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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.)

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.