Random Primers Research Articles

Detection of replication-competent circular single stranded DNA from Deoni cattle

Bovine meat and milk factors (BMMF) having structural similarity to plasmids of Acinetobacter baumanni and some single stranded (ss) DNA viruses are free circulating replication-competent circular ssDNA molecules which were first isolated from serum of Bos taurus animals. Isolation of BMMF from tissues of multiple sclerosis as well as some cancers led researchers to conclude that the consumption of these agents through milk and meat have a positive correlation with these diseases and that the absence of such molecules in B. indicus animals could account to the low occurrence of cancer in those areas consuming products of indigenous animals. Hence, the present study was designed to investigate its presence in Deoni, one of the indigenous cattle breeds of India. The DNA isolated from milk samples were subjected to rolling circle amplification (RCA) using exo-resistant random primers. The products of RCA were then used as template for PCR for amplification of circular DNA. The amplicons obtained of size around 2000bp were cloned in E. coli and sequenced. The BLASTn, multiple sequence alignment and phylogenetic analysis of the obtained sequences were carried out to identify the similarities of these isolates. The sequence showed maximum homology with either BMMF2 or plasmids of Acinetobactor spp. Replication initiation protein (Rep) which showed similarity to bacterial plasmids was present in the sequence. This is the pioneer report on the presence of BMMF in milk of indigenous cattle of India. Keywords: BMMF, replication-competent, circular ssDNA, Deoni

Prevalence and Diversity of Plant Parasitic Nematodes in Irish Peatlands

The prevalence of plant parasitic nematodes (PPN) in the Irish peatlands was investigated in five different peatland habitats—raised bog, cutover scrub/woodlands, fens and peat grasslands, which were further sub-categorised into fourteen different sub-habitats. Within the raised bog habitat were healthy bog hummock (HBH), healthy bog lawn (HBL), degraded bog hummock (DBH) and degraded bog lawn (DBL) and the fen habitats were fen peat (FP) and rich fen peat (R-FP). Cutover scrub or woodland habitat included cutover scrub rewetted (C-RW), cutover scrub non-rewetted (C-NRW), woodlands rewetted (W-RW) and woodlands non-rewetted (W-NRW). Grassland included wasted peat (WP), rough grazing (RG-I) and improved fen peat grassland (IFPG-RW and IFPG-NRW). Soil samples from peatlands were all collected between July and December 2023 when the temperature ranged from 12 to 20 °C. One half of each sample was used for molecular nematode analysis and the other half for morphological identification of nematodes. For the morphological identification, a specific nematode extraction protocol was optimised for peatland soils, and the extracted nematodes were fixed onto slides to be studied under a high-power light microscope. Subsequently, the other part of the soil was processed to isolate total DNA, from which the 18S rRNA gene was sequenced for the identification of nematode taxa. The extracted DNA was also used for randomly amplified polymorphic DNA (RAPD) fingerprinting analysis to determine banding patterns that could classify different bog habitats based on PPN random primers. Compared to that in the climax habitats (HBH, HBL, DBH, DBL, FP, R-FP), PPN prevalence was recorded as being higher in grasslands (WP, RG-I, IFPG-RW and IFPG-NRW) and scrub/woodland ecosystems (C-RW, C-NRW, W-RW, W-NRW). The results indicate that nematode populations are different across the various bog habitats. Emerging and current quarantine PPN belonging to the families Pratylenchidae, Meloidogynidae, Anguinidae and Heteroderidae were noted to be above the threshold limits mentioned under EPPO guidelines, in grassland and wooded peatland habitats. Future actions for PPN management may need to be considered, along with the likelihood that these PPN might impact future paludiculture and other crops and trees growing in nearby agricultural lands.

Point-of-care testing of rpoB in Mycobacterium tuberculosis using multiply-primed-RCA coupled with CRISPR/Cas12a

PurposeDue to the serious threat of tuberculosis to global health and limitations of existing diagnostic methods, this study combined the CRISPR/Cas12a system with Multiply-primed-RCA (MRCA) technology for Mycobacterium tuberculosis Point-of-care Testing (POCT). MethodWe utilized T4 and Taq DNA ligases, compared the effects of specific primers and random 6NS primers on the method, and integrated MRCA and the CRISPR-Cas12a system in one tube. By optimizing conditions such as the concentration of DNA ligase, the concentration of padlock probes, and the number of cycles, we finally established T4-MRCA-Cas12a and Taq-MRCA-Cas12a methods for both stepwise and one-step. ResultsThe limits of detection of the one-step T4/Taq-MRCA-Cas12a were 104aM and 103aM. With no cross-reactivity with DNA from other bacterial strains. The accuracy and specificity were 88 % and 100 % for T4-MRCA-Cas12a, and 96 % and 100 % for Taq-MRCA-Cas12a, respectively. ConclusionWe developed a POCT method that can directly identify MTB through the naked eye.

Optimizing the method for expressing human monoclonal antibodies from a single peripheral blood cell from vaccinated donors

Human monoclonal antibodies are essential diagnostic and research tools and one of the most promising therapeutics. In the past years, single B cell technologies have evolved and over-come conventional methods' limitations, enabling the isolation of scarce B cell populations with desirable characteristics. In this study, we describe a simple and efficient method to isolate anti-gen-specific plasmablasts and memory B cells from hepatitis B virus vaccinated donors' peripheral blood and consequently amplification of immunoglobulin variable region genes. Amplified immunoglobulin variable region genes were cloned into expression vectors and transfected into a human cell line to produce human recombinant monoclonal antibodies. Major challenges in this protocol were isolation of antigen-specific B cells based on surface markers, recovering mRNA from a single cell for efficient amplification, and cloning the correct insert into a desired expression vector. The essential feature of our protocol was the separation of B cells from peripheral blood mononuclear cells before sorting. We identified antigen-specific binding B cells based on the expression of surface markers CD19, CD27, IgG, and binding to hepatitis B surface antigen. Efficient single-cell reverse transcription and polymerase chain reaction (RT-PCR) were achieved using a random primer mix and Kapa Hifi Hot Start Polymerase. Amplification efficiency differed among heavy and light chain variable regions (highest at heavy chain (68 %) and lowest at lambda light chain (22 %)). After co-transfection of HEK293T/17 with successfully cloned heavy and light chain vectors, 70 % of transfected cells produced recombinant monoclonal antibodies at a concentration ∼ 4 μg/ml and 7 % of them showed binding to HBsAg. Human monoclonal antibodies from peripheral blood have advantages over antibodies of mouse origin or phage display libraries, because of their high specificity and self-tolerance. Using the described protocol, we can generate fully human monoclonal antibodies to any other antigen for application in immunotherapy or basic research.

Isolation and characterization of two novel phages, ?Rspv1 and ?Rspv2, with a potential for biocontrol of Ralstonia solanacearum.

Bacteriophages offer an effective biocontrol strategy, presenting an environmentally friendly alternative to combat the issues of antibiotic and copper-based bactericide resistance in bacterial phytopathogens. In this study, two phages targeting Ralstonia solanacearun (designated as ?Rspv1 and ?Rspv2) were identified as potential biocontrol agents against solanaceous wilt, a highly destructive disease causing significant economic losses globally. Both phages demonstrated resistance to chloroform, while ?Rspv1 exhibited a broader host range compared to ?Rspv2 across five isolates of R. solanaceraum. The replication cycle for ?Rspv2 was determined to be 90 min. for a single growth cycle, with a burst size of 85 PFU per cell. Morphologically, ?Rspv1 featured icosahedral heads with long tails, characteristic of the Myoviridae family, whereas ?Rspv2 displayed a filamentous particle shape typical of Inoviridae viruses. Additionally, a Random Amplified Polymorphic DNA (RAPD) analysis using four random primers revealed a diverse nature of the two phages, suggesting their potential utility in disease management strategies. This research unveils two novel virulent phages, and their characterization highlights promising for phage therapy with future investigations aim to field level evaluation against R. solanacearum.

CiRS-7 circular RNA overexpression in plasma cells is a promising molecular biomarker of unfavorable prognosis in multiple myeloma.

Several non-coding RNAs are known to be associated with the pathobiology and progression of multiple myeloma (MM). ciRS-7 (also known as CDR1-AS), a key oncogenic circular RNA (circRNA) that sponges miR-7-5p and other cancer-related microRNAs, was recently found to be downregulated in malignant plasma cells resistant to immunomodulatory drugs. Considering that various circRNAs have a strong potential as molecular biomarkers, we aimed to investigate the expression of ciRS-7 in plasma cell disorders, assess its prognostic importance in MM, and compare these findings with those of individuals with smoldering MM (SMM) and monoclonal gammopathy of unknown significance (MGUS). This study included 171 patients (110 newly diagnosed MM, 34 SMM, and 27 MGUS cases), from which bone marrow aspirate samples were collected for CD138+ plasma cell selection. Total RNA was reversely transcribed using random hexamer primers, and the expression levels of ciRS-7 were quantified using an in-house-developed protocol that includes pre-amplification and real-time quantitative polymerase chain reaction. ciRS-7 levels were found to significantly differ among CD138+ plasma cells of MM, SMM, and MGUS patients. ROC analysis indicated that ciRS-7 expression effectively distinguishes between MM and SMM patients. Moreover, high levels of ciRS-7 were associated with unfavorable prognosis in MM, independently of MM patients' age and Revised International Staging System stage. Additionally, in silico analysis predicted the binding of 85 microRNAs to ciRS-7. In conclusion, this study provides novel insights into the role of ciRS-7 as a promising molecular marker able to distinguish MM from SMM and predict prognosis in MM.

Exploring the impact of primer length on efficient gene detection via high-throughput sequencing

Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.

Streamlined Full-Length Total RNA Sequencing of Paraformaldehyde-Fixed Brain Tissues.

Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.

Exploration of Anethum graveolens diversity from North Western Himalayan

Anethum graveolens L., an aromatic plant grown during winter season belongs to the family Apiaceae. The leaves and seeds of the plants are widely consumed in India. The seeds of the plants are rich in aroma, due to the richness of essential oils whereas the leaves hold immense potential due to their antioxidant and heavy metal stress alleviation properties. However, the genetic diversity of A. graveolens from North Western Himalayan has not been explored so far. Therefore, the genetic diversity of A. graveolens was investigated using 33 random amplified polymorphic (RAPD) and 27 inter sequence specific region (ISSR) markers. Out of the 33 random primers, only 4 RAPD markers resulted in PCR amplification whereas 18 ISSR markers resulted in reproducible PCR amplification. Out of these ISSR markers, 11 markers accounted for 75–100 % of the polymorphism across all the accessions. Additionally, protein profiling was also used to investigate the diversity of A. graveolens landraces. Overall, the cluster analysis using Neighbor joining (N-J) and unweighted pair group method with arithmetic mean (UPGMA) method followed by principal component analysis (PCA) revealed that the accessions of A. graveolens irrespective to their geographic distribution shared the common group. Furthermore, the antioxidant potential of the local landraces was also investigated based on the free radical scavenging potential using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The present current study on exploration of A. graveolens diversity can be helpful in developing conservation strategies of its local germplasm prevalent in North Western Himalayan region.

Comparison of RNA-Sequencing Methods for Degraded RNA.

RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

First Report of Sweet Potato Virus E(SPVE) Infecting Sweet Potato in China.

Sweet potato (Ipomoea batatas [L.] Lam.) is a versatile crop, cultivated in the subtropical and tropical areas, as food, fodder, and industrial raw material crop. In China, sweet potato has been used as a health-care food in recent years, as it contains a wide range of nutrients and xenobiotic phytochemicals. However, viral diseases are major constraint for the sweet potato yield and quality, especially the seed production and quality. Over 30 species of viruses infect sweet potato worldwide (Clark et al. 2012). More recently, a few new viruses infected sweet potato were identified, such as sweet potato virus E (SPVE), which was reported in Korea(Jo et al. 2020). In May 2022, a sweet potato sample (JSXZ1) with virus-like symptom, such as mosaic and vein clearing were collected from sweet potato germplasm Xuzhou resource nursery, Jiangsu Province, China (N34˚16', E117˚18') (Fig. S1A). To investigate the virus disease, the sample JSXZ1 showing the typical symptoms of disease was prepared for Small-RNA (sRNA) deep-sequencing. The sRNA library was constructed using TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA) and sequenced using the Illumine Hiseq 2500 platform by LC-Bop Technologies (Hangzhou) CO., LTD. The sample was sequenced to obtain 26, 358, 439 raw reads and 22, 969, 139 clean reads after quality control trimming and analysis. The Velvet 1.0.5 software was used to de novo assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm. Viruses found in the sample were sweet potato latent virus (SPLV), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato badnavirus A (SPBV-A) and sweet potato badnavirus B (SPBV-B). Surprisingly, besides the viruses listed above, 28 contigs matched sequences of SPVE isolate GS (MH388502). To verify the result, total RNA was extracted from the sample JSXZ1 and from other leave samples (JSXZ2-JSXZ5) that contained SPFMV, SPVC, SPLV, SPVG respectively stored in lab using FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., LTD, Nanjing, China). cDNA was synthesized using random primer (hexadeoxyribonucleotide mixture; pd(N)6). The cDNA serves as template in PCR using a newly designed primer pairs based on SPVE p1 gene (SPVE-F: 5'- TCACCAAAAAGAATGCTACAAC-3'/SPVE-R: 5'-GAAATCCTCCCACTCTCCATA-3'). An expected ~500-bp PCR fragment was obtained in JSXZ1, while none of the fragment was obtained from JSXZ2-JSXZ5 (Fig. S1B). The PCR fragment was cloned into pMD18-T vector (Takara Bio Inc., Beijing, China) and plasmid DNA from transformed Escherichia coli DH5α cell (n=3) were commercially sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the three fragment clones we obtained were 100% identical when compared. A BLASTN analysis of the sequences revealed that they are specific to SPVE and shared 98.62% nucleotide identity to SPVE GS isolate (MH388502) and one sequence was submitted to GenBank (Accession number OQ948331). To determine the occurrence of SPVE in infected sweet potato plants, a total of 37 leaves samples with viral symptom collected from Shandong Province (n=6) and Jiangsu Province (n=31) were indexed by RT-PCR as described before. Only 9 (24.3%) out of 37 from Shandong (n=1) and Jiangsu (n=8) were positive to SPVE respectively. In addition, five additional viruses (SPFMV, SPVC, SPVG, SPLV, SPCSV) were detected among these 37 samples and always in a mixed infection of two or more viruses. To our knowledge, this is the first report of SPVE infecting sweet potato in China. Sweet potato is an important crop in China and other countries (Zhang et al. 2023). China is the largest sweet potato producer all over the world. In addition, as sweet potato is produced through the vegetative propagation mode, thus, more attention should be paid to detection and monitoring of occurrence of SPVE in China.

The development and utilization of two SCAR markers linked to the resistance of banana (Musa spp. AAA) to Fusarium oxysporum f. sp. cubense race 4

Banana (Musa spp.) production worldwide is seriously threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). The best way to control this disease is to grow resistant cultivars. However, it requires large-scale field evaluations and labor- and time-consuming to obtain disease-resistant germplasm. Development of early, reliable, and reproducible selection strategies are considered as the efficient approach which could speed up the selection procedure. In this study, two pairs of sequence-related amplified polymorphism (SRAP) primers related to banana Foc resistance/susceptibility were screened from 100 pairs of random primers. Correspondingly, two pairs of sequence characterized amplified region (SCAR) markers (namely SC4-F/SC4-R and SC14-F/SC14-R, respectively) were successfully generated from these two SRAP markers using 30 cultivars either resistant or susceptible to Foc. Both SCAR markers were located in mitochondrion genome and showed discriminatory power of 96.67% and 100%, respectively. Mitochondrial proteins possibly play a very important role in banana resistance to Foc. In additional, these two SCAR markers were employed simultaneously to screen potential resistant germplasm from 53 accessions with unknown resistance to Foc, and the results revealed a consistency of 83.0% with each other, further indicating their high reliability and reproducibility. These results suggest that both SCAR markers could be used in molecular marker-assisted selection for banana germplasm resistant to Fusarium.

High-Throughput Host-Microbe Single-Cell RNA Sequencing Reveals Ferroptosis-Associated Heterogeneity during Acinetobacter baumannii Infection.

Interactions between host and bacterial cells are integral to human physiology. The complexity of host-microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high-resolution approaches to capture their full complexity. The latest breakthroughs in single-cell RNA sequencing (scRNA-seq) have opened up a new era of studies in host-pathogen interactions. Here, we first report a high-throughput cross-species dual scRNA-seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom-seq). Using reference cells, scRandom-seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom-seq witnessed polarization of THP-1 derived-macrophages and the intracellular A.b-induced ferroptosis-stress in host cells. The inhibition of ferroptosis by Ferrostatin-1 (Fer-1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom-seq provides a high-throughput cross-species dual single-cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host-microbe interactions in infection systems and tumor micro-environments.

High‐Throughput Host–Microbe Single‐Cell RNA Sequencing Reveals Ferroptosis‐Associated Heterogeneity during Acinetobacter baumannii Infection

AbstractInteractions between host and bacterial cells are integral to human physiology. The complexity of host–microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high‐resolution approaches to capture their full complexity. The latest breakthroughs in single‐cell RNA sequencing (scRNA‐seq) have opened up a new era of studies in host–pathogen interactions. Here, we first report a high‐throughput cross‐species dual scRNA‐seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom‐seq). Using reference cells, scRandom‐seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom‐seq witnessed polarization of THP‐1 derived‐macrophages and the intracellular A.b‐induced ferroptosis‐stress in host cells. The inhibition of ferroptosis by Ferrostatin‐1 (Fer‐1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom‐seq provides a high‐throughput cross‐species dual single‐cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host–microbe interactions in infection systems and tumor micro‐environments.

Simultaneous identification of DNA and RNA pathogens using metagenomic sequencing in cases of severe acute respiratory infection.

Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.

First report of saffron latent virus in Crocus sativus from Hungary.

In early April of 2018 we sampled asymptomatic autumn flowering Crocus plants (Fig. S1.) in a private collection in Hajdú-Bihar county, Hungary. From each species (Cr. kotschyanus subsp. kotschyanus, Cr. sativus, Cr. speciosus) 200 mg leaf sample was collected from 5 neighboring shoot, which were treated as one sample. ELISA tests were carried out in duplicates using potyvirus-specific MAb PTY1 antibodies (Jordan and Hammond 1991) on the samples (Agdia, Elkhart, IN, USA). A sample was considered positive if the absorbance was at least three times greater than that of the negative control. Only one sample tested positive; the absorbance values of Cr. sativus leaves were 0.013 and 0.014, while the negative controls were 0.002 and 0.003, respectively. The samples were further tested by RT-PCR for potyviruses (Salamon and Palkovics 2005), tomato spotted wilt virus (TSWV) (Nemes and Salánki 2020) and nepovirus subgroup A (Digiaro et al. 2007). Total nucleic acid was extracted with the phenol-chloroform method of White and Kaper (1989), and reverse transcription was carried out with Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) using random hexamer primer. The samples were negative for TSWV and nepovirus subgroup A, but a single PCR product of ~ 1700 nucleotide (nt) was amplified with potyvirus specific primers and cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). The 1726 nt long insert sequence, including the complete coat protein region was determined and deposited in the NCBI GenBank database (Accession No: OR425160). Digestion of the original PCR products with restriction enzyme SacI yielded only the predicted restriction fragments (364 / 1362 bp), indicating the presence of only a single potyvirus in the infected sample. BLASTn analysis of the CP cistron revealed the highest nt identities to saffron latent virus (SaLV) Iranian isolates (GenBank AccNo.: MN990394 - 85.44%, MN990415 - 85.39% and RefSeq: NC_036802 - 84.05%). For phylogenetic analyses MEGA11 (Tamura et al. 2021) was used. The resulting Maximum Likelihood tree (Fig. S2) showed that all Iranian SaLV isolates grouped together, while the Hungarian isolate is on an adjacent branch, separate from other virus species, and supported with 100% bootstrap values. From these results, it appears that the Hungarian isolate has been separated from the Iranian clade, and has evolved separately as a distinct lineage. We were unable to fulfill Koch's postulates as all available Crocus sativus plants were infected with SaLV. Latent potyvirus infection of Crocus species, by bean yellow mosaic virus (BYMV), iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV) and turnip mosaic virus (TuMV) has been reported by Grilli Caiola and Faoro (2011). SaLV was first reported from Iran (Parizad et al. 2017), but to our knowledge has never been reported from Europe or from any current EPPO member state. Since Crocus species can be asymptomatic virus reservoirs, it is important that any certification scheme for production should require laboratory tests to prove the health of the plants; or advise growers to keep possible high value susceptible crops such as breeding material and nuclear stocks at a distance from crocuses to mitigate virus transmission between stocks. It is also advisable to grow infected lots far from healthy stocks and protected wild hosts. To our knowledge, this is the first report of SaLV from Hungary and from Europe.

Characterization of three citrullus lanatus (thunb.) Matsum. &amp; nakai varieties by fluorescent chromosome staining and rapd analysis

Three commercial Citrullus lanatus varieties, namely Sonya, Kanya and Sweet black-2, were investigated with cyto-molecular techniques for characterization. Despite having similar somatic chromosome numbers and karyotype formulae (2n = 22), these three varieties had considerable diversities in Chromomycin A3 (CMA)- and 4′, 6-diamidino-2-phenylindole (DAPI)-banding patterns. The total somatic chromosomal length was longest in Sweet black-2 (71.44 ± 2.94 µm) and shortest in Sonya (42.31 ± 2.45 µm). Each variety's unique GC- and AT-rich chromatin composition was represented by a variety of numbers, percentages, and patterns of distribution. Eleven random primers were employed to evaluate the genetic diversity among three varieties. Each variety showed a distinct fingerprinting pattern with 88.72% polymorphism while having 10 of the same RAPD bands. Bangladesh J. Bot. 52(4): 1033-1040, 2023 (December)

Pan-Cancer Single-Nucleus Total RNA Sequencing Using snHH-Seq.

Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high-throughput and high-sensitivity method called snHH-seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full-length RNA-seq data is also established. snHH-seq is applied to more than 730000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

Genetic relatedness among interspecific hybrids in the genus Capsicum and their implications in breeding King chilli

The present study explores the potentiality of interspecific crosses using three different Capsicum species, comprising of two C. annuum (Krishna and Capsicum), five C. chinense (King chilli A1, A5, A18, A19, A20) and one C. frutescens (Mem) genotypes, with the objective to identify the incompatibility barriers, accomplish the morpho-molecular characterization of parents and hybrids and assess the genetic relatedness between the genotypes.Amongst the three species used for the crossing programme, 'Krishna' (C. annuum) and 'Mem' (C. frutescens) are local cultivars of Assam (India) and possess excellent adaptability. Guinness World Records certified King chilli, or 'Bhut Jolokia' (C. chinense), as the world's hottest chilli (10,001,304 SHU) in September 2007. King chilli has tremendous export potential but is prone to viral diseases, thereby necessitating the transfer of desirable disease resistance from local cultivars.It was observed from the study that the reciprocal crosses involving C. chinense and C. frutescens were compatible, but C. frutescens (Mem) was crossable with only accession A5, suggesting genotype specificity in the interspecific crosses. The usual Euclidean distance between Mem and A5 was 716.605Similarly reciprocal crosses involving C. annuum and C. chinense were partially incompatible. Both the C. annuum (Krishna, Capsicum) genotypes successfully crossed with King chilli accession A18 thereby exhibiting genotype specificity. It was observed that in the cross A18 x Krishna the F1 seeds failed to germinate and in the cross A18 x Capsicum the F1 plants exhibited dwarfism. Hence, unilateral incompatibility was observed when King chilli accession A18 was used as the female parent. In the reciprocal crosses, germination and plant development was normal.The pollen viability ranged from 72.64 to 94.92 % for parents and 23.55 to 81.02 % for the hybrids. Thus, hybrids exhibited lower pollen viability compared to their parents. Significant morphological differences were visible between the parents and their combinations; some features were similar to the male or female parent, and others were either intermediate or novel.Parental polymorphism was detected using thirty random SSR primers, and the markers showing polymorphic bands were used to confirm the hybridity of F1s. Therefore, an attempt was made in this study to assess the possibility of interspecific crosses to transfer disease resistance from the related species to enhance the quality of King chilli, thus improving its commercial valve.