Treatment-naïve people living with HIV aged 50 years or older in Beijing, China, 2010-2020: joinpoint regression model analysis of surveillance data.

INTRODUCTION: As they age, people living with HIV (PLWH) must face new challenges, such as accelerated ageing and higher rates of comorbidities. This study described the characteristics of HIV acquisition among treatment-naïve PLWH aged ≥50 years and <50 years in Beijing from 2010 to 2020, exploring associated risk factors for comorbidities. METHODS: In this cross-sectional study, differences in HIV-related and non-HIV-related characteristics were compared using the t-test, Mann-Whitney U test and chi-square test. Temporal trend data were analysed via joinpoint regression. A multivariate logistic regression model was conducted to analyse the associated factors with PLWH having one or more comorbidities. RESULTS: The proportion of PLWH aged ≥50 years has significantly increased since 2013, with a corresponding increase in homosexual transmission in this age group over the past decade. The proportion of individuals with CD4 counts <200 cells/µl significantly decreased from 2010 to 2013 among PLWH aged ≥50 years and from 2010 to 2014 among those aged <50 years. Delayed initiation of antiretroviral therapy (ART) improved for both age groups over the course of the decade, especially from 2014 to 2020. Compared to PLWH aged <50 years, those aged ≥50 years had a higher proportion of CD4 counts <200 cells/µl, higher levels of plasma HIV RNA load and a higher prevalence of non-HIV-related risk factors. Multivariate analysis revealed that PLWH aged ≥50, male, not single, transmission through heterosexual contact or drug injection, WHO Stage IV, coinfection with hepatitis B virus/hepatitis C virus and CD4 counts <200 cells/µl at the initiation of ART were associated with higher risk of the presence of an HIV comorbidity. CONCLUSIONS: Due to the persistent burden of HIV-related characteristics or symptoms and the increasing prevalence of coexisting comorbidities among treatment-naïve PLWH aged ≥50 years, physicians should provide the highest-quality screening, prevention, treatment and management of coexisting comorbidities, adopting a multidisciplinary approach.

Internet use, cardiometabolic multimorbidity, and death in older adults: a multi-cohort study spanning developing and developed countries.

BACKGROUND: Internet use is a double-edged sword for older adults' health. Whether internet use can prevent cardiometabolic diseases and death in older adults remains controversial. METHODS: Four cohorts across China, Mexico, the United States, and Europe were utilized. Internet use was defined using similar questions. Cardiometabolic diseases included diabetes, heart diseases, and stroke, with 2 or more denoting cardiometabolic multimorbidity. Depressive symptoms were assessed using the Center for Epidemiological Studies Depression Scale and Europe-depression scale. The competing risk analysis based on subdistribution hazard regression, random-effects meta-analysis, and mediation analysis were utilized. RESULTS: A total of 104,422 older adults aged 50 or older were included. Internet users (vs. digital exclusion) were at lower risks of diabetes, stroke, and death, with pooled sHRs (95% CIs) of 0.83 (0.74-0.93), 0.81 (0.71-0.92), and 0.67 (0.52-0.86), respectively, which remained significant in sensitivity analyses. The inverse associations of internet use with new-onset cardiometabolic diseases and death were progressively significant in Mexico, China, the United States, and Europe. For instance, older internet users in Europe were at 14-30% lower cardiometabolic risks and 40% lower risk of death. These associations were partially mediated by reduced depressive symptoms and were more pronounced in those with high socioeconomic status and women. Furthermore, patients with prior cardiometabolic conditions were at about 30% lower risk of death if they used the internet, which was also mediated by reduced depressive symptoms. However, certain cardiometabolic hazards of internet use in those aged < 65 years, with low socioeconomic status, men, and single ones were also observed. CONCLUSION: Enhancing internet usage in older adults can reduce depressive symptoms and thus reduce the risks of cardiometabolic diseases and death. The balance of internet use, socioeconomic status, and health literacy should be considered when popularizing the internet in older adults.

Do visual and step height factors cause imbalance during bipedal and unipedal stances? A plantar pressure perspective.

Objective: The plantar pressure analysis technique was used to explore the static balance ability and stability of healthy adult males under the influence of visual and step height factors during bipedal and unipedal stances. Methods: Thirty healthy adult males volunteered for the study. Experiments used the F-scan plantar pressure analysis insoles to carry out with eyes open (EO) and eyes closed (EC) at four different step heights. The plantar pressure data were recorded for 10 s and pre-processed to derive kinematic and dynamic parameters. Results: For unipedal stance, most of kinematic parameters of the subjects' right and left feet were significantly greater when the eyes were closed compared to the EO condition and increased with step height. The differences in toe load between right and left feet, open and closed eyes were extremely statistically significant (p < 0.001). The differences in midfoot load between the EO and EC conditions were statistically significant (p = 0.024) and extremely statistically significant between the right and left feet (p < 0.001). The difference in rearfoot load between EO and EC conditions was extremely statistically significant (p < 0.001) and statistically significant (p = 0.002) between the right and left feet. For bipedal stance, most of kinematic parameters of the subjects' EO and EC conditions were statistically significant between the right and left feet and increased with step height. The overall load's difference between EO and EC states was statistically significant (p = 0.003) for both feet. The overall load's difference between the right and left feet was extremely statistically significant (p < 0.001) in the EC state. The differences between the right and left feet of the forefoot and rearfoot load with EO and EC suggested that the right foot had a smaller forefoot load, but a larger rearfoot load than the left foot (p < 0.001). The differences between the forefoot and rearfoot load of the subjects' both feet with EO and EC were extremely statistically significant (p < 0.001). Conclusion: Both visual input and step height factors, even the dominant foot, act on kinematic and dynamic parameters that affect the maintenance of static balance ability.

Prevalence and Trends of Birth Defects - Five Counties, Shanxi Province, China, 2003-2022.

What is already known about this topic?: The Shanxi Province, located in northern China, holds the highest prevalence of birth defects (BDs) across the country. Following the implementation of a nationwide folic acid supplementation program in 2009, a significant reduction of 53.89% in the prevalence of neural tube defects (NTDs) was observed in Shanxi from 2012 to 2017. However, despite this decrease, the prevalence rate for congenital heart defects (CHDs) in 2017 was over four times that of the 2012 rate. Since 2014, CHDs have emerged as the most predominant BD in Shanxi. What is added by this report?: The present study has identified a marked reduction in the prevalence of both total BDs and NTDs in five counties within Shanxi over the past two decades. As of 2017-2022, NTDs continue to be the most prevalent BDs recorded in this region. Contrarily, there has been a noteworthy increase in the prevalence of CHDs, ranking them among the top five most common BDs in the region between 2017 and 2022, though their rate remains below the national average. Additionally, the proportion of external anomalies remains high. Nevertheless, due to constrained access to primary healthcare services and diagnostic facilities, the early detection rate for internal anomalies, particularly CHDs, may be underestimated in the region. What are the implications for public health practice?: The results of this study underscore the necessity for augmented efforts in promoting folic acid supplementation as a preventive measure for NTDs. Moreover, improvements in the distribution of medical resources within this region are recommended, particularly the introduction and enforcement of local training programs aimed at enhancing CHD screening and diagnostic processes in these respective counties.

Trends in the Prevalence of Births with Chromosomal Abnormalities - Haidian District, Beijing Municipality, China, 2013-2022.

What is already known about this topic?: The primary causes of early miscarriage and stillbirth are chromosomal abnormalities (CAs) whose prevalence has been observed to increase in recent years. What is added by this report?: According to data received from a hospital-based birth-defect surveillance system in the Haidian District, Beijing, there was a significant increase in the prevalence of CAs along with most subtypes from 2013 to 2022. This noted increase in the reported prevalence is potentially attributable to factors such as a rise in maternal age, alongside the enhanced detection efficacy resulting from the utilization of noninvasive prenatal testing. What are the implications for public health practice?: The escalating prevalence of sex CAs and other previously rare CAs pose novel challenges for genetic counseling and healthcare practitioners. These professionals are tasked with the accurate evaluation and interpretation of detection data, which must then be conveyed appropriately to patients. Furthermore, it is imperative to intensify health education efforts to assist women in making informed treatment decisions, considering the diverse prognoses associated with different CAs.

Temporal Trends of Clinical Characteristics and Treatments in People Living with HIV at the Initiation of Antiretroviral Therapy - Beijing Municipality, China, 2010-2020.

What is already known about this topic?: Antiretroviral therapy (ART) eligibility criteria and treatment regimens were updated in national guidelines. However, whether treatment was timely and followed guidelines was under-assessed. What is added by this report?: Among 22,591 people living with human immunodeficiency virus (PLWH) who initiated ART in Beijing between 2010 and 2020, the time from diagnosis to initiating ART decreased, the clinical condition of PLWH improved, and ART regimens changed in accordance with guidelines. What are the implications for public health practice?: Over the past decade, improvements in clinical status have been observed among PLWH; however, a proportion of PLWH remain who started ART late. Early linkage to human immunodeficiency virus (HIV) care should be further improved.

Effect of a blended learning design in an undergraduate nursing electrocardiogram course: A quasi-experimental study.

AIM: The study aimed to explore learning achievement and students' satisfaction with a blended learning (BL) electrocardiogram (ECG) education programme in undergraduate nursing education. DESIGN: This was a quasi-experimental post-test design. METHODS: The study was conducted during semester one of the academic year 2020/2021. Participants were divided into two groups: the BL group and face-to-face (FTF) group. The BL group received the newly designed BL programme. The FTF group received the traditional learning methods. Post-test measures of the study variables such as knowledge, study time and satisfaction were conducted. Chi-square (χ2 ) test was used to evaluate categorical variables. The Mann-Whitney test was used to analyse continuous variables. RESULTS: Participants in BL group had significantly higher scores in ECG interpretation and total score. No significant differences in foundational knowledge between the two groups were found. Study time was significantly longer in the BL group. Regarding satisfaction, significant differences were found in structure rationality and promotion of self-learning.

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review.

Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.

Pectin: a critical component in cell-wall-mediated immunity.

Contrary to the classical cell-wall model, pectin metabolism may play a crucial role in cell-wall integrity, detection of plant pathogens, and defense response. Here we discuss the evidence and propose a new metabolic and regulatory model linking pectin to cell-wall-mediated immunity, including ripening-associated disease susceptibility in the tomato.

A fungal-modified material with high uranium (VI) adsorption capacity and strong anti-interference ability.

With open-chain polyether as the bridge chain, a new fungal-modified material with diamidoxime groups was prepared by a series of uncomplex synthesis reaction. The orthogonal experiment obtained its optimized adsorption conditions as follows: the initial pH value of 6.5, the initial uranyl concentration of 40 mg L-1, the contact time of 130 min, and the a solid-liquid ratio of 25 mg L-1. The maximum adsorption capacity of target material was 446.20 mg g-1, and it was much greater than that of the similar monoamidoxime material (295.48 mg g-1). The linear Langmuir (R2 = 0.9856) isotherm models and the linear pseudo-second-order kinetic model (R2 = 0.9931) fit the experimental data of uranium (VI) adsorption better, indicating the adsorption mechanism should mainly be the monolayer adsorption and chemical process. In addition, the relevant experiments exhibited the prepared material had the good reusability, which reached 84.25% of the maximum capacity after five cycles, and the excellent anti-interference performance. The above features suggest the modified fungus material will have the good application prospect in the future.

The Effects of Nine Compounds on Aldehyde-Oxidase-Related Genes in Bactrocera dorsalis (Hendel).

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) (B. dorsalis) is an important agricultural, major invasive, and quarantine pest that can cause significant damage to the economic value of the fruit and vegetable industry. Male bait is one of the most effective methods of surveying, monitoring, and controlling B. dorsalis. In our study, we constructed cDNA libraries using total RNA extracted independently from the antennae, mouthparts, and thoracic legs of male and female adults and the ovipositors of female adults and screened out four aldehyde-oxidase-related genes (AOX-related), C58800, C66700, C67485, and C67698. Molecular docking predictions showed that eight compounds, including 3,4-dimethoxycinnamyl alcohol, 3,4-dimethoxy-cinnamaldehyde, deet, ethyl N-acetyl-N-butyl-ß-alaninate, n-butyl butyrate, n-butyl butyrate, ethyl butyrate, methyl eugenol, and ethyl acetate, could combine with proteins encoded by the four B. dorsalis AOX-related genes. Furthermore, QPCR was performed to confirm that four compounds, including 3,4-dimethoxy cinnamic aldehyde, butyl levulinic acid ethyl ester (mosquito repellent), butyl butyrate, and methyl eugenol, induced significant changes in the AOX-related genes of B. dorsalis. These results provide useful information and guidance for the batch screening of potentially useful compounds and the search for effective attractants of B. dorsalis.

Genome-wide identification of the pectin methylesterase inhibitor genes in Brassica napus and expression analysis of selected members.

Pectin methylesterase inhibitors (PMEIs) modulate the status of pectin methylesterification by inhibiting the activity of pectin methylesterase (PME). Recent advances indicate PMEIs play an important role in regulating plant cell wall properties and defense responses. In this study, a genome-wide analysis of PMEI gene family in Brassica napus (B. napus) was conducted and the expression patterns of PMEI genes in response to Sclerotinia sclerotiorum (S. sclerotiorum) was investigated. A total of 190 PMEI proteins were identified from the genome of B. napus. Chromosomal location, gene structure and properties of the PMEI family were analyzed, and these features were compared with Arabidopsis thaliana (A. thaliana). A total of 123 syntenic ortholog pairs were detected from BnPMEI family by synteny analysis. Results showed the expansion of BnPMEI genes was likely predominately from whole-genome duplication (WGD) or segmental duplications. Multiple cis-elements related to plant growth and development, environmental stress responses, hormone responses were detected in the promoters of BnPMEI genes, implying they were regulated by both internal and external factors. Furthermore, expression analysis of transcriptome data combined with quantitative RT-PCR (qRT-PCR) validation identified several candidates that were strongly responsive to S. sclerotiorum infection. These BnPMEI genes are candidates for manipulation to breed novel and improved genotypes that are more resistant to sclerotinia stem rot (SSR). Extensive interactions were detected among 30 BnPMEI proteins, forming complex protein-protein interaction networks. Besides, 48 BnPMEIs showed interactions with other proteins including a range of cell wall structure-related enzymes. This study provides new insights into the evolution and function of PMEIs in B. napus and lays a foundation for breeding novel genotypes for crop improvement.

Detection of arrhythmia in 12-lead varied-length ECG using multi-branch signal fusion network.

Objective.Automatic detection of arrhythmia based on electrocardiogram (ECG) plays a critical role in early prevention and diagnosis of cardiovascular diseases. With the increase in widely available digital ECG data and the development of deep learning, multi-class arrhythmia classification based on automatic feature extraction of ECG has become increasingly attractive. However, the majority of studies cannot accept varied-length ECG signals and have limited performance in detecting multi-class arrhythmias.Approach.In this study, we propose a multi-branch signal fusion network (MBSF-Net) for multi-label classification of arrhythmia in 12-lead varied-length ECG. Our model utilizes the complementary power between different structures, which include Inception with depthwise separable convolution (DWS-Inception), spatial pyramid pooling (SPP) Layer, and multi-scale fusion Resnet (MSF-Resnet). The proposed method can extract features from each lead of 12-lead ECG recordings separately and then effectively fuse the features of each lead by integrating multiple convolution kernels with different receptive fields, which can achieve the information of complementation between different angles of the ECG signal. In particular, our model can accept 12-lead ECG signals of arbitrary length.Main results.The experimental results show that our model achieved an overall classification F1 score of 83.8% in the 12-lead ECG data of CPSC-2018. In addition, the F1 score of the MBSF-Net performed best among the MBF-Nets which are removed the SPP layer from MBSF-Net. In comparison with the latest ECG classification algorithms, the proposed model can be applied in varied-length signals and has an excellent performance, which not only can fully retain the integrity of the original signals, but also eliminates the cropping/padding signal beforehand when dealing with varied-length signal database.Significance.MBSF-Net provides an end-to-end multi-label classification model with outperfom performance, which allows detection of disease in varied-length signals without any additional cropping/padding. Moreover, our research is beneficial to the development of computer-aided diagnosis.

Molecular and biochemical basis of softening in tomato.

We review the latest information related to the control of fruit softening in tomato and where relevant compare the events with texture changes in other fleshy fruits. Development of an acceptable texture is essential for consumer acceptance, but also determines the postharvest life of fruits. The complex modern supply chain demands effective control of shelf life in tomato without compromising colour and flavour.The control of softening and ripening in tomato (Solanum lycopersicum) are discussed with respect to hormonal cues, epigenetic regulation and transcriptional modulation of cell wall structure-related genes. In the last section we focus on the biochemical changes closely linked with softening in tomato including key aspects of cell wall disassembly. Some important elements of the softening process have been identified, but our understanding of the mechanistic basis of the process in tomato and other fruits remains incomplete, especially the precise relationship between changes in cell wall structure and alterations in fruit texture.

Assessing Ecological Vulnerability under Climate Change and Anthropogenic Influence in the Yangtze River Estuarine Island-Chongming Island, China.

Understanding and assessing ecological vulnerability for estuarine islands are important for maintaining estuarine island ecosystem services and its sustainable development. However, due to its complex fresh water-sea-land interaction mechanism and multiple stressors from both climate change and anthropogenic influence, a comprehensive evaluation of ecological vulnerability for estuarine islands has been limited. Therefore, taking the typical estuary island of Chongming Island as an example, we developed a comprehensive evaluation system of ecological vulnerability for an estuarine island ecosystem based on the pressure-state-response (PSR) conceptual model, and explored the spatial and temporal distribution of ecological vulnerability in 2005 and 2015. The results indicated that the main pressures of Chongming Island from saltwater intrusion intensity and land use intensity were mainly distributed in northern coastal areas and eastern areas of wetland; the ecological vulnerability index (EV) of Chongming Island showed a slight decrease from 2005 to 2015; and three categories of towns based on ecological vulnerability assessment for an eco-island planning and environmental management were identified. Our study provides an effective evaluation system of ecological vulnerability for estuarine islands, which could be helpful for planners and decision makers in improving eco-island planning and environmental management.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane.

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane (x = 10-13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I (MgCh) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

Host susceptibility factors render ripe tomato fruit vulnerable to fungal disease despite active immune responses.

The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical processes associated with defense and susceptibility in tomato (Solanum lycopersicum) fruit. Using unripe and ripe fruit inoculated with three fungal pathogens, we identified common pathogen responses reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. We established that the magnitude and diversity of defense responses do not significantly impact the interaction outcome, as susceptible ripe fruit mounted a strong immune response to pathogen infection. Then, to distinguish features of ripening that may be responsible for susceptibility, we utilized non-ripening tomato mutants that displayed different susceptibility patterns to fungal infection. Based on transcriptional and hormone profiling, susceptible tomato genotypes had losses in the maintenance of cellular redox homeostasis, while jasmonic acid accumulation and signaling coincided with defense activation in resistant fruit. We identified and validated a susceptibility factor, pectate lyase (PL). CRISPR-based knockouts of PL, but not polygalacturonase (PG2a), reduced susceptibility of ripe fruit by >50%. This study suggests that targeting specific genes that promote susceptibility is a viable strategy to improve the resistance of tomato fruit against fungal disease.

The Microstructural Difference and Its Influence on the Ballistic Impact Behavior of a Near ß-Type Ti5.1Al2.5Cr0.5Fe4.5Mo1.1Sn1.8Zr2.9Zn Titanium Alloy.

In this work, a near ß-type Ti5.1Al2.5Cr0.5Fe4.5Mo1.1Sn1.8Zr2.9Zn alloy was hot-rolled at the temperature of 800-880 °C with a thickness reduction of 87.5% and then heat-treated with the strategy of 880 °C/1 h/air cooling (AC) + 650 °C/3 h/AC. The microstructure difference between the hot-rolled and heat-treated titanium alloys and its influence on the ballistic impact behavior of the hot-rolled and heat-treated titanium alloys were analyzed. The microstructural investigation revealed that the average size of the acicular secondary α phase (αs) dropped from 75 to 42 nm, and the corresponding amount of this phase increased significantly after heat treatment. In addition, the dislocation density of the α and ß phases decreased from 0.3340 × 1015/m2 and 4.6746 × 1015/m2 for the hot-rolled titanium alloy plate to 0.2806 × 1015/m2 and 1.8050 × 1015/m2 for the heat-treated one, respectively. The high strength of the heat-treated titanium alloy was maintained, owing to the positive contribution of the acicular secondary α phase. Furthermore, the critical fracture strain increased sharply from 19.9% for the hot-rolled titanium alloy plate to 23.1% for the heat-treated one, thereby overcoming (to some extent) the constraint of the strength-ductility trade-off. This is mainly attributed to the fact that the dislocation density and the difference between the dislocation densities of the α and ß phases decreased substantially, and deformation localization was effectively suppressed after heat treatment. Damage to the hot-rolled and heat-treated titanium alloy plates after the penetration of a 7.62 mm ordinary steel core projectile at a distance of 100 m was assessed via industrial computer tomography and microstructure observation. The results revealed that a large crack (volume: 2.55 mm3) occurred on the rear face and propagated toward the interior of the hot-rolled titanium alloy plate. The crack tip was connected to a long adiabatic shear band with a depth of 3 mm along the thickness direction. However, good integrity of the heat-treated titanium alloy plate was maintained, owing to its excellent deformation capability. Ultimately, the failure mechanism of the hot-rolled and heat-treated titanium alloy plates was revealed by determining the crack-forming reasons in these materials.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

KEY MESSAGE: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.