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Background: Although the incidence of Cancer of Unknown Primary (CUP) is estimated to be 1-2 % of all cancers worldwide, no international standards for diagnostic workup are yet established. Such an international guideline would facilitate international comparison, provide adequate incidence and survival rates, and ultimately improve care of patients with CUP., Methods: Participants for a four round modified Delphi study were selected via a CUP literature search in PubMed and an international network of cancer researchers. A total of 90 CUP experts were invited, and 34 experts from 15 countries over four continents completed all Delphi survey rounds., Findings: The Delphi procedure resulted in a multi-layer CUP classification for the diagnostic workup. Initial diagnostic workup should at least consist of history and physical examination, full blood count, analysis of serum markers, a biopsy of the most accessible lesion, a CT scan of chest/abdomen/pelvis, and immunohistochemical testing. Additionally, the expert panel agreed on the need of an ideal diagnostic lead time for CUP patients. There was no full consensus on the place in diagnostic workup of symptom-guided MRI or ultrasound, a PET/CT scan, targeted gene panels, immunohistochemical markers, and whole genome sequencing., Interpretation: Consensus was reached on the contents of the first diagnostic layer of a multi-layer CUP classification. This is a first step towards full consensus on CUP diagnostics, that should also include supplementary and advanced diagnostics., Competing Interests: Conflict of interest statements The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Citrus greening, also known as Huanglongbing (HLB), is caused by the unculturable bacterium Candidatus Liberibacter spp. (e.g., C Las), and has caused a devastating decline in citrus production in many areas of the world. As of yet, there are no definitive treatments for controlling the disease. Antimicrobial peptides (AMPs) that have the potential to block secretion-dependent effector proteins at the outer-membrane domains were screened in silico. Predictions of drug-receptor interactions were built using multiple in silico techniques, including molecular docking analysis, molecular dynamics, molecular mechanics generalized Born surface area analysis, and principal component analysis. The efflux pump TolC of the Type 1 secretion system interacted with natural bacteriocin plantaricin JLA-9, blocking the β barrel. The trajectory-based principal component analysis revealed the possible binding mechanism of the peptides. Furthermore, in vitro assays using two closely related culturable surrogates of C Las ( Liberibacter crescens and Rhizobium spp.) showed that Plantaricin JLA-9 and two other screened AMPs inhibited bacterial growth and caused mortality. The findings contribute to designing effective therapies to manage plant diseases associated with Candidatus Liberibacter spp.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kulshreshtha, Sharma, Padilla and Mandadi.)

Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens -based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., β-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes ( vir G and vir E), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dominguez, Padilla and Mandadi.)

Sugarcane ( Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein ( ShGPCR1 ) from sugarcane and energy cane ( Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca 2+ ) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca 2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT , DEHYDRIN , DROUGHT RESPONSIVE 4 , GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3 , SALT OVERLY SENSITIVE 1 , VACUOLAR Na+/H+ ANTIPORTER 1 , NAM / ATAF1/2 / CUC2 , COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3 . We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ramasamy, Damaj, Vargas-Bautista, Mora, Liu, Padilla, Irigoyen, Saini, Sahoo, DaSilva and Mandadi.)

The green peach aphid (Myzus persicae) is a phloem-feeding insect that causes economic damage on a wide array of crops. Using a luminol-based assay, a superoxide-responsive reporter gene (Zat12::luciferase), and a probe specific to hydrogen peroxide (HyPer), we demonstrated that this aphid induces accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. Similar to the apoplastic oxidative burst induced by pathogens, this response to aphids was rapid and transient, with two peaks occurring within 1 and 4 hr after infestation. Aphid infestation also induced an oxidative response in the cytosol and peroxisomes, as measured using a redox-sensitive variant of green fluorescent protein (roGFP2). This intracellular response began within minutes of infestation but persisted 20 hr or more after inoculation, and the response of the peroxisomes appeared stronger than the response in the cytosol. Our results suggest that the oxidative response to aphids involves both apoplastic and intracellular sources of ROS, including ROS generation in the peroxisomes, and these different sources of ROS may potentially differ in their impacts on host suitability for aphids., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Sugarcane and energy cane ( Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg -1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 ( Ubi ) promoter. Co-expression of recombinant GNA from stacked multiple promoters ( pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus ) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg -1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg -1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg -1 tissue). Purification by mannose-agarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications., (Copyright © 2020 Padilla, Damaj, Yang, Molina, Berquist, White, Solís-Gracia, Da Silva and Mandadi.)

The conversion of oleic acid (C18:1) to linoleic acid (C18:2) in the endoplasmic reticulum is critical to the accumulation of polyunsaturated fatty acids in seeds and other tissues, and this reaction is catalyzed by a Δ12-desaturase, FATTY ACID DESATURASE2 (FAD2). Here, we report that the tomato ( Solanum lycopersicum ) genome harbors two genes, SlFAD2-1 and SlFAD2-2 , which encode proteins with in vitro Δ12-desaturase activity. In addition, tomato has seven divergent FAD2 members that lack Δ12-desaturase activity and differ from canonical FAD2 enzymes at multiple amino acid positions important to enzyme function. Whereas SlFAD2-1 and SlFAD2-2 are downregulated by biotic stress, the majority of divergent FAD2 genes in tomato are upregulated by one or more stresses. In particular, SlFAD2-7 is induced by the potato aphid ( Macrosiphum euphorbiae ) and has elevated constitutive expression levels in suppressor of prosystemin-mediated responses2 ( spr2 ), a tomato mutant with enhanced aphid resistance and altered fatty acid profiles. Virus-induced gene silencing of SlFAD2-7 in spr2 results in significant increases in aphid population growth, indicating that a divergent FAD2 gene contributes to aphid resistance in this genotype. Thus, the FAD2 gene family in tomato is important both to primary fatty acid metabolism and to responses to biotic stress., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Referees and assistant referees are submitted to high physical stress during matches. Pressure to make decisions in front of large crowds is another potential stressor. These two stressors can impair attention executive control, depending on physical fitness and individual vulnerability or resilience to situational pressure. Error percentage for referees and assistants may reach around 14% during a soccer match. Although previous studies have suggested that soccer referees and assistants should take cognitive assessments, they are only required by Fédération Internationale de Football Association (FIFA) to demonstrate knowledge of the rules and pass annually in a fitness test (FIFA-Test). This study aimed to assess attention performance in referees and assistants before and after the mandatory FIFA-Test. It is hypothesized that the high physical demands associated with the pressure to pass the FIFA-Test would interfere with attention performance. The sample included 33 referees and 20 assistants. The Continuous Visual Attention Test (CVAT) consisted of a 15-min Go/No-go task. Performance in the CVAT is based on four variables: omission and commission errors, reaction time, and variability of reaction time (VRT). Failure in the CVAT was defined by a performance below the 5th percentile of the age- and sex-matched normative data in at least one variable of the CVAT. Before the FIFA-Test all participants performed the CVAT. The second CVAT began 3-7 min directly following completion of the FIFA-test. Considering only the officials who passed both the FIFA-Test and the first CVAT (19 referees and 15 assistants), 44% (9 referees and 6 assistants) exhibited a performance decline in the second CVAT. A significant increase in VRT was found after the high intensity exercise. As increase in VRT is thought to reflect executive dysfunctions and lapses of attention, we concluded that physical fitness alone may not be enough to help officials cope with the physical and contextual stresses associated with the FIFA-Test. These data suggest that over 35% of soccer referees and their assistants who were considered physically able to referee matches may not be mentally prepared for the attentional demands of refereeing soccer matches.

Background: Obstructive sleep apnea (OSA) is characterized by apnea-hypopnea during sleep. Overnight polysomnography (PSG) is usually used to detect the frequency of apneic and hypopneic events. Attention and executive deficits are commonly reported in OSA patients. Previous investigations suggested that cognitive impairments were dependent on attention deficits. However, attention is not a unitary domain and consists of different subdomains such as alertness, sustained attention, focused attention, and executive attention (impulsivity/hyperactivity). Little is known about the attention subdomains affected in OSA. Attention is commonly assessed using continuous performance tests, such as the continuous visual attention test (CVAT). Distinct variables can be derived from the CVAT. Each CVAT variable is associated with a specific attention subdomain. Objective: This study aimed to examine the variables of the CVAT that are affected by OSA and to identify the most reliable CVAT variable that distinguishes OSA from controls via discriminant analysis. Method: Patients scheduled to perform a PSG were invited to participate in this study. Immediately before the PSG, they performed the CVAT. Based on the PSG results, 27 treatment-naïve OSA patients were sampled. The same number of healthy controls were selected to match the two groups by age and gender. Five CVAT variables were examined: commission errors, omission errors, reaction time (RT), variability of reaction time (VRT), and coefficient of variability (VRT/RT). Results: ANCOVAs indicated that RT and VRT were affected by OSA. No difference in accuracy (omission and commission errors) was observed between healthy controls and OSA patients. When the VRT measurements were corrected for their respective RT values (VRT/RT), the mean difference on this coefficient did not reach significance. The discriminant analysis indicated that the two groups could be best differentiated by the RT variable. Conclusions: Attention problems, commonly observed in OSA patients, may reflect a primary problem on the alertness subdomain. The CVAT was able to detect the primary (alertness-RT parameter) and the secondary deficits (sustained attention-VRT parameter) associated with OSA. As there is no learning effect in the condition of retests, the CVAT can be used to assess the cognitive recovery in OSA patients during treatment.

Sugarcane and energycane ( Saccharum spp. hybrids) are prominent sources of sugar, ethanol, as well as high-value bioproducts globally. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, limited germplasm resources, long breeding cycle, as well as recalcitrance to genetic transformation. Here, we present a biolistic-based transformation and bioreactor-based micro-propagation system that has been utilized successfully to transform twelve elite cane genotypes, yielding transformation efficiencies of up to 39%. The system relies on the generation of embryogenic callus from sugarcane and energycane apical shoot tissue, followed by DNA bombardment of embryogenic leaf roll discs (approximately one week) or calli (approximately 4 weeks). We present optimal criteria and practices for selection and regeneration of independent transgenic lines, molecular characterization, as well as a bioreactor-based micro-propagation technique, which can aid in rapid multiplication and analysis of transgenic lines. The cane transformation and micro-propagation system described here, although built on our previous protocols, has significantly accelerated the process of producing and multiplying transgenic material, and it is applicable to other varieties. The system is highly reproducible and has been successfully used to engineer multiple commercial sugarcane and energycane varieties. It will benefit worldwide researchers interested in genomics and genetics of sugarcane photosynthesis, cell wall, and bioenergy related traits.

Background: Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications., Results: We developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection., Conclusion: To facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.