OsCPK12 phosphorylates OsCATA and OsCATC to regulate H2O2 homeostasis and improve oxidative stress tolerance in rice.

Calcium-dependent protein kinases (CPKs), the best-characterized calcium sensors in plants, regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. However, how CPKs regulate the antioxidant defense system remains largely unknown. We previously found that impaired function of OsCPK12 leads to oxidative stress in rice, with more H2O2, lower catalase (CAT) activity, and lower yield. Here, we explored the roles of OsCPK12 in oxidative stress tolerance in rice. Our results show that OsCPK12 interacts with and phosphorylates OsCATA and OsCATC at Ser11. Knockout of either OsCATA or OsCATC leads to an oxidative stress phenotype accompanied by higher accumulation of H2O2. Overexpression of the phosphomimetic proteins OsCATAS11D and OsCATCS11D in oscpk12-cr reduced the level of H2O2 accumulation. Moreover, OsCATAS11D and OsCATCS11D showed enhanced catalase activity in vivo and in vitro. OsCPK12-overexpressing plants exhibited higher CAT activity as well as higher tolerance to oxidative stress. Our findings demonstrate that OsCPK12 affects CAT enzyme activity by phosphorylating OsCATA and OsCATC at Ser11 to regulate H2O2 homeostasis, thereby mediating oxidative stress tolerance in rice.

Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Sparse Phenotyping and Haplotype-Based Models for Genomic Prediction in Rice.

The multi-environment genomic selection enables plant breeders to select varieties resilient to diverse environments or particularly adapted to specific environments, which holds a great potential to be used in rice breeding. To realize the multi-environment genomic selection, a robust training set with multi-environment phenotypic data is of necessity. Considering the huge potential of genomic prediction enhanced sparse phenotyping on the cost saving of multi-environment trials (MET), the establishment of a multi-environment training set could also benefit from it. Optimizing the genomic prediction methods is also crucial to enhance the multi-environment genomic selection. Using haplotype-based genomic prediction models is able to capture local epistatic effects which could be conserved and accumulated across generations much like additive effects thereby benefitting breeding. However, previous studies often used fixed length haplotypes composed by a few adjacent molecular markers disregarding the linkage disequilibrium (LD) which is of essential role in determining the haplotype length. In our study, based on three rice populations with different sizes and compositions, we investigated the usefulness and effectiveness of multi-environment training sets with varying phenotyping intensities and different haplotype-based genomic prediction models based on LD-derived haplotype blocks for two agronomic traits, i.e., days to heading (DTH) and plant height (PH). Results showed that phenotyping merely 30% records in multi-environment training set is able to provide a comparable prediction accuracy to high phenotyping intensities; the local epistatic effects are much likely existent in DTH; dividing the LD-derived haplotype blocks into small segments with two or three single nucleotide polymorphisms (SNPs) helps to maintain the predictive ability of haplotype-based models in large populations; modelling the covariances between environments improves genomic prediction accuracy. Our study provides means to improve the efficiency of multi-environment genomic selection in rice.

Photoperiod and gravistimulation-associated Tiller Angle Control 1 modulates dynamic changes in rice plant architecture.

KEY MESSAGE: TAC1 is involved in photoperiodic and gravitropic responses to modulate rice dynamic plant architecture likely by affecting endogenous auxin distribution, which could explain TAC1 widespread distribution in indica rice. Plants experience a changing environment throughout their growth, which requires dynamic adjustments of plant architecture in response to these environmental cues. Our previous study demonstrated that Tiller Angle Control 1 (TAC1) modulates dynamic changes in plant architecture in rice; however, the underlying regulatory mechanisms remain largely unknown. In this study, we show that TAC1 regulates plant architecture in an expression dose-dependent manner, is highly expressed in stems, and exhibits dynamic expression in tiller bases during the growth period. Photoperiodic treatments revealed that TAC1 expression shows circadian rhythm and is more abundant during the dark period than during the light period and under short-day conditions than under long-day conditions. Therefore, it contributes to dynamic plant architecture under long-day conditions and loose plant architecture under short-day conditions. Gravity treatments showed that TAC1 is induced by gravistimulation and negatively regulates shoot gravitropism, likely by affecting auxin distribution. Notably, the tested indica rice containing TAC1 displayed dynamic plant architecture under natural long-day conditions, likely explaining the widespread distribution of TAC1 in indica rice. Our results provide new insights into TAC1-mediated regulatory mechanisms for dynamic changes in rice plant architecture.

Genomic prediction of rice mesocotyl length indicative of directing seeding suitability using a half-sib hybrid population.

Direct seeding has been widely adopted as an economical and labor-saving technique in rice production, though problems such as low seedling emergence rate, emergence irregularity and poor lodging resistance are existing. These problems are currently partially overcome by increasing seeding rate, however it is not acceptable for hybrid rice due to the high seed cost. Improving direct seeding by breeding is seen as the ultimate solution to these problems. For hybrid breeding, identifying superior hybrids among a massive number of hybrids from crossings between male and female parental populations by phenotypic evaluation is tedious and costly. Contrastingly, genomic selection/prediction (GS/GP) could efficiently detect the superior hybrids capitalizing on genomic data, which holds a great potential in plant hybrids breeding. In this study, we utilized 402 rice inbred varieties and 401 hybrids to investigate the effectiveness of GS on rice mesocotyl length, a representative indicative trait of direct seeding suitability. Several GP methods and training set designs were studied to seek the optimal scenario of hybrid prediction. It was shown that using half-sib hybrids as training set with the phenotypes of all parental lines being fitted as a covariate could optimally predict mesocotyl length. Partitioning the molecular markers into trait-associated and -unassociated groups based on genome-wide association study using all parental lines and hybrids could further improve the prediction accuracy. This study indicates that GS could be an effective and efficient method for hybrid breeding for rice direct seeding.

Mutation of DEFECTIVE EMBRYO SAC1 results in a low seed-setting rate in rice by regulating embryo sac development.

The seed-setting rate has a significant effect on grain yield in rice (Oryza sativa L.). Embryo sac development is essential for seed setting; however, the molecular mechanism underlying this process remains unclear. Here, we isolated defective embryo sac1 (des1), a rice mutant with a low seed-setting rate. Cytological examination showed degenerated embryo sacs and reduced fertilization capacity in des1. Map-based cloning revealed a nonsense mutation in OsDES1, a gene that encodes a putative nuclear envelope membrane protein (NEMP)-domain-containing protein that is preferentially expressed in pistils. The OsDES1 mutation disrupts the normal formation of functional megaspores, which ultimately results in a degenerated embryo sac in des1. Reciprocal crosses showed that fertilization is abnormal and that the female reproductive organ is defective in des1. OsDES1 interacts with LONELY GUY (LOG), a cytokinin-activating enzyme that acts in the final step of cytokinin synthesis; mutation of LOG led to defective female reproductive organ development. These results demonstrate that OsDES1 functions in determining the rice seed-setting rate by regulating embryo sac development and fertilization. Our study sheds light on the function of NEMP-type proteins in rice reproductive development.

OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice.

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/ß Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1-1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

Evaluation and validation of an RT-PCR assay for specific detection of monkeypox virus (MPXV).

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus within the Poxviridae family. MPXV is endemic to Central and West Africa. However, the world is currently witnessing an international outbreak with no clear epidemiological links to travel or animal exposure and with ever-increasing numbers of reported cases worldwide. Here, we evaluated and validated a new, sensitive, and specific real-time PCR-assay for MPXV diagnosis in humans and compare the performance of this novel assay against a Food & Drug Administration-cleared pan-Orthopox RT-PCR assay. We determined specificity, sensitivity, and analytic performance of the PKamp™ Monkeypox Virus RT-PCR assay targeting the viral F3L-gene. In addition, we further evaluated MPXV-PCR-positive specimens by viral culture, electron microscopy, and viral inactivation assays. The limit of detection was established at 7.2 genome copies/reaction, and MPXV was successfully identified in 20 clinical specimens with 100% correlation against the reference method with 100% sensitivity and specificity. Our results demonstrated the validity of this rapid, robust, and reliable RT-PCR assay for specific and accurate diagnosis of MPXV infection in human specimens collected both as dry swabs and in viral transport media. This assay has been approved by NYS Department of Health for clinical use.

The clock component OsLUX regulates rice heading through recruiting OsELF3-1 and OsELF4s to repress Hd1 and Ghd7.

INTRODUCTION: Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination. OBJECTIVES: In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s-OsELF3-1-OsLUX complex directly regulates flowering-related genes to mediate rice heading. METHODS: We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s-OsELF3-1-OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s-OsELF3-1-OsLUX) complex. RESULTS: The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s-OsELF3-1-OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element. CONCLUSION: Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s-OsELF3-1-OsLUX)-Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.

Mapping and Validation of qHD7b: Major Heading-Date QTL Functions Mainly under Long-Day Conditions.

Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow-up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short-day and two long-day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7-kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well-known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.

qHD5 encodes an AP2 factor that suppresses rice heading by down-regulating Ehd2 expression.

Heading date is crucial for rice reproduction and the geographical expansion of cultivation. We fine-mapped qHD5 and identified LOC_Os05g03040, a gene that encodes an AP2 transcription factor, as the candidate gene of qHD5 in our previous study. In this article, using two near-isogenic lines NIL(BG1) and NIL(XLJ), which were derived from the progeny of the cross between BigGrain1 (BG1) and Xiaolijing (XLJ), we verified that LOC_Os05g03040 represses heading date in rice through genetic complementation and CRISPR/Cas9 gene-editing experiments. Complementary results showed that qHD5 is a semi-dominant gene and that the qHD5XLJ and qHD5BG1 alleles are both functional. The homozygous mutant line generated from knocking out qHD5XLJ in NIL(XLJ) headed earlier than NIL(XLJ) under both short-day and long-day conditions. In addition, the homozygous mutant line of qHD5BG1 in NIL(BG1) also headed slightly earlier than NIL(BG1). All of these results show that qHD5 represses the heading date in rice. Transient expression showed that the qHD5 protein localizes to the nucleus. Transactivation activity assays showed that the C-terminus is the critical site that affects self-activation in qHD5XLJ. qRT-PCR analysis revealed that qHD5 represses flowering by down-regulating Ehd2. qHD5 may have been selected during indica rice domestication.

Deletion of Diterpenoid Biosynthetic Genes CYP76M7 and CYP76M8 Induces Cell Death and Enhances Bacterial Blight Resistance in Indica Rice '9311'.

Lesion mimic mutants (LMMs) are ideal materials for studying cell death and resistance mechanisms. Here, we identified and mapped a novel rice LMM, g380. The g380 exhibits a spontaneous hypersensitive response-like cell death phenotype accompanied by excessive accumulation of reactive oxygen species (ROS) and upregulated expression of pathogenesis-related genes, as well as enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). Using a map-based cloning strategy, a 184,916 bp deletion on chromosome 2 that overlaps with the diterpenoid biosynthetic gene cluster was identified in g380. Accordingly, the content of diterpenoids decreased in g380. In addition, lignin, one of the physical lines of plant defense, was increased in g380. RNA-seq analysis showed 590 significantly differentially expressed genes (DEG) between the wild-type 9311 and g380, 585 of which were upregulated in g380. Upregulated genes in g380 were mainly enriched in the monolignol biosynthesis branches of the phenylpropanoid biosynthesis pathway, the plant-pathogen interaction pathway and the phytoalexin-specialized diterpenoid biosynthesis pathway. Taken together, our results indicate that the diterpenoid biosynthetic gene cluster on chromosome 2 is involved in immune reprogramming, which in turn regulates cell death in rice.

STRIPE3, encoding a human dNTPase SAMHD1 homolog, regulates chloroplast development in rice.

Chloroplast is an important organelle for photosynthesis and numerous essential metabolic processes, thus ensuring plant fitness or survival. Although many genes involved in chloroplast development have been identified, mechanisms underlying such development are not fully understood. Here, we isolated and characterized the stripe3 (st3) mutant which exhibited white-striped leaves with reduced chlorophyll content and abnormal chloroplast development during the seedling stage, but gradually produced nearly normal green leaves as it developed. Map-based cloning and transgenic tests demonstrated that a splicing mutation in ST3, encoding a human deoxynucleoside triphosphate triphosphohydrolase (dNTPase) SAMHD1 homolog, was responsible for st3 phenotypes. ST3 is highly expressed in the third leaf at three-leaf stage and expressed constitutively in root, stem, leaf, sheath, and panicle, and the encoded protein, OsSAMHD1, is localized to the cytoplasm. The st3 mutant showed more severe albino leaf phenotype under exogenous 1-mM dATP/dA, dCTP/dC, and dGTP/dG treatments compared with the control conditions, indicating that ST3 is involved in dNTP metabolism. This study reveals a gene associated with dNTP catabolism, and propose a model in which chloroplast development in rice is regulated by the dNTP pool, providing a potential application of these results to hybrid rice breeding.

Pan-stage real-time PCR for quantitation of Trypanosoma cruzi parasitic loads in blood samples.

Chagas disease is a complex zoonosis caused by Trypanosoma cruzi. The diagnosis of this infection is complex and molecular tools are suggested to detect the parasite in blood samples. A long-standing question arises in Chagas disease molecular diagnostics and is related to the feasibility of using epimastigotes in standard curves to quantify parasitic loads. Herein, we conducted experiments running standard curves with all the known life stages of T. cruzi. Our results indicate that regardless of the life stage employed, there are no statistically significant differences when calculating parasitic loads in blood samples. Our results have practical implications from a laboratory perspective in terms of the usability of epimastigotes to build standard curves for T. cruzi pan-stage assessment. Future studies are needed to further improve T. cruzi molecular diagnostic methods and enhance their impact in clinical practice.

RT-PCR/MALDI-TOF Diagnostic Target Performance Reflects Circulating SARS-CoV-2 Variant Diversity in New York City.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we use a data set of 256 SARS-CoV-2-positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. During this time frame, two targets in the N gene (N2 and N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3'-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk, 20.02; 95% CI, 11.36 to 35.72; P < 0.0001). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3'-mismatch to the N2 probe PBS and increases target dropout risk (relative risk, 11.92; 95% CI, 8.17 to 14.06; P < 0.0001). These findings highlight the robust capability of MassARRAY SARS-CoV-2 Panel target results to reveal circulating virus diversity, and they underscore the power of multitarget design to capture variants of concern.

Tiller Angle Control 1 Is Essential for the Dynamic Changes in Plant Architecture in Rice.

Plant architecture is dynamic as plants develop. Although many genes associated with specific plant architecture components have been identified in rice, genes related to underlying dynamic changes in plant architecture remain largely unknown. Here, we identified two highly similar recombinant inbred lines (RILs) with different plant architecture: RIL-Dynamic (D) and RIL-Compact (C). The dynamic plant architecture of RIL-D is characterized by 'loosetiller angle (tillering stage)-compact (heading stage)-loosecurved stem (maturing stage)' under natural long-day (NLD) conditions, and 'loosetiller angle (tillering and heading stages)-loosetiller angle and curved stem (maturing stage)' under natural short-day (NSD) conditions, while RIL-C exhibits a compact plant architecture both under NLD and NSD conditions throughout growth. The candidate locus was mapped to the chromosome 9 tail via the rice 8K chip assay and map-based cloning. Sequencing, complementary tests, and gene knockout tests demonstrated that Tiller Angle Control 1 (TAC1) is responsible for dynamic plant architecture in RIL-D. Moreover, TAC1 positively regulates loose plant architecture, and high TAC1 expression cannot influence the expression of tested tiller-angle-related genes. Our results reveal that TAC1 is necessary for the dynamic changes in plant architecture, which can guide improvements in plant architecture during the modern super rice breeding.

CRISPR-Based Genome Editing: Advancements and Opportunities for Rice Improvement.

To increase the potentiality of crop production for future food security, new technologies for plant breeding are required, including genome editing technology-being one of the most promising. Genome editing with the CRISPR/Cas system has attracted researchers in the last decade as a safer and easier tool for genome editing in a variety of living organisms including rice. Genome editing has transformed agriculture by reducing biotic and abiotic stresses and increasing yield. Recently, genome editing technologies have been developed quickly in order to avoid the challenges that genetically modified crops face. Developing transgenic-free edited plants without introducing foreign DNA has received regulatory approval in a number of countries. Several ongoing efforts from various countries are rapidly expanding to adopt the innovations. This review covers the mechanisms of CRISPR/Cas9, comparisons of CRISPR/Cas9 with other gene-editing technologies-including newly emerged Cas variants-and focuses on CRISPR/Cas9-targeted genes for rice crop improvement. We have further highlighted CRISPR/Cas9 vector construction model design and different bioinformatics tools for target site selection.

Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection.

Saliva is a promising specimen for the detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and noninvasive collection. However, together with intrinsic enzymes and oral microbiota, children's unique dietary habits may introduce substances that interfere with diagnostic testing. To determine whether children's dietary choices impact SARS-CoV-2 molecular detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n = 5) who self-collected saliva at home before and at 0, 20, and 60 min after eating 20 foods they selected. Each of 72 specimens was split into two volumes and spiked with SARS-CoV-2-negative or SARS-CoV-2-positive clinical standards before side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay. Detection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 min after eating 11 of 20 foods. Interference resolved at 20 and 60 min after eating all foods except hot dogs in one participant. This represented a significant improvement in the detection of nucleic acids compared to saliva collected at 0 min after eating (p = 0.0005). We demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting for 20 min after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

Disruption of OsPHD1, Encoding a UDP-Glucose Epimerase, Causes JA Accumulation and Enhanced Bacterial Blight Resistance in Rice.

Lesion mimic mutants (LMMs) have been widely used in experiments in recent years for studying plant physiological mechanisms underlying programmed cell death (PCD) and defense responses. Here, we identified a lesion mimic mutant, lm212-1, which cloned the causal gene by a map-based cloning strategy, and verified this by complementation. The causal gene, OsPHD1, encodes a UDP-glucose epimerase (UGE), and the OsPHD1 was located in the chloroplast. OsPHD1 was constitutively expressed in all organs, with higher expression in leaves and other green tissues. lm212-1 exhibited decreased chlorophyll content, and the chloroplast structure was destroyed. Histochemistry results indicated that H2O2 is highly accumulated and cell death is occurred around the lesions in lm212-1. Compared to the wild type, expression levels of defense-related genes were up-regulated, and resistance to bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) was enhanced, indicating that the defense response was activated in lm212-1, ROS production was induced by flg22, and chitin treatment also showed the same result. Jasmonic acid (JA) and methyl jasmonate (MeJA) increased, and the JA signaling pathways appeared to be disordered in lm212-1. Additionally, the overexpression lines showed the same phenotype as the wild type. Overall, our findings demonstrate that OsPHD1 is involved in the regulation of PCD and defense response in rice.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.