Gene Primers Research Articles

Protective Effect of Crocin on Cadmium Induced-Overexpression of Matrix Metalloproteinases (MMPs) Gene Family in Rat Liver

Background: Metal pollutants such as Cadmium are known as carcinogenic agents and correlated with metastatic potential and tumor aggressiveness. Matrix metalloproteinases (MMPs) are taken into account as the focal points of metastasis. Crocin, a carotenoid chemical compound in Saffron, has been proved to have anti-cancerous properties. However, there is no reliable evidence yet to prove its anti-metastatic activity. Objective: The evaluation of the protective effect of Crocin against Cadmium through monitoring the expression of MMP genes. Materials and Methods: The in-silico analysis based on the GEO database was done for purposefully choosing MMPs. At the wet lab step, after 8 weeks of treatment of the animal (40 female Wistar rats divided into four groups of 10) with Cadmium, Crocin, complex (Cadmium+Crocin) and placebo (control), RNA extraction followed by cDNA synthesizing was done from rats’ liver tissues. Using primers for MMP genes (MMP-1, 3, 7, 12 and 13), the relative fold change of gene expression was studied. Ultimately, the statistical analysis determined the difference in gene expression between groups. Results: Cadmium significantly upregulated the selected MMP genes in comparison with controls. In contrast, Crocin significantly downregulated the expression of all studied MMP genes. Complex administration of Cadmium and Crocin displayed a controlled increase of expression in all MMPs except MMP-1, which was lower than the Cadmium group. Conclusion: These results indicated that the elevation of MMPs expression level by Cadmium was significantly suppressed by Crocin. This could promisingly demonstrate that Crocin possesses a modulating role in inhibiting overexpression of MMPs that might be considered as the anti-metastatic agent.

First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 μl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 μl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.

On-chip screening of SARS-CoV-2 cDNA by LAMP-integrated rotational diffusometry

The unprecedented pandemic has raised the demand for prompt, precise, and large-scale virus detection techniques to control the transmission of contagious illnesses. In this study, a loop-mediated isothermal amplification (LAMP) based on-chip platform was developed to address this challenge using rotational diffusometry and functionalized Janus particles. A recombinant plasmid containing a cDNA sequence of the SARS-CoV-2 non-structural protein 2 (nsp2) gene was employed here as a proof-of-concept for COVID-19 detection. Specifically, designed primers and the functionalized Janus particles were simultaneously loaded on a microfluidic chip to perform the LAMP reaction on a hot plate. The optimal Janus particle concentrations for diffusometric analysis were thoroughly validated, and the performance of the on-chip LAMP reaction was assessed using thermal image analysis. Utilization of the highly sensitive rotational diffusometry achieved a limit of detection of 1 pg/μL in just 10 min with a sample volume of 20 μL. Our method delivered a tenfold higher sensitivity than the conventional method by utilizing only half of its usual required time. Overall, this study proposes a potential nucleic acid (NA) amplification device to aid the rapid diagnosis of various diseases by modifying the primers for different target genes.

A multiplex PCR forensic protocol for the molecular certification of sea catfishes (Ariidae – Siluriformes) from coastal Amazon, Brazil

The marine catfishes (family Ariidae) are important fisheries resources, particularly along the Amazon coast in northern Brazil. Whole fresh specimens and fish fillets are commercialized in both national and international markets, including several records of species substitution and frauds in the trade of processed products. In addition, the life traits of marine catfish species make them particularly vulnerable to overexploitation. Therefore, we developed a molecular certification system for the authentication of these species based on DNA markers. Specific and universal primers were used for the amplification of the mitochondrial Cytochrome b (Cytb) gene and combined with primers for the 16S ribosomal RNA gene (control marker) to provide the first forensic molecular protocol based on multiplex PCR for marine catfishes from Brazilian Amazon coast. Accordingly, distinct band profiles were obtained for each of the most exploited species along this region: Bagre bagre, Notarius grandicassis, Sciades couma, Sciades herzbergii, Sciades parkeri, Sciades passany, and Sciades proops. To amplify the band pattern required for species identification, a concentration of 20 ng/ul was required. In the cross-amplification assay, other fish species were discriminated from the Ariidae bands. The multiplex PCR was about 35x cheaper and saved 13 h of labor to identify the seven species of Ariidae when compared to DNA sequencing. Thus, this protocol represents a viable and efficient tool to the certification of processed products by inspection agencies and to assure the consumer’s rights, eventually leading to an improved management in fisheries. The fishing industry might benefit from the multiplex protocol for the authentication of commercial products, adding market value to them based on DNA certification labels.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

EVALUATION OF SECRETED FRIZZLED-RELATED PROTEIN-2 METHYLATION STATUS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

BackgroundChronic Myeloid Leukemia (CML) is a myeloproliferative disorder defined by the chromosomal translocation (t) (9;22) encoding for the BCR-ABL fusion gene. Tumor development and progression may be affected by hypermethylation of promoter regions of certain genes. Secreted Frizzled-Related Proteins (SFRPs) genes produce Wnt signaling antagonists, and their epigenetic silencing has been detected in multiple types of leukaemia. While epigenetic silencing has been broadly reported for SFRPs, SFRP2 is a family member less studied in leukaemia.
 ObjectivesTo evaluate SFRP2 gene methylation patterns in CML patients compared with healthy individuals.
 Materials and MethodsSodium bisulfite was applied to the extracted DNA from blood samples of 75 CML patients and 25 healthy subjects who served as the control group. The DNA was then analyzed by methylation-specific high-resolution melting (MS-HRM) using specific primers for the SFRP2 gene in promoter sequence.
 ResultsForty-five out of 75 CML patients investigated were shown to have SFRP2 methylation percentages between 50% to 100%, whereas 19 out of 25 healthy subjects had a methylation level of less than 50%.
 ConclusionThe current study demonstrated that SFRP2 promoter hypermethylation exists in CML, as in several other solid tumours. Hence, methylation of this gene may play a part in initiating leukemogenesis.

Surveillance of Vermont wildlife in 2021–2022 reveals no detected SARS-CoV-2 viral RNA

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American’s wildlife populations.

Evaluating five primer pairs for environmental DNA metabarcoding of Central European fish species based on mock communities

Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. Prior to the introduction of eDNA-based assessments into regulatory monitoring contexts (e.g., EU Water Framework Directive), there is a demand for methodological standardization. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for comprehensive fish monitoring is the choice of suitable primer pairs to accurately identify the fishes present in a given water body. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate the performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial 'mock' community composed of DNA from 45 Central European fish species and examined the detection ability and reproducibility of five primer pairs. Our study highlights the effect of primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair was the best choice for eDNA metabarcoding of Central European freshwater fish. Also, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs displayed good detection ability and reproducibility. However, less specific primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how the careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.

New Record: Molecular Depiction of Rhamnolipids (rhlA) Gene in Locally Isolated Strains of Pseudomonas aeruginosa

Soil samples were collected from oil-contaminated sites which were located in west Qurna, Basrah, Iraq. Pseudomonas species were initially isolated on mineral salts and Pseudomonas agar media and identified using morphological and biochemical characterizations. Then, specific primers for the rhlA gene belonging to Pseudomonas aeruginosa were designed based on the primer design conditions, and PCR was performed to amplify the 888 bp size fragment of the rhlA gene; additionally, the primary PCR products were purified and sent for sequencing. The band of about 888bp was determined on the gel, the amplified rhlA gene sequencing findings were revised, only 366 bp were ready to analyze using the (BLAST) software, and the final result was identified as a partial sequence of chromosomal rhlA gene related to Pseudomonas aeruginosa with percent identity of 99.45%. The query gene’s incomplete matching with another partial rhlA record on NCBI was caused by variations in two base pair sequences (T in sequence 348 and C in sequence 353, respectively), and despite the small difference, this results in variation in the amino acids produced; so that a new record number, ON637169, was assigned when the sequence was deposited in GenBank. The relation among the new record of partial rhlA gene with the same number of the other rhlA gene sequences (60 records) was demonstrated by creating a phylogenetic tree.

Influence of alleles of the Ppd-D1 gene and different duration of requirement in yarovization on economic valuable signs of winter bread wheat

Aim. To determine the influence of alternative alleles of the Ppd-D1 gene and phenotypic differences in the duration of the need for vernalization and their interaction on earing time and grain yield of recombinant lines of bread winter wheat. Methods. Growing plants in the field and on the vegetation site, PCR with specific primers for Ppd-D1 and Ppd-B1c genes, phenological observations, artificial vernalization, statistical analysis. Results. 136 recombinant F7 lines from crossing [F1 (Chaika//Cappelle Desperez/2В Chinese Spring) / Kharkivska 81] F2 / Obryi identified by alleles of the Ppd-D1 gene, characterized by the duration of the need for vernalization, duration of the period to earing, plant height, weight 1000 grains and grain harvest. Conclusions. Genotypes with the presence of the Ppd-D1a allele eared 2.9 days earlier than those with the Ppd-B1b allele, and the lines with a 30-day need – one day earlier than those with a 40-day need for vernalization. Different combinations of alternative alleles of the Ppd-D1 gene and characteristics of the lines regarding the need for vernalization significantly influenced the differences in the duration of the period before heading and the weight of 1000 grains. Among the lines that significantly exceeded the control varieties Antonivka and Kuyalnik in terms of yield, those with a different combination of two traits were found, but the share of lines with a combination of the Ppd-D1a gene and 30 daily need for vernalization was greater and they formed the largest yield.

Laboratory Diagnosis of Mpox, Central African Republic, 2016-2022.

During 2016-2022, PCR testing confirmed 100 mpox cases among 302 suspected cases in the Central African Republic. The highest detection rates were from active lesions (40%) and scabs (36%); cycle thresholds were lower (≈18) than those for blood samples (≈33). Results were consistent for generic primer- and clade I primer-specific PCR tests.

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

Optimization of early diagnosis of fibrosis in patients with non-alcoholic fatty liver disease with obesity and overweight

Objective — to optimize of early diagnosis of fibrosis in patients with NAFLD with obesity and overweight based on ROC analysis of non‑invasive laboratory and instrumental diagnostic markers. Materials and methods. The study included 120 patients with NAFLD (main group) with 1 — 3 degrees of obesity or overweight (BMI ≥25.0 — 39.9 kg/m2) and 20 practically healthy volunteers (control group) 23.50 [21.35; 24.78] kg/m2. Patients in the main group were divided into two subgroups depending on BMI. The subgroup I included 85 patients with NAFLD with concomitant obesity, BMI 36.50 [32.00; 40.60] kg/m2, and the subgroup II included 35 patients with NAFLD and overweight 28.00 [27.10; 29.35] kg/m2. Determination of the degree of fibrosis according to the METAVIR scale by measuring the average stiffness of the liver parenchyma, in the mode of shear wave elastography (ultrasound scanning system Soneus P7). The composition of the gut microbiota at the level of the main phylotypes was studied by identifying total bacterial DNA and DNA of Bacteroidetes, Firmicutes, as well as their ratio (F/B ratio), by quantitative real‑time polymerase chain reaction using universal primers for the 16S rRNA gene and taxon‑specific primers. Statistical processing was performed using Statistica 13.1. The data are presented in the form of Me [LQ; UQ], where Me is the median, LQ and UQ are the lower and upper quartiles, respectively. Results. Determination of the characteristic curves obtained as a result of the analysis allowed us to identify the most informationally significant additional diagnostic indicators: CRP, TNF‑α, microRNA‑122, microRNA‑34a (RU), the percentage of visceral fat, and F/B ratio. The analysis of ROC curves allowed us to determine the cut‑off value for each of the additional diagnostic criteria: cut‑off value for serum CRP concentration — 4.5 mg/l (AUC=0.99, sensitivity 1.00, specificity 0.030; p <0.05); for the serum concentration of TNF‑α — 5.5 pg/mL (AUC=0.97, sensitivity 0.98, specificity 0.025; p <0.05). The cut‑off value for the serum level of ­microRNA‑122 — 12.50 RU (AUC=0.99, sensitivity 0.98, specificity 0.048; p <0.05); for the serum level of microRNA‑34a — 5.50 RU (AUC=0.98, sensitivity 0.98, specificity 0.106; p <0.05). The cut‑off value for the proportion of VF — 8.5% (AUC=0.99, sensitivity 1.00, specificity 0.175; p <0.05); for F/B ratio — 1.51 (AUC=0.95, sensitivity 0.83, specificity 0.021; p <0.05). The analysis of the studied parameters allowed us to develop an algorithm for early comprehensive diagnosis of fibrosis in patients with NAFLD with obesity and overweight. The algorithm we propose consists of the following 4 stages. Stage I includes assessment of hepatopathy and interviewing the patient to determine complaints and anamnesis to exclude etiologic factors of secondary fatty liver. Stage II includes the determination of cytolysis markers, namely ALT and GGT. Stage III includes the determination of metabolic and proinflammatory parameters: HOMA index; TC and LDL cholesterol; CRP and TNF‑α. Stage IV includes the assessment of genetic and biological factors: determination of the main phylotypes of the gut microbiota and measurement of serum concentrations of microRNA‑122 and microRNA‑34a. The algorithm allows screening patients with NAFLD with obesity and overweight and identifying patients with early stages of fibrosis through a phased examination. Conclusions. The generalization of traditional diagnostic algorithms with the results of our search for additional diagnostic criteria made it possible to create a 4‑stage algorithm for early comprehensive diagnosis of fibrosis in patients with NAFLD with concomitant obesity or overweight. The use of the developed algorithm allows stratifying patients with NAFLD with obesity and overweight by the presence of fibrosis in the early stages, including by level of care, and timely prescribing preventive and therapeutic measures to reduce the risk of development, improve prognosis and prevent progression.

Insight into Trophic Interactions of Spiders in Olive Groves with Integrated and Ecological Pest Management Using DNA Metabarcoding

Spiders act as one of the major predators among arthropods in agroecosystems and are crucial to the control of pest densities. As they are almost exclusively predators, they have developed a number of hunting strategies, which have consequently impacted their complex trophic interactions. The development of molecular biology methods for environmental DNA analysis has made it easier to analyze such complex trophic networks. In order to ascertain the trophic interactions of spiders, a sampling of spider species was carried out in two olive groves in Zadar County, Croatia, under integrated and ecological pest management. To construct the trophic networks of spiders, we performed DNA metabarcoding. The combined general and spider-excluding primers were able to recognize prey from 12 distinct orders in the guts of the 57 spiders. According to our results, spiders have a complex trophic network that exhibits seasonal and site-specific variations. The results obtained from both sites having different pest management also confirmed that spiders consumed phytophagous insects in the highest ratio, including some important pests, in comparison to other prey and that management and guild type had an impact on the predation of phytophagous insects.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

Identification of Cotylophoron cotylophorum (Fischoeder, 1901) in cattle on St. Kitts, West Indies and its relationship with African and Asian populations

There is limited information about the species of rumen fluke (Family Paramphistomidae) in the Caribbean. However, knowledge of species distribution is needed to better understand disease risk and epidemiology. Morphological identification is challenging with more recent DNA sequencing enabling a better understanding of rumen fluke distribution. In this study, rumen fluke specimens, collected between 2015 and 2016 from cattle on the island of St. Kitts, West Indies, were analysed. The ribosomal internal transcribed spacer 2 (ITS-2) region of rDNA was amplified using generic trematode primers. Results from Sanger sequencing were compared to reference sequences in GenBank and indicated the species was Cotylophoron cotylophorum with 100% sequence identity and 91% query cover. The ITS2 sequences were then compared to previously published ITS2 sequences for the Cotylophoron genus. When all the St. Kitts C. cotylophorum ITS2 sequences were compared with all other Cotylophoron sequences from India, Kenya, and Zimbabwe, three variable nucleotide sites, resulting in five unique haplotypes, were identified. Nine ITS2 sequences shared haplotype 1, which included all those from St. Kitts and single representatives from India and Kenya, potentially indicating global movement of this species.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/β-Sandy-R for the β-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

Unveiling the Role of MX1 and MX2 Gene Expression in Early Pregnancy of Kari Ewes: Insights into INF-tau Signaling and Potential as a Predictive Biomarker for Pregnancy Detection in Domestic Ruminants

Myxovirus resistance (MX) genes are relevant to a family of interferon (IFN)-stimulated genes. In domestic ruminants such as ewes and cattle, the MX genes have two isoforms called the Myxovirus Resistance 1 (MX1) gene and the Myxovirus Resistance 2 (MX2) gene. INF-tau (INFt) is released from embryonic trophoblast cells in the early days of pregnancy and induces expression of MX genes in the uterine endometrium. In the present study, we summarized the relative expression of the MX1 and MX2 genes during the early days of pregnancy in Kari ewes. Blood samples were taken from inseminated and non-inseminated (control) ewes on different days. Peripheral Blood Monocytes (PBMC) were isolated, and with the addition of MX1 and MX2 gene primers, complementary deoxyribonucleic acid (cDNA) was synthesized by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The cDNA was then amplified by rounds of PCR amplification. The amplified product was run on a 2% Agarose gel. The results showed expression of the MX1 and MX2 genes only in pregnant ewes, while no expression was observed in non-pregnant or control ewes. Expression in pregnant ewes was observed on days 14, 16, 17, and 18 after insemination. When comparing the MX1 and MX2 genes, the expression of the MX2 gene was relatively higher than that of the MX1 gene on all experimental days. The result suggested that INF-tau MX genes are expressed during the early days of pregnancy and are involved in the establishment of pregnancy. These results justify the development of this biomarker as a predictive tool for the early detection of pregnancy in ewes and other domestic ruminants.

Screening of Microorganisms Producing Polymer (PHB) from Dump Sites Soil in Ilorin Metropolis, Kwara State, Nigeria

This study was aimed at screening microorganisms isolated from dump sites for polymer production. Five samples of dump site soil were gathered from each of 10 distinct sites, totaling fifty samples, throughout the collection process. Microorganisms were isolated using the pour plate method. Bacteria isolates were identified using biochemical tests and molecular analysis using 16S rRNA gene primer while morphological characteristics are 18SrRNA gene primer were used to identify fungi isolate. Screening of the organisms for polymer production was done using Sudan Black Band Nile Blue A. The polymer extracted were analyzed using Fourier Transform Infrared Spectroscopy(FTIR) and Scanning Electron Microscope (SEM). Results revealed the presence of five fungi and seven bacterial species identified to be Aspergillus niger, Penicillium chrysogenum, Penicillium marneffei, Cladosporium tenuissimum and Rhizopus sp., while bacterial isolates were identified as Bacillus licheniformis, Bacillus cereus, Paenibacillus polymyxa, Bacillus megaterium, Bacillus mycoides, Bacillus subtillis and Enterobacter species. Only four bacterial isolates were positive for polymer production and molecularly identified as Bacillus subtilis strain AGB1 (OM273871), Bacillus megaterium strain AGB2 (OM216844), Paenibacillus polymyxa strain AGB3 (OM273889) and Bacillus licheniformis strain AGB4 (OP703543).The polymer has rough surfaces and crystalline in nature, while FTIR result indicated that the polymer had C=O carbonyl functional group and identified as polyhydrxybutyrate (PHB) polymer while SEM showed that it has crystalline surface. It can be concluded that bacterial isolates which were polymer producers can be utilized for large-scale production of polyhydroxybutyrate for industrial purposes

Strategies and software support for the management of hardware performance counters

AbstractHardware performance counters (HPCs) are facilities offered by most off‐the‐shelf CPU architectures. They are a vital support to post‐mortem performance profiling and are exploited by standard tools such as Linux or Intel V‐Tune. Nevertheless, an increasing number of application domains (e.g., simulation, task‐based high‐performance computing, or cybersecurity) are exploiting them to perform different activities, such as self‐tuning, autonomic optimization, and/or system inspection. This repurposing of HPCs can be difficult, for example, because of the overhead for extracting relevant information. This overhead might render any online or self‐tuning activity ineffective. This article discusses various practical strategies to exploit HPCs beyond post‐mortem profiling, suitable for different application contexts. The presented strategies are accompanied by a general primer on HPCs usage on Linux. We also provide reference x86 (both Intel and AMD) implementations targeting the Linux kernel, upon which we present an experimental assessment of the viability of our proposals.