Coding Sequence Research Articles

Polarization insensitive non-interleaved frequency multiplexed dual-band Terahertz coding metasurface for independent control of reflected waves

Independent control of electromagnetic (EM) waves by metasurfaces for multiple tasks are highly desired and is the recent hot topic of research. In this work we contribute a polarization insensitive frequency multiplexed 2-bit coding metasurface to control the Terahertz (THz) waves in the two operating bands independently. In this regard, as a first step a cascaded meta-atom composed of square rings and/or square metallic patches separated by two polyimide substrates is designed and optimized that provides sixteen independent distinct discrete phases in the reflection geometry. These meta-atoms are then distributed with distinct coding sequences in the two-dimensional spatial plane to realize various bi-functional metasurfaces. As a proof of the concept various full structures are designed and simulated to realize a series of bi-functionalities including anomalous reflection/beam shaping, beam shaping/anomalous reflection, beam deflection/Orbital angular momentum (OAM) beam generation with distinct modes and propagating wave to surface wave (PW–SW) conversion/PW beam manipulation in the lower and higher THz bands, respectively. All the simulation results are in excellent agreement with their theoretical equivalents. We envision that the proposed meta-designs have potential applications for the multi-spectral control of EM waves in THz band. The idea can be further extended to design frequency dependent tri-functional and multi-functional THz meta-devices.

Genomic Sequence of a Czech Isolate of Erysimum Latent Virus from Sisymbrium altissimum.

The Erysimum latent virus (ELV), a tymovirus, was first isolated from several wild and cultivated brassicas in Germany. Its virions were shown to be serologically distinct from those of the turnip yellow mosaic virus (TYMV), which is also found in wild and cultivated plants in several European countries but also in other parts of the world. TYMV and ELV were among the first plant viruses to have had their genomes sequenced, and when other tymovirus genomes were sequenced, it was found that, in phylogenies, ELV is probably the basal outlier to all other tymoviruses. Here, we report the near-complete genomic sequence of another isolate of ELV from Czechia. This isolate was found in 1990 in Sisymbrium altissimum plants showing mosaic symptoms. It was detected using ELISA tests and electron microscopy. We have now sequenced the full coding sequence of this isolate using contemporary high throughput methods and found that the German and Czech isolates of ELV are closely related and are of the same virus species.

An independent base composition of each rate class for improved likelihood-based phylogeny estimation; the 5rf model.

The combination of a time reversible Markov process with a "hidden" mixture of gamma distributed relative site rates plus invariant sites have become the most favoured options for likelihood and other probabilistic models of nucleotide evolution (e.g., tr4gi which approximates a gamma with four rate classes). However, these models assume a homogeneous and stationary distribution of nucleotide (character or base) frequencies. Here, we explore the potential benefits and pitfalls of allowing each rate category (rate class) of a 4gi mixture model to have its own base frequencies. This is achieved by starting each of the five rate classes, at the tree's root, with its own free choice of nucleotide frequencies to create a 4gi5rf model or a 5rf model in shorthand. We assess the practical identifiability of this approach with a BEAST 2 implementation, aiming to determine if it can accurately estimate credibility intervals and expected values for a wide range of plausible parameter values. Practical identifiability, as distinguished from mathematical identifiability, gauges the model's ability to identify parameters in real-world scenarios, as opposed to theoretically with infinite data. One of the most common types of phylogenetic data is mitochondrial DNA (mtDNA) protein coding sequence. It is often assumed current models analyse robustly such data and that higher likelihood/posterior probability models do better. However, this abstract shows that vertebrate mtDNA remains a very difficult type of data to fully model, and that dramatically higher likelihoods do not mean a model is measurably more accurate with respect to recovering key parameters of biological interest (e.g., monophyletic groups, their support and their ages). The 4gi5rf model considerably improves marginal likelihoods and seems to reverse some apparent errors exacerbated by the 4gi model, while introducing others. Problems appear to be linked to non-stationary DNA repair processes that alter the mutation/substitution spectra across lineages and time. We also show such problems are not unique to mtDNA and are encountered in analysing nuclear sequences. Non-stationarity of DNA repair processes mutation/substitution spectra thus pose an active challenge to obtaining reliable inferences of relationships and divergence times near the root of placental mammals, for example. An open source implementation is available under the LGPL 3.0 license in the beastbooster package for BEAST 2, available from https://github.com/rbouckaert/beastbooster.

The Small Auxin-Up RNA 50 (SAUR50) Gene from Ammopiptanthus nanus Negatively Regulates Drought Tolerance.

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response.

Identification and functional analysis of recent IS transposition events in rhizobia

Rhizobia are alpha- and beta- Proteobacteria that, through the establishment of symbiotic interactions with leguminous plants, are able to fix atmospheric nitrogen as ammonium. The successful establishment of a symbiotic interaction is highly dependent on the availability of nitrogen sources in the soil, and on the specific rhizobia strain. Insertion sequences (ISs) are simple transposable genetic elements that can move to different locations within the host genome and are known to play an important evolutionary role, contributing to genome plasticity by acting as recombination hot-spots, and disrupting coding and regulatory sequences. Disruption of coding sequences may have occurred either in a common ancestor of the species or more recently. By means of ISComapare, we identified Differentially Located ISs (DLISs) in nearly related rhizobial strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium. Our results revealed that recent IS transposition could have a role in adaptation by enabling the activation and inactivation of genes that could dynamically affect the competition and survival of rhizobia in the rhizosphere.Graphical

Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.

Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences.

N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.

Comparative plastome analyses and evolutionary relationships of 25 East Asian species within the medicinal plant genus Scrophularia (Scrophulariaceae)

BackgroudScrophularia L., a genus of the Scrophulariaceae, is a group of important medicinal plants used for eliminating heat and detoxifying. East Asia has an abundance of potentially medicinal Scrophularia species, and it serves as a secondary diversity center of the genus. However, the genomic resources available for germplasm identification and pharmaceutical exploration of East Asian Scrophularia are insufficient, hindering its commercial and industrial development. Additionally, the interspecific relationships of most East Asian Scrophularia species remain unclear.MethodsIn this study, we sequenced the leaves of 25 East Asian species of the genus Scrophularia, assembled and annotated the complete chloroplast genomes, and subsequently performed comparative and phylogenetic analyses on these genomes.Results and discussionThe conserved plastome length of these 25 species ranged from 151,582 bp to 153,239 bp, containing a total of 132 coding genes, including 18 duplicated genes and 114 unique genes. Through genome alignment of these 25 species, 38-53 repeated sequences and 7 shared SSRs were identified, along with regions with high nucleotide polymorphism (Pi), which could potentially serve as molecular markers for species identification. The genome structure, gene content, and arrangement showed conservation, while variations were observed in the IR boundary regions and IGS. Phylogenetic inferences based on whole plastomes or on coding sequences (CDS) only yielded congruent results. We categorized the 25 East Asian Scrophularia species into six distinct clades and further explored their interspecies relationships using morphological characteristics, such as flower color, the relative position of stamens and corolla, and plant height. This could lay a genetic basis for future resource development of Scrophularia in East Asia.

Deciphering regulation of FOXP3 expression in human conventional T cells.

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals composition dynamics in embryonic tissue, cancer cells, and macrophages

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.

Genomic analysis of Bacillus subtilis sub sp. subtilis GEB5 reveals its genetic assets for nematicidal and plant growth promoting mechanisms

The immense benefit of chemical nematicides in controlling plant parasitic nematodes in high-value crops is counterbalanced by the development of resistance in nematodes due to their overuse. Bacillus subtilis is a promising bacterium that not only confers resistance against plant parasitic nematodes but also promotes plant growth through nutrient acquisition. Considering the multifaceted action of B. subtilis attempts were made to sequence the whole genome of endophytic B. subtilis GEB5 to find the genome complexity of the potential bio-control agent isolated from the guava. The results confirmed the isolate as B. subtilis sub sp. subtilis with a circular genome of 8,341,276 bp (8.3 Mb) length and 41.8% GC content. The results of genome annotation revealed the presence of 8209 coding sequences (CDS) genes, 136 transfer RNA (tRNA) genes, and 6 ribosomal RNA (rRNA) genes. Nonribosomal peptide synthetase (NRPS) gene clusters in the genome of GEB5 included fengycin, bacillaene, plipastatin, subtilin, subtilosin A, bacilysin, surfactin, sporulation killing factor, bacillibactin. Moreover, the GEB5 genome assembly had 21 homologs corresponding to nematode-virulent proteases. Furthermore, the results of greenhouse experiments showcased that GEB5 quenched the population of M. enterolobii (72% reduction) and also boosted the plant growth of guava. A plurality of the putatively encoded nematode virulence protease and its ability to boost plant growth suggests its environmentally robust bio-control potential in protecting plants against invading plant parasitic nematodes.

T1 and T2 measurements across multiple 0.55T MRI systems using open-source vendor-neutral sequences.

To compare T1 and T2 measurements across commercial and prototype 0.55T MRI systems in both phantom and healthy participants using the same vendor-neutral pulse sequences, reconstruction, and analysis methods. Standard spin echo measurements and abbreviated protocol measurements of T1, B1, and T2 were made on two prototype 0.55 T systems and two commercial 0.55T systems using an ISMRM/NIST system phantom. Additionally, five healthy participants were imaged at each system using the abbreviated protocol for T1, B1, and T2 measurement. The phantom measurements were compared to NMR-based reference measurements to determine accuracy, and both phantom and in vivo measurements were compared to assess reproducibility and differences between the prototype and commercial systems. Vendor-neutral sequences were implemented across all four systems, and the code for pulse sequences and reconstruction is freely available. For participants, there was no difference in the mean T1 and T2 relaxation times between the prototype and commercial systems. In the phantom, there were no significant differences between the prototype and commercial systems for T1 and T2 measurements using the abbreviated protocol. Quantitative T1 and T2 measurements at 0.55T in phantom and healthy participants are not statistically different across the prototype and commercial systems.

A Radiological Clip Design Using Ultrasound Identification to Improve Localization.

We demonstrate the use of ultrasound to receive an acoustic signal transmitted from a radiological clip designed from a custom circuit. This signal encodes an identification number and is localized and identified wirelessly by the ultrasound imaging system. We designed and constructed the test platform with a Teensy 4.0 microcontroller core to detect ultrasonic imaging pulses received by a transducer embedded in a phantom, which acted as the radiological clip. Ultrasound identification (USID) signals were generated and transmitted as a result. The phantom and clip were imaged using an ultrasonic array (Philips L7-4) connected to a Verasonics™ Vantage 128 system operating in pulse inversion (PI) mode. Cross-correlations were performed to localize and identify the code sequences in the PI images. USID signals were detected and visualized on B-mode images of the phantoms with up to sub-millimeter localization accuracy. The average detection rate across 30,400 frames of ultrasound data was 98.1%. The USID clip produced identifiable, distinguishable, and localizable signals when imaged. Radiological clips are used to mark breast cancer being treated by neoadjuvant chemotherapy (NAC) via implant in or near treated lesions. As NAC progresses, available marking clips can lose visibility in ultrasound, the imaging modality of choice for monitoring NAC-treated lesions. By transmitting an active signal, more accurate and reliable ultrasound localization of these clips could be achieved and multiple clips with different ID values could be imaged in the same field of view.

Polymorphism Identification in the Coding Sequences (ORFs) of the Porcine Pregnancy-Associated Glycoprotein 2-like Gene Subfamily in Pigs.

Pregnancy-associated glycoproteins (PAGs) are a polygenic family with many scattered genes and pseudogenes resulting from the duplication or fusion of a pseudogene with expression beginning in the trophoblast during the peri-implantation period and continuing in the trophectoderm. In this study, single-nucleotide polymorphism (SNP) and insertion/deletion (InDels) in the open reading frame (nine exons) of crossbreed pigs are reported for the first time. Novel SNPs/InDels were researched using genomic DNA templates isolated from the leukocytes of crossbreed pigs (N = 25), which were amplified, gel-out-purified, and sequenced. Sixteen SNPs and one InDel (g.6961_6966 Ins TGCCAA) were identified in the crossbreed pigs. In silico analysis revealed that among 16 SNPs, only 10 SNPs cause amino acid (aa) substitutions, and InDel codes asparagine (N298) and alanine (A299). The results provide a novel broad-based database (main pattern) that will be critical for future research into the possible correlations between the SNP genotypes of the pPAG2-L subfamily in pigs of various breeds whose reproductive traits are known.

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport.

Programmed ribosomal frameshifting is a translational recoding phenomenon in which a proportion of ribosomes are stimulated to slip backwards or forwards on an mRNA1, rephasing the ribosome relative to the mRNA. While frameshifting is often employed by viruses2, very few phylogenetically conserved examples are known in vertebrate genes and the evidence for some of these is controversial3,4. Here we report a +1 frameshifting signal in the coding sequence of the human gene PLEKHM2, encoding the ARL8-dependent, lysosome-kinesin-1 adaptor protein PLEKHM25. This +1 frameshifting signal, UCC_UUU_CGG, is highly conserved in vertebrates and exhibits an influenza virus-like frameshift motif with similar efficiency6,7. Purification and mass spectrometry of GFP-tagged trans-frame protein from cells confirms frameshifting. Structure prediction shows that the new C-terminal domain generated by this frameshift forms an alpha-helix. This additional domain relieves PLEKHM2 from autoinhibition, allowing it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Thus, the frameshift proteoform generates a constitutively active adaptor of kinesin-1.

DNA damage drives antigen diversification through mosaic Variant Surface Glycoprotein (VSG) formation in Trypanosoma brucei.

Antigenic variation, using large genomic repertoires of antigen-encoding genes, allows pathogens to evade host antibody. Many pathogens, including the African trypanosome Trypanosoma brucei, extend their antigenic repertoire through genomic diversification. While evidence suggests that T. brucei depends on the generation of new variant surface glycoprotein (VSG) genes to maintain a chronic infection, a lack of experimentally tractable tools for studying this process has obscured its underlying mechanisms. Here, we present a highly sensitive targeted sequencing approach for measuring VSG diversification. Using this method, we demonstrate that a Cas9-induced DNA double-strand break within the VSG coding sequence can induce VSG recombination with patterns identical to those observed during infection. These newly generated VSGs are antigenically distinct from parental clones and thus capable of facilitating immune evasion. Together, these results provide insight into the mechanisms of VSG diversification and an experimental framework for studying the evolution of antigen repertoires in pathogenic microbes.

Characterization of the Coding Sequence of the MC1R (Melanocortin 1 Receptor) Gene of Ayam Cemani Black Chickens.

Plumage color is one of the most important traits characterizing chicken breeds. Black-boned chickens constitute a specific group of breeds with a unique phenotype. One of the representatives is the Indonesian Ayam Cemani. The extraordinary black phenotype results from a specific chromosomal rearrangement. We used complete CDS of crucial color-related gene MC1R, which plays a key role in melanin distribution but has not been previously studied in Ayam Cemani. It turned out that Ayam Cemani individuals possess a newly found non-synonymous mutation G355A resulting in amino acid substitution D119N. Together with the presence of G274A (E92K), the new missense variant enabled us to distinguish a new extended black allele at the E locus. All of the investigated birds were heterozygous in terms of the new mutation. Previous studies and our own results indicate a high level of genetic variation within the MC1R gene within and between chicken breeds. Besides the key mutations that make it possible to distinguish particular major alleles, there are also numerous substitutions that give haplotypes more characteristics for individual breeds.

Further diversity in the origins of fish antifreeze proteins.

Shifts in environmental conditions can impose strong selection for adaptive traits. During the Cenozoic era, as the oceans cooled, many marine teleost fish species were at risk of freezing. This led to the independent emergence of distinct ice-binding antifreeze proteins (AFPs). The report in this issue by Graham and Davies reveals the development of AFP genes in shorthorn and longhorn sculpin from a copy of the lunapark gene. The predicted sculpin AFP sequences are unrelated to that of lunapark; the coding sequences for the AFPs appear to have arisen from small portions of the lunapark gene by codon frameshifting along with a series of mutations.

Transcriptome Analysis of Multiple Plant Parts in the Woody Oil Tree Camellia drupifera Loureiro

Camellia drupifera is mainly used in forestry for its high-value industrial products; however, limited information is available on its transcriptome. This study aimed to construct a full-length transcriptome sequence based on the PacBio sequencing platform for various plant parts of C. drupifera, including flower buds, leaves, leaf buds, branches, the pericarp, and seed kernels. The transcriptomes were annotated with 23,207 genes, with 58 subgroups in the GO classification. The KEGG database revealed 10,407 genes involved in the metabolic pathway analysis, with 68,192 coding sequences, 3352 TF families, 48,541 SSRs, 1421 IncRNAs, and 2625 variable shears predicted. The transcriptomes of different parts were analyzed and compared. The majority of differentially expressed genes (DEGs) were found between the pericarp and seed kernels, followed by leaves and the pericarp with 5662 DEGs, and flower buds and leaf buds with 1616 DEGs. GO and KEGG enrichment analyses showed that KEGG differential genes were significant in microbial metabolism, carbon metabolism, and other functions. The data annotation and analysis of the full-length transcriptome and the comparative analysis between different plant parts provided a theoretical basis for studying gene function, metabolic pathway regulation, and gene expression analysis in KEGG.

In-silico identification of putatively functional intergenic small open reading frames in the cucumber genome and their predicted response to biotic and abiotic stresses.

The availability of high-throughput sequencing technologies increased our understanding of different genomes. However, the genomes of all living organisms still have many unidentified coding sequences. The increased number of missing small open reading frames (sORFs) is due to the length threshold used in most gene identification tools, which is true in the genic and, more importantly and surprisingly, in the intergenic regions. Scanning the cucumber genome intergenic regions revealed 420 723 sORF. We excluded 3850 sORF with similarities to annotated cucumber proteins. To propose the functionality of the remaining 416 873 sORF, we calculated their codon adaptation index (CAI). We found 398 937 novel sORF (nsORF) with CAI ≥ 0.7 that were further used for downstream analysis. Searching against the Rfam database revealed 109 nsORFs similar to multiple RNA families. Using SignalP-5.0 and NLS, identified 11 592 signal peptides. Five predicted proteins interacting with Meloidogyne incognita and Powdery mildew proteins were selected using published transcriptome data of host-pathogen interactions. Gene ontology enrichment interpreted the function of those proteins, illustrating that nsORFs' expression could contribute to the cucumber's response to biotic and abiotic stresses. This research highlights the importance of previously overlooked nsORFs in the cucumber genome and provides novel insights into their potential functions.