Three de novo assembled wild cacao genomes from the Upper Amazon.

Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83-0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Utilization of a New Hundred-Genomes Pipeline to Design a Rapid Duplex LAMP Detection Assay for Xanthomonas euvesicatoria and X. vesicatoria in Tomato.

Xanthomonas euvesicatoria and X. vesicatoria are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. Therefore, this work aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of X. euvesicatoria and X. vesicatoria by loop-mediated isothermal amplification. In total, 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg µl-1 for both pathogens with an assay duration of 40 min on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with X. euvesicatoria and X. vesicatoria pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

Oral bacterial decontamination using an innovative prototype for photocatalytic disinfection.

OBJECTIVES: The aim of this study was to evaluate the effectiveness of a prototype photocatalytic device for bacterial decontaminations of the oral cavity. METHODS: Sixty-four subjects (18-65) were selected and randomly assigned to eight groups (n = 8), according to oral disinfection protocol: (G1): distilled water (control); (G2): 1.5% hydrogen peroxide (HP); (G3): 3.0% HP; (G4): 0.12% chlorhexidine (CHX); (G5): Germinator; (G6): 1.5% HP + Germinator; (G7): 3.0%HP + Germinator; (G8): 0.12% CHX + Germinator. Stimulated saliva was collected before and after a 3-min mouthwash and/or Germinator application. The patients were kept relaxed and retained saliva 5-10 min, spitting out into the tube for 3 min. The percentage bacterial reduction was checked by counting the colony-forming units (CFUs) after culturing on blood agar plates. Data were subjected to one-way ANOVA followed by Tukey's post hoc test (α = 5%) for statistical significance. RESULTS: The highest bacterial reduction was observed in groups 3 (3.0% HP), 6 (1.5% HP + Germinator), and 7 (3.0% + Germinator), with no statistically significant difference between them (p > 0.05). Groups 6 (1.5% HP + Germinator) and 8 (0.12% CHX + Germinator) showed higher bacterial reduction than groups 2 (1.5% HP) and 4 (0.12% CHX) (p < 0.05). Finally, group 5 (Germinator) showed higher bacterial reduction than control group (DW) and group 4 (0.12% CHX) (p < 0.05). CONCLUSIONS: The photocatalytic disinfection was effective against oral bacteria and improved the antimicrobial action of 1.5% HP and 0.12%. CLINICAL SIGNIFICANCE: The photocatalytic disinfection can be an alternative protocol to provide the oral decontamination.

Push-out bond strength and marginal adaptation of apical plugs with bioactive endodontic cements in simulated immature teeth.

OBJECTIVES: This study evaluates the bond strength and marginal adaptation of mineral trioxide aggregate (MTA) Repair HP and Biodentine used as apical plugs; MTA was used as reference material for comparison. MATERIALS AND METHODS: A total of 30 single-rooted teeth with standardized, artificially created open apices were randomly divided into 3 groups (n = 10 per group), according to the material used to form 6-mm-thick apical plugs: group 1 (MTA Repair HP); group 2 (Biodentine); and group 3 (white MTA). Subsequently, the specimens were transversely sectioned to obtain 2 (cervical and apical) 2.5-mm-thick slices per root. Epoxy resin replicas were observed under a scanning electron microscope to measure the gap size at the material/dentin interface (the largest and smaller gaps were recorded for each replica). The bond strength of the investigated materials to dentin was determined using the push-out test. The variable bond strengths and gap sizes were evaluated independently at the apical and cervical root dentin slices. Data were analyzed using descriptive and analytic statistics. RESULTS: The comparison between the groups regarding the variables' bond strengths and gap sizes showed no statistical difference (p > 0.05) except for a single difference in the smallest gap at the cervical root dentin slice, which was higher in group 3 than in group 1 (p < 0.05). CONCLUSIONS: The bond strength and marginal adaptation to root canal walls of MTA HP and Biodentine cement were comparable to white MTA.

Root Canal Filling Quality Comparison of a Premixed Calcium Silicate Endodontic Sealer and Different Carrier-Based Obturation Systems.

BACKGROUND: The number of voids within the root canal obturation is a relevant parameter to describe the quality of the technique, as well as to predict long-term prognosis. The aim of this study was to evaluate the quality of root canal obturation in curved root canals filled with Thermafil, GuttaCore, GuttaFusion and lateral compaction with AH Plus and EndoSequence BC sealer, by means of percentage of gutta-percha and sealer filled area. METHODS: 200 curved canals of mandibular molars were divided in five groups (n = 40). Each specimen was evaluated at 3, 6 and 9 mm from the apex. A total of 600 samples were analyzed. Obturation was performed with either Thermafil (TH), GuttaCore (GC), GuttaFusion (GF) or lateral compaction (LC), using AH Plus as sealer. In another group EndoSequence BC sealer (BC) was used. Subgroups (n = 20) were made depending on the use of sonic activation during the placement of the sealer. The percentage of total obturation material was analyzed as an indirect measurement of percentage of voids. RESULTS: GF showed a better performance at the apical level, while GC showed the higher percentage of total obturation at the coronal area. No differences were observed for the activation of sealers in any of the groups. Statistical analysis was performed by using two-way ANOVA. Statistical significance was set at CI: 95% (p < 0.05). CONCLUSIONS: Sonic activation of sealers did not show any benefit to avoid the presence of voids within obturation of curved canals. Following the manufacturer recommendations, we found that TH did not show benefits when applied to curved canals.

KEC: unique sequence search by K-mer exclusion.

SUMMARY: Searching for amino acid or nucleic acid sequences unique to one organism may be challenging depending on size of the available datasets. K-mer elimination by cross-reference (KEC) allows users to quickly and easily find unique sequences by providing target and non-target sequences. Due to its speed, it can be used for datasets of genomic size and can be run on desktop or laptop computers with modest specifications. AVAILABILITY AND IMPLEMENTATION: KEC is freely available for non-commercial purposes. Source code and executable binary files compiled for Linux, Mac and Windows can be downloaded from https://github.com/berybox/KEC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rubisco activase A (RcaA) is a central node in overlapping gene network of drought and salinity in Barley (Hordeum vulgare L.) and may contribute to combined stress tolerance.

Co-occurrence of abiotic stresses, especially drought and salinity, is a natural phenomenon in field conditions and is worse for crop production than any single stress. Nowadays, rigorous methods of meta-analysis and systems biology have made it possible to perform cross-study comparisons of single stress experiments, which can uncover main overlapping mechanisms underlying tolerance to combined stress. In this study, a meta-analysis of RNA-Seq data was conducted to obtain the overlapping gene network of drought and salinity stresses in barley (Hordeum vulgare L.), which identified Rubisco activase A (RcaA) as a hub gene in the dual-stress response. Thereafter, a greenhouse experiment was carried out using two barley genotypes with different abiotic stress tolerance and evaluated several physiochemical properties as well as the expression profile and protein activity of RcaA. Finally, machine learning analysis was applied to uncover relationships among combined stress tolerance and evaluated properties. We identified 441 genes which were differentially expressed under both drought and salinity stress. Results revealed that the photosynthesis pathway and, in particular, the RcaA gene are major components of the dual-stress responsive transcriptome. Comparative physiochemical and molecular evaluations further confirmed that enhanced photosynthesis capability, mainly through regulation of RcaA expression and activity as well as accumulation of proline content, have a significant association with combined drought and salinity stress tolerance in barley. Overall, our results clarify the importance of RcaA in combined stress tolerance and may provide new insights for future investigations.

The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.

Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.

Coronal Tooth Discoloration After the Use of White Mineral Trioxide Aggregate.

Mineral trioxide aggregate (MTA) has been used in regenerative procedures but observations have indicated that with its use there is dark staining of the tooth structure. This case report describes the treatment of tooth discoloration caused by white mineral trioxide aggregate (WMTA). Revascularization was performed with the use of WMTA in the upper left central incisor. Grayish discoloration appeared 9 months later, the tooth necessitating internal bleaching of the tooth. A significant improvement was observed in the crown after internal bleaching. The choice of material should not rely solely on biological and functional aspects, but aesthetic considerations should also be taken into account. The patient needs to be advised of risks, benefits, and alternatives to internal bleaching, eg, a veneer or a crown. Coronal discoloration is a significant distraction and embarrassment for many people in achieving an aesthetic smile; thus, it must be corrected at the earliest possible stage of discoloration.

Antimicrobial photodynamic therapy and guided endodontics: A case report.

A 35-year-old woman with a history of pulp canal obliteration (PCO) and apical periodontitis was treated with guided endodontics combined with antimicrobial photodynamic therapy (aPDT). The use of aPDT was paramount to the approach performed, and its application should be encouraged in cases of severely calcified canals when apical patency is unachievable. The combination of these techniques ensured successful clinical, radiographic, and tomographic outcomes at the 1-year follow-up. Key Words: Antimicrobial PDT, guided endodonics, periodontitis, pulp canal obliteration.

Genome-Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

Promoter Proximal Pausing Limits Tumorous Growth Induced by the Yki Transcription Factor in Drosophila.

Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step in the regulation of gene expression. Regulation of PPP is brought about by complexes 7SK snRNP, P-TEFb (Cdk9/cycT), and the negative elongation factor (NELF), which are highly conserved from Drosophila to humans. Here, we show that RNAi-mediated depletion of bin3 or Hexim of the 7SK snRNP complex or depletion of individual components of the NELF complex enhances Yki-driven growth, leading to neoplastic transformation of Drosophila wing imaginal discs. We also show that increased CDK9 expression cooperates with Yki in driving neoplastic growth. Interestingly, overexpression of CDK9 on its own or in the background of depletion of one of the components of 7SK snRNP or the NELF complex necessarily, and specifically, needed Yki overexpression to cause tumorous growth. Genome-wide gene expression analyses suggested that deregulation of protein homeostasis is associated with tumorous growth of wing imaginal discs. As both Fat/Hippo/Yki pathway and PPP are highly conserved, our observations may provide insights into mechanisms of oncogenic function of YAP-the ortholog of Yki in humans.

Development of Real-Time and Colorimetric Loop Mediated Isothermal Amplification Assay for Detection of Xanthomonas gardneri.

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/µL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls.

High temperature-induced plant disease susceptibility: more than the sum of its parts.

Higher temperatures associated with climate change often increase the severity of plant diseases. An understanding of how plants respond to pathogens during high temperature stress is required for crop improvement, but the molecular mechanisms underlying this response are largely unknown. Mechanistic research has primarily focused on plant responses during either single stresses or heat-induced loss of single gene resistance. Transcriptome analyses of plant responses to a single stress compared to combined-stresses reveal significant differences showing that single-stress response studies are inadequate for determining the mechanisms of high temperature-induced disease susceptibility. To combat plant disease in light of climate change, future research will require comprehensive study designs and analyses.

High-Quality Genome Resource of Xanthomonas hyacinthi Generated via Long-Read Sequencing.

The bacterial plant pathogen Xanthomonas hyacinthi is the causal agent of yellow disease of Hyacinthus and other ornamental plant genera. There is no available complete genome for X. hyacinthi, limiting basic research for this pathogen. Here, we release a high-quality complete genome sequence for the X. hyacinthi type strain, CFBP 1156. Single-molecule real-time (SMRT) sequencing with a mean coverage of 306× revealed two contigs of 4,918,645 and 44,381 bp in size. This was the first characterized plant-disease-causing species of Xanthomonas and this genome provides a resource to better understand the biology of yellow disease of hyacinth.

Irreversible Pulpitis - A Source of Antibiotic Over-Prescription?

Antibiotic resistance is a growing public health concern. Antibiotics continue to be prescribed by some clinicians to resolve dental pain even though research indicates that antibiotics are not effective for treating conditions such as irreversible pulpitis. The objective of this study was to determine the extent to which current research and evidence around irreversible pulpitis has been translated into dental practice and the gaps in dentists' knowledge. An on-line clinical vignette format survey questionnaire about treatment of irreversible pulpitis was distributed to the members of the Academy of Operative Dentistry and Academy of General Dentistry (US based international dental bodies). Their responses were recorded and evaluated. A total of 403 dentists participated in the survey. Over a third (39.3%) indicated they would prescribe antibiotics for symptomatic irreversible pulpitis in a permanent tooth occurring without any signs of systemic infection. The rest indicated they would not prescribe antibiotics; most of them would prescribe an analgesic combined with pulpectomy. Those who had undertaken advanced education training achieved a significantly higher mean knowledge score compared to those with just a primary dental degree (p=0.011). Similarly, full or part time academicians had a higher mean knowledge score than the clinicians who work only in private practice (p=0.014). Some dentists continue to prescribe antibiotics inappropriately for alleviating pain due to irreversible pulpitis. Antibiotic prescribing practices of dentists with advanced education or academic engagement were better as compared to the other participants. There is clear evidence of antibiotic over-prescribing for irreversible pulpitis which needs to be addressed urgently.

Irreversible pulpitis - a source of antibiotic over-prescription?

Abstract Antibiotic resistance is a growing public health concern. Antibiotics continue to be prescribed by some clinicians to resolve dental pain even though research indicates that antibiotics are not effective for treating conditions such as irreversible pulpitis. The objective of this study was to determine the extent to which current research and evidence around irreversible pulpitis has been translated into dental practice and the gaps in dentists' knowledge. An on-line clinical vignette format survey questionnaire about treatment of irreversible pulpitis was distributed to the members of the Academy of Operative Dentistry and Academy of General Dentistry (US based international dental bodies). Their responses were recorded and evaluated. A total of 403 dentists participated in the survey. Over a third (39.3%) indicated they would prescribe antibiotics for symptomatic irreversible pulpitis in a permanent tooth occurring without any signs of systemic infection. The rest indicated they would not prescribe antibiotics; most of them would prescribe an analgesic combined with pulpectomy. Those who had undertaken advanced education training achieved a significantly higher mean knowledge score compared to those with just a primary dental degree (p=0.011). Similarly, full or part time academicians had a higher mean knowledge score than the clinicians who work only in private practice (p=0.014). Some dentists continue to prescribe antibiotics inappropriately for alleviating pain due to irreversible pulpitis. Antibiotic prescribing practices of dentists with advanced education or academic engagement were better as compared to the other participants. There is clear evidence of antibiotic over-prescribing for irreversible pulpitis which needs to be addressed urgently.

Resumo A resistência aos antibióticos é uma preocupação crescente para a saúde pública. Os antibióticos continuam a ser prescritos por alguns dentistas para resolver a dor dentária, embora pesquisas indiquem que os antibióticos não são eficazes no tratamento de condições como a pulpite irreversível. O objetivo deste estudo foi determinar em que medida as pesquisas atuais e as evidências em torno da pulpite irreversível foram traduzidas em prática odontológica e as lacunas existentes no conhecimento dos dentistas. Um questionário de pesquisa em formato de vinheta clínica on-line sobre o tratamento da pulpite irreversível foi distribuído para os membros da Academia de Odontologia Operatória e da Academia de Odontologia Geral (órgãos dentários internacionais dos EUA). Suas respostas foram registradas e avaliadas. Um total de 403 dentistas participou da pesquisa. Mais de um terço (39,3%) indicaram que prescreveriam antibióticos para pulpite irreversível sintomática em um dente permanente sem qualquer sinal de infecção sistêmica. O restante respondeu que eles não prescreveriam antibióticos; a maioria deles prescreveria um analgésico combinado com pulpectomia. Aqueles que realizaram o treinamento de educação avançada obtiveram uma pontuação de conhecimento médio significativamente maior em comparação com aqueles com apenas um grau primário de conhecimento odontológico (p=0,011). Da mesma forma, acadêmicos em tempo integral ou parcial tiveram uma pontuação média de conhecimento maior do que os clínicos que trabalham apenas em consultório particular (p=0,014). Alguns dentistas continuam a prescrever antibióticos inadequadamente para aliviar a dor decorrente de pulpite irreversível. Práticas de prescrição de antibióticos por dentistas com educação avançada ou envolvimento acadêmico foram melhores em comparação com os outros participantes. Há evidências claras de excesso de prescrição de antibiótico para pulpite irreversível que precisa ser tratada com urgência.