer than intra-kingdom associations due to the nature of the data involved using standard network inference techniques. We quantify the challenges of cross-kingdom network inference from both theoretical and practical points of view using synthetic and real-world microbiome data. We detail the theoretical issue presented by combining compositional data sets drawn from the same environment, e.g. 16S and ITS sequencing of a single set of samples, and we survey common network inference techniques for their ability to handle this error. We then test these techniques for the accuracy and usefulness of their intra- and inter-kingdom associations by inferring networks from a set of simulated samples for which a ground-truth set of associations is known. We show that while the two methods mitigate the error of cross-kingdom inference, there is little difference between techniques for key practical applications including identification of strong correlations and identification of possible keystone taxa (i.e. hub nodes in the network). Furthermore, we identify a signature of the error caused by transkingdom network inference and demonstrate that it appears in networks constructed using real-world environmental microbiome data.

5.
Int J Biol Sci ; 20(4): 1218-1237, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38385082

RESUMO

MCJ (Methylation-Controlled J protein), an endogenous repressor of the mitochondrial respiratory chain, is upregulated in multiple liver diseases but little is known about how it is regulated. S-adenosylmethionine (SAMe), the biological methyl donor, is frequently depleted in chronic liver diseases. Here, we show that SAMe negatively regulates MCJ in the liver. While deficiency in methionine adenosyltransferase alpha 1 (MATα1), enzyme that catalyzes SAMe biosynthesis, leads to hepatic MCJ upregulation, MAT1A overexpression and SAMe treatment reduced MCJ expression. We found that MCJ is methylated at lysine residues and that it interacts with MATα1 in liver mitochondria, likely to facilitate its methylation. Lastly, we observed that MCJ is upregulated in alcohol-associated liver disease, a condition characterized by reduced MAT1A expression and SAMe levels along with mitochondrial injury. MCJ silencing protected against alcohol-induced mitochondrial dysfunction and lipid accumulation. Our study demonstrates a new role of MATα1 and SAMe in reducing hepatic MCJ expression.


Assuntos
Hepatopatias Alcoólicas , S-Adenosilmetionina , Humanos , S-Adenosilmetionina/metabolismo , Transporte de Elétrons , Fígado/metabolismo , Mitocôndrias/metabolismo , Hepatopatias Alcoólicas/metabolismo
6.
Prehosp Emerg Care ; 28(2): 215-220, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37171895

RESUMO

OBJECTIVE: Prehospital clinicians need a practical means of providing adequate preoxygenation prior to intubation. A bag-valve-mask (BVM) can be used for preoxygenation in perfect conditions but is likely to fail in emergency settings. For this reason, many airway experts have moved away from using BVM for preoxygenation and instead suggest using a nonrebreather (NRB) mask with flush rate oxygen.Literature on preoxygenation has suggested that a NRB mask delivering flush rate oxygen (on a 15 L/min O2 regulator, maximum flow, ∼50 L/min) is noninferior to BVM at 15 L/min held with a tight seal. However, in the prehospital setting, where emergency airway management success varies, preoxygenation techniques have not been deeply explored. Our study seeks to determine whether preoxygenation can be optimally performed with NRB at flush rate oxygen. METHODS: We performed a crossover trial using healthy volunteers. Subjects underwent 3-min trials of preoxygenation with NRB mask at 25 L/min oxygen delivered from a portable tank, NRB at flush rate oxygen from a portable tank, NRB with flush rate oxygen from an onboard ambulance tank, and BVM with flush rate oxygen from an onboard ambulance tank. The primary outcome was the fraction of expired oxygen (FeO2). We compared the FeO2 of the BVM-flush to other study groups, using a noninferiority margin of 10%. RESULTS: We enrolled 30 subjects. Mean FeO2 values for NRB-25, NRB-flush ambulance, NRB-flush portable, and BVM-flush were 63% (95% confidence interval [CI] 58-68%), 74% (95%, CI 70-78%), 78% (95%, CI 74-83%), and 80% (95%, CI 75-84%), respectively. FeO2 values for NRB-flush on both portable tank and ambulance oxygen were noninferior to BVM-flush on the ambulance oxygen system (FeO2 differences of 1%, 95% CI -3% to 6%; and 6%, 95% CI 1-10%). FeO2 for the NRB-25 group was inferior to BVM-flush (FeO2 difference 16%, 95% CI 12-21%). CONCLUSIONS: Among healthy volunteers, flush rate preoxygenation using NRB masks is noninferior to BVM using either a portable oxygen tank or ambulance oxygen. This is significant because preoxygenation using NRB masks with flush rate oxygen presents a simpler alternative to the use of BVMs. Preoxygenation using NRB masks at 25 L/min from a portable tank is inferior to BVM at flush rate.


Assuntos
Serviços Médicos de Emergência , Máscaras , Humanos , Manuseio das Vias Aéreas/métodos , Oxigênio , Respiração Artificial/métodos , Estudos Cross-Over
7.
Ann Emerg Med ; 83(2): 91-99, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37725022

RESUMO

STUDY OBJECTIVE: To determine what patient characteristics were associated with the application of physical restraints in our emergency department (ED). METHODS: This was a retrospective analysis of encounters in the ED of an urban, Level I academic trauma center. We included ED encounters of adult patients (aged ≥18 years) during a 5-year period starting in 2017. We evaluated the independent association of restraint application during an encounter using a generalized estimating equation model. RESULTS: There were 464,031 ED encounters during the time period from 162,244 unique patients, including 34,798 (7.5%) with restraint application, comprising 18,166 unique patients. Several variables were associated with an increased likelihood of restraint use during an encounter. The variable with the highest odds ratio was intoxication with drugs or alcohol (adjusted odds ratio [aOR] 8.29; 95% confidence interval (CI) 7.94 to 8.65). American Indian race was associated with increased odds of restraint application (aOR 1.42; 95% CI 1.31 to 1.54) compared to the reference value of White race. Black race (aOR 0.58; 95% CI 0.55 to 0.61) and Hispanic ethnicity (aOR 0.42; 95% CI 0.37 to 0.48) were associated with lower odds of restraint application. CONCLUSIONS: Drug and alcohol intoxication were most closely associated with restraint. Encounters in which the patient was American Indian had higher odds of restraint, but this study does not replicate prior findings regarding other racial disparities in restraint.


Assuntos
Etnicidade , Grupos Raciais , Restrição Física , Adulto , Humanos , Serviço Hospitalar de Emergência , Estudos Retrospectivos , Indígena Americano ou Nativo do Alasca
8.
Front Fungal Biol ; 4: 1285531, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38155707

RESUMO

Members of the fungal genus Morchella are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various Morchella isolates. Genome sequences obtained from these Pseudomonas isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

10.
Commun Biol ; 6(1): 948, 2023 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-37723238

RESUMO

Diverse members of early-diverging Mucoromycota, including mycorrhizal taxa and soil-associated Mortierellaceae, are known to harbor Mollicutes-related endobacteria (MRE). It has been hypothesized that MRE were acquired by a common ancestor and transmitted vertically. Alternatively, MRE endosymbionts could have invaded after the divergence of Mucoromycota lineages and subsequently spread to new hosts horizontally. To better understand the evolutionary history of MRE symbionts, we generated and analyzed four complete MRE genomes from two Mortierellaceae genera: Linnemannia (MRE-L) and Benniella (MRE-B). These genomes include the smallest known of fungal endosymbionts and showed signals of a tight relationship with hosts including a reduced functional capacity and genes transferred from fungal hosts to MRE. Phylogenetic reconstruction including nine MRE from mycorrhizal fungi revealed that MRE-B genomes are more closely related to MRE from Glomeromycotina than MRE-L from the same host family. We posit that reductions in genome size, GC content, pseudogene content, and repeat content in MRE-L may reflect a longer-term relationship with their fungal hosts. These data indicate Linnemannia and Benniella MRE were likely acquired independently after their fungal hosts diverged from a common ancestor. This work expands upon foundational knowledge on minimal genomes and provides insights into the evolution of bacterial endosymbionts.


Assuntos
Micorrizas , Tenericutes , Filogenia , Genômica , Micorrizas/genética , Tamanho do Genoma
11.
Microbiome ; 11(1): 192, 2023 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-37626434

RESUMO

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.


Assuntos
Microbiota , Multiômica , Análise de Dados , Eucariotos , Microbiota/genética , Células Procarióticas
12.
Fungal Biol ; 127(5): 1005-1009, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37142360

RESUMO

Research on bacterial-fungal interactions (BFIs) has revealed that fungi and bacteria frequently interact with one another within diverse ecosystems and microbiomes. Assessing the current state of knowledge within the field of BFI research, particularly with respect to what interactions between bacteria and fungi have been previously described, is very challenging and time consuming. This is largely due to a lack of any centralized resource, with reports of BFIs being spread across publications in numerous journals using non-standardized text to describe the relationships. To address this issue, we have developed the BFI Research Portal, a publicly accessible database of previously reported interactions between bacterial and fungal taxa to serve as a centralized resource for the field. Users can query bacterial or fungal taxa to see what members from the other kingdom have been observed as interaction partners. Search results are accompanied by interactive and intuitive visual outputs, and the database is a dynamic resource that will be updated as new BFIs are reported.


Assuntos
Fungos , Microbiota , Bactérias
13.
Resuscitation ; 186: 109726, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36764570

RESUMO

AIM: Tracheal intubation is associated with interruption in cardiopulmonary resuscitation (CPR). Current knowledge of tracheal intubation during active CPR focuses on the out-of-hospital environment. We aim to describe characteristics of tracheal intubation during active CPR in the emergency department (ED) and determine whether first attempt success was associated with CPR being continued vs paused. MEASUREMENTS: We reviewed overhead video from adult ED patients receiving chest compressions at the start of the orotracheal intubation attempt. We recorded procedural detail including method of CPR, whether CPR was continued vs paused, and first attempt intubation success (primary outcome). We performed logistic regression to determine whether continuing CPR was associated with first attempt success. RESULTS: We reviewed 169 instances of tracheal intubation, including 143 patients with continued CPR and 26 patients with paused CPR. Those with paused CPR were more likely to be receiving manual rather than mechanical chest compressions. Video laryngoscopy and bougie use were common. First attempt success was higher in the continued CPR group (87%, 95% CI 81% to 92%) than the interrupted CPR group (65%, 95% CI 44% to 83%, difference 22% [95% CI 3% to 41%]). The multivariable model demonstrated an adjusted odds ratio of 0.67 (95% CI 0.17 to 2.60) for first attempt intubation success when CPR was interrupted vs continued. CONCLUSIONS: It was common to continue CPR during tracheal intubation, with success comparable to that achieved in patients without cardiac arrest. It is reasonable to attempt tracheal intubation without interrupting CPR, pausing only if necessary.


Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca , Parada Cardíaca Extra-Hospitalar , Adulto , Humanos , Reanimação Cardiopulmonar/métodos , Parada Cardíaca/terapia , Serviço Hospitalar de Emergência , Intubação Intratraqueal/métodos , Tórax , Parada Cardíaca Extra-Hospitalar/terapia
14.
iScience ; 26(2): 105987, 2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36756374

RESUMO

Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a -/- mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a -/- mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a -/- mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a -/- mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a -/- mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.

15.
Ann Emerg Med ; 81(6): 667-676, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36841658

RESUMO

STUDY OBJECTIVE: Bougie use during emergency tracheal intubation has not been well studied in children. METHODS: This was a 10-year observational study of pediatric intubations (<18 years of age) in the emergency department (ED) of an academic institution. Bougie training and use are standard in our ED, including for emergency medicine residents. Study data were collected by a combination of charts and video reviews. We compare first-attempt intubation success and procedural complications between pediatric patients with and without bougie use during tracheal intubation in the ED. In addition, we evaluate the independent association of bougie use with first-attempt intubation success using multivariable logistic regression. RESULTS: We collected data on intubation success and bougie use for 195 pediatric patients over more than 10 years. On the first tracheal intubation attempt, a pediatric bougie was used in 126 patients (65%). Median patient age was 5 years (interquartile range 1.7 to 9) in the bougie group and 1.7 years (interquartile range 0.2 to 5) in the no bougie group. Intubation was successful on the first attempt in 72% of intubations with a bougie versus 78% without a bougie (absolute difference -6%, 95% confidence interval [CI] -19 to 6%); the adjusted odds of first-attempt success with a bougie were 0.54 (95% CI 0.24 to 1.19). A procedural complication occurred for 38% of patients in the bougie group versus 51% in the no bougie group (-13%, 95% CI -27% to 2%). Two neonates, one in each group, experienced a potential injury to the airway or lower respiratory tract. CONCLUSION: In an academic ED where the bougie is commonly used, bougie use in children was not associated with procedural success or complications. Our study suggests that a randomized clinical trial is needed to determine the effect of bougie use during emergency pediatric intubation.


Assuntos
Estado Terminal , Intubação Intratraqueal , Recém-Nascido , Humanos , Criança , Lactente , Pré-Escolar , Estado Terminal/terapia , Intubação Intratraqueal/efeitos adversos , Sistema de Registros , Serviço Hospitalar de Emergência , Modelos Logísticos , Laringoscopia
17.
Prehosp Emerg Care ; 27(6): 826-831, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-35952352

RESUMO

Massive pulmonary embolism (hemodynamically unstable, defined as systolic BP <90 mmHg) has significant morbidity and mortality. Point of care ultrasound (POCUS) has allowed clinicians to detect evidence of massive pulmonary embolism much earlier in the patient's clinical course, especially when patient instability precludes computerized tomography confirmation. POCUS detection of massive pulmonary embolism has traditionally been performed by physicians. This case series demonstrates four cases of massive pulmonary embolism diagnosed with POCUS performed by non-physician prehospital personnel.


Assuntos
Serviços Médicos de Emergência , Embolia Pulmonar , Humanos , Ultrassonografia , Embolia Pulmonar/diagnóstico por imagem , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos
18.
J Imaging ; 8(10)2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-36286360

RESUMO

The benefits of autonomous image segmentation are readily apparent in many applications and garners interest from stakeholders in many fields. The wide range of benefits encompass applications ranging from medical diagnosis, where the shape of the grouped pixels increases diagnosis accuracy, to autonomous vehicles where the grouping of pixels defines roadways, traffic signs, other vehicles, etc. It even proves beneficial in many phases of machine learning, where the resulting segmentation can be used as inputs to the network or as labels for training. The majority of the available image segmentation algorithmic development and results focus on visible image modalities. Therefore, in this treatment, the authors present the results of a study designed to identify and improve current semantic methods for infrared scene segmentation. Specifically, the goal is to propose a novel approach to provide tile-based segmentation of occlusion clouds in Long Wave Infrared images. This work complements the collection of well-known semantic segmentation algorithms applicable to thermal images but requires a vast dataset to provide accurate performance. We document performance in applications where the distinction between dust cloud tiles and clear tiles enables conditional processing. Therefore, the authors propose a Gray Level Co-Occurrence Matrix (GLCM) based method for infrared image segmentation. The main idea of our approach is that GLCM features are extracted from local tiles in the image and used to train a binary classifier to provide indication of tile occlusions. Our method introduces a new texture analysis scheme that is more suitable for image segmentation than the solitary Gabor segmentation or Markov Random Field (MRF) scheme. Our experimental results show that our algorithm performs well in terms of accuracy and a better inter-region homogeneity than the pixel-based infrared image segmentation algorithms.

19.
IMA Fungus ; 13(1): 14, 2022 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-35996182

RESUMO

Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type (MAT) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit (RPB2) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.

20.
Front Microbiol ; 13: 892227, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35722318

RESUMO

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

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