Internal Genes Research Articles

Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation in Camellia impressinervis.

Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.

Novel influenza A viruses in pigs with zoonotic potential, Chile.

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) have recently been identified in Chile. The objective of this study was to evaluate their zoonotic potential. We perform phylogenetic analyses to determine the genetic origin and evolution of these viruses, and a serological analysis to determine the level of cross-protective antibodies in the human population. Eight genotypes were identified, all with pandemic H1N1 2009-like internal genes. H1N1 and H1N2 were the subtypes more commonly detected. Swine H1N2 and H3N2 IAVs had hemagglutinin and neuraminidase lineages genetically divergent from IAVs reported worldwide, including human vaccine strains. These genes originated from human seasonal viruses were introduced into the swine population since the mid-1980s. Serological data indicate that the general population is susceptible to the H3N2 virus and that elderly and young children also lack protective antibodies against the H1N2 strains, suggesting that these viruses could be potential zoonotic threats. Continuous IAV surveillance and monitoring of the swine and human populations is strongly recommended.IMPORTANCEIn the global context, where swine serve as crucial intermediate hosts for influenza A viruses (IAVs), this study addresses the pressing concern of the zoonotic potential of novel reassortant strains. Conducted on a large scale in Chile, it presents a comprehensive account of swine influenza A virus diversity, covering 93.8% of the country's industrialized swine farms. The findings reveal eight distinct swine IAV genotypes, all carrying a complete internal gene cassette of pandemic H1N1 2009 origin, emphasizing potential increased replication and transmission fitness. Genetic divergence of H1N2 and H3N2 IAVs from globally reported strains raises alarms, with evidence suggesting introductions from human seasonal viruses since the mid-1980s. A detailed serological analysis underscores the zoonotic threat, indicating susceptibility in the general population to swine H3N2 and a lack of protective antibodies in vulnerable demographics. These data highlight the importance of continuous surveillance, providing crucial insights for global health organizations.

Genetic Diversity and Biological Characteristics of H3 Avian Influenza Virus Isolated from China in 2021–2022 Showed the Emerging H3N8 Posed a Threat to Human Health

The H3 influenza viruses are widespread in domestic poultry but have been ignored because their pathogenicity in poultry is low. Three human infections with H3N8 influenza viruses have been reported in China since 2022, raising public concern. Here, we comprehensively analyzed 30 H3 subtype avian influenza viruses isolated from live poultry markets in China between 2021 and 2022. Genetic and phylogenetic analyses showed that the H3 viruses have undergone frequent reassortment and have formed complex genotypes. Notably, the viruses that caused human infections in 2022–2023 were highly homologous to the H3N8 viruses circulating in poultry in 2022, with internal genes derived from the H9N2 viruses. The analysis of chicken infections indicated that the novel H3N8 viruses were more infectious in chickens than those that do not carry H9N2 genes, whereas the H3 viruses detected in China in 2021–2022 showed low pathogenicity in mice. Our findings suggest that the novel H3N8 viruses bearing internal H9N2 genes have adapted to and circulated in chickens and pose a threat to human health. These results highlight the need for continued surveillance of the H3 influenza viruses and their impact on the poultry industry.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Assessment of Reference Genes Stability in Cortical Bone of Obese and Diabetic Mice.

Bone, a pivotal structural organ, is susceptible to disorders with profound health implications. The investigation of gene expression in bone tissue is imperative, particularly within the context of metabolic diseases such as obesity and diabetes that augment the susceptibility to bone fractures. The objective of this study is to identify a set of internal control genes for the analysis of gene expression. This study employs reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) to assess gene expression in bone tissue. We selected fourteen housekeeping genes and assessed their stability in the cortical bone of mouse models for obesity and diabetes using four well-established algorithms (GeNorm, BestKeeper, NormFinder, and the comparative Delta Ct method). We identified Rpl13a as the mostly stably expressed reference gene in cortical bone tissue from mouse models of obesity and diabetes (db/db), while Gapdh was found to be the most stable reference gene in another diabetes model, KKAy mice. Additionally, Ef1a, Ppia, Rplp0, and Rpl22 were identified as alternative genes suitable for normalizing gene expression in cortical bone from obesity and diabetes mouse models. These findings enhance RT-qPCR accuracy and reliability, offering a strategic guide to select reference gene for studying bone tissue gene expression in metabolic disorders.

Comprehensive Assessment of Reference Gene Expression within the Whitefly Dialeurodes citri Using RT-qPCR.

The citrus whitefly, Dialeurodes citri, is a destructive pest that infests citrus plants. It is a major vector in transmitting plant viruses such as citrus yellow vein clearing virus (CYVCV), which has caused severe economic losses worldwide, and therefore efficient control of this pest is economically important. However, the scope of genetic studies primarily focused on D. citri is restricted, something that has potentially limited further study of efficient control options. To explore the functionalities of D. citri target genes, screening for specific reference genes using RT-qPCR under different experimental conditions is essential for the furtherance of biological studies concerning D. citri. The eight candidate reference genes were evaluated by dedicated algorithms (geNorm, Normfinder, BestKeeper and ΔCt method) under five specific experimental conditions (developmental stage, sex, tissue, population and temperature). In addition, the RefFinder software, a comprehensive evaluation platform integrating all of the above algorithms, ranked the expression stability of eight candidate reference genes. The results showed that the best reference genes under different experimental settings were V-ATP-A and RPS18 at different developmental stages; α-tubulin, 18S and V-ATP-A in both sexes; EF1A and α-tubulin in different tissues; Actin and Argk under different populations; and RPS18 and RPL13 in different temperatures. The validation of selected reference genes was further identified using heat shock protein (Hsp) 70 as a reporter gene. Our study, for the first time, provides a detailed compilation of internal reference genes for D. citri that are suitable for RT-qPCR analysis, which is robust groundwork for comprehensive investigation of the functional target genes of D. citri.

Swine influenza A virus infection dynamics and evolution in intensive pig production systems.

Swine influenza A virus (swIAV) is one of the main viral pathogens responsible for respiratory disease in farmed pigs. While outbreaks are often epidemic in nature, increasing reports suggest that continuous, endemic infection of herds is now common. The move towards larger herd sizes and increased intensification in the commercial pig industry may promote endemic infection; however, the impact that intensification has on swIAV infection dynamics and evolution is unclear. We carried out a longitudinal surveillance study for over 18 months on two enzootically infected, intensive, indoor, and multi-site pig production flows. Frequent sampling of all production stages using individual and group sampling methods was performed, followed by virological and immunological testing and whole-genome sequencing. We identified weaned pigs between 4 and 12-weeks old as the main reservoir of swIAV in the production flows, with continuous, year-round infection. Despite the continuous nature of viral circulation, infection levels were not uniform, with increasing exposure at the herd level associated with reduced viral prevalence followed by subsequent rebound infection. A single virus subtype was maintained on each farm for the entire duration of the study. Viral evolution was characterised by long periods of stasis punctuated by periods of rapid change coinciding with increasing exposure within the herd. An accumulation of mutations in the surface glycoproteins consistent with antigenic drift was observed, in addition to amino acid substitutions in the internal gene products as well as reassortment exchange of internal gene segments from newly introduced strains. These data demonstrate that long-term, continuous infection of herds with a single subtype is possible and document the evolutionary mechanisms utilised to achieve this.

Occurrence and genotypic identification of Blastocystis sp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Heilongjiang Province, China

Blastocystis sp., Enterocytozoon bieneusi, and Giardia duodenalis are three common zoonotic intestinal parasites, and cattle are important hosts of these three intestinal protozoa. In this study, 1632 fecal samples were collected from dairy farms in Heilongjiang Province, China, and screened for Blastocystis sp., E. bieneusi, and G. duodenalis using polymerase chain reaction. Of these, 149 (9.13%) were positive for three zoonotic pathogens, including 104 (6.40%), 22 (1.35%), and 23 (1.41%) for Blastocystis sp., E. bieneusi, and G. duodenalis, respectively. Based on partial SSU rRNA gene sequencing analysis, 104 positive samples of Blastocystis sp. were found, and a total of nine known subtypes were identified, including ST10 (61), ST3 (18), ST14 (6), ST26 (7), ST24 (3), ST25 (2), ST1 (2), ST5 (2), and ST21 (1). Among these, three subtypes (ST1, ST3, and ST5) were recognized as zoonotic subtypes, and two subtypes (ST10 and ST14) were specific to animals. All 23 Giardia duodenalis-positive samples belonged to assemblage E (n = 23) based on sequenced beta-giardin (bg) and triosephosphate isomerase (tpi) genes. Three known genotypes of E. bieneusi, namely J (n = 9), I (n = 6), and BEB4 (n = 7), were identified by sequence analysis of the internal transcriptional spacer region gene. Our study provides basic data for prevention and control in Heilongjiang Province; however, further research is required to better understand the prevalence and public health significance of these pathogens in the Heilongjiang region.

Ecological and Genetic Landscapes of Global H12 Avian Influenza Viruses and Biological Characteristics of an H12N5 Virus Isolated from Wild Ducks in Eastern China

Wild migratory birds are considered the central reservoirs of avian influenza viruses. H12 viruses are one of the 16 hemagglutinin (HA) subtypes of avian influenza viruses and are rarely reported because they are infrequently detected in birds. Consequently, the ecological and genetic profiles of H12 viruses and their adaptation in domestic birds and mammals remain unclear. Here, we found that H12N5 viruses were predominant in the nine identified H12NX subtypes, with the HA (H12) and neuraminidase (NA) (N5) genes showing combination bias in the categorized analysis of subtype combinations (H12 and N1–N9; H1–H12, H14, H15, and N5). These identified H12N5 viruses were primarily detected in birds of Anatidae and Scolopacidae in North America, excluding their possible characterization as chicken or mammalian viruses. The H12N5 viruses were divided into the North American lineage and Eurasian lineage according to their genetic differences, including the HA and NA surface genes and internal genes, although reassortment was observed between the two lineages. We isolated an Eurasian-lineage H12N5 virus from wild ducks in Eastern China, which was one of the 12 identified H12 viruses in China. Infectivity studies indicated that the H12N5 virus is poorly adapted to domestic ducks and chickens, although viral shedding could be detected in both inoculated and contact birds. Additionally, the naturally isolated H12N5 virus did not achieve good replication in mice. These results indicate that the rare subtype of H12 viruses was mainly pooled in wild migratory birds and has an established phylogeography, with low risks of spillover into domestic birds and mammals.

The Molecular Epidemiology of Epizootic Hemorrhagic Disease Viruses Identified in Israel between 2015 and 2023.

Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease seriously affecting cattle and some wild ruminants and has a worldwide distribution. All viruses can be subdivided into "Eastern" and "Western" topotypes according to geographic distribution via the phylogenetic analysis of internal genes. In Israel, during the last decade, three outbreaks were registered: caused by EHDV-6 in 2015, by EHDV-1 in 2016, and by EHDV-7 in 2020. Additionally, RNA of EHDV-8 was found in imported calves from Portugal in 2023. During the same period in other countries of the region, non-Israeli-like EHDV-6 and EHDV-8 were identified. Full genome sequencing, BLAST, and phylogenetic analyses of the locally and globally known EHDV genomes allowed us to presume the probable route and origin of these viruses detected in Israel. Thus, EHDV-6 has probably been circulating in the region for a long period when EHDV-1 and -8 appeared here for the last years, while their route of introduction into the new areas was probably natural; all of them belonged to the "Western" topotype. In contrast, EHDV-7 probably had the "Eastern", anthropogenic origin. Data from the study can facilitate the evaluation of the appearance or reappearance of EHDVs in the Mediterranean area and enhance the planning of prevention measures.

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-β. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

One-Health Challenge in H9N2 Avian Influenza: Novel Human-Avian Reassortment Virus in Guangdong Province, China

China is one of the highest producers of poultry meat output in the world, with a large scale of chicken rearing. Statistically analyzed H9N2-subtype avian influenza viruses (AIVs) have become the dominant subtype in China’s live poultry market, with the highest detection rate. Although H9N2 AIV is of low pathogenicity and tends not to cause serious disease and high mortality in poultry, it poses a great challenge to the domestic poultry farming industry by causing a decrease in appetite, a decline in egg production, and deaths caused by mixed infections with another pathogenic microorganism. Moreover, novel influenza viruses (H7N9 and H3N8) infecting humans have emerged in China, and the H9N2 AIV provides all or part of the internal genes to the new recombinant viruses, posing a potential threat to public health and safety and human health. In this research, six H9N2 AIVs were isolated from feces or oropharyngeal swabs collected from live poultry markets and duck farms in Zhanjiang. After epidemiological investigations, phylogenetic analyses, and molecular characterization, we found that the ZJ81 strain was a chicken–human–mink recombinant virus, the ML3 strain was a chicken-human recombinant virus, and all six virus strains of the virus had a bias for the human receptor-binding site and a mutation that could cause an increase in virulence in mice. Therefore, surveillance and control of H9N2 AIV should be strengthened to provide data support for cross-species transmission of H9N2 AIV.

Hsa-miR-194-5p and hsa-miR-195-5p are down-regulated expressed in high dysplasia HPV-positive Pap smear samples compared to normal cytology HPV-positive Pap smear samples

BackgroundThe human papillomavirus (HPV) infection may affect the miRNA expression pattern during cervical cancer (CC) development. To demonstrate the association between high-risk HPVs and the development of cervix dysplasia, we examined the expression patterns of hsa-miR-194-5p and hsa-miR-195-5p in Pap smear samples from southeast Iranian women. We compared samples that were HPV-positive but showed no abnormality in the cytological examination to samples that were HPV-positive and had severe dysplasia.MethodsPap smear samples were obtained from 60 HPV-positive (HPV-16/18) patients with histologically confirmed severe dysplasia (cervical intra-epithelial neoplasia (CIN 3) or carcinoma in situ) and the normal cytology group. The expression of hsa-miR-194-5p and hsa-miR-195-5p was analyzed by real-time quantitative PCR, using specific stem-loop primers and U6 snRNA as the internal reference gene. Clinicopathological features were associated with miRNA expression levels. Furthermore, functional enrichment analysis was conducted using in silico tools. The Kaplan–Meier survival method was also obtained to discriminate survival-significant candidate miRNAs in CC, and receiver operating characteristic (ROC) curves were constructed to assess the diagnostic value.ResultsCompared to HPV-positive cytologically normal Pap smear samples, hsa-miR-194-5p and hsa-miR-195-5p relative expression decreased significantly in HPV-positive patients with a severe dysplasia Pap smear. Kaplan–Meier analysis indicated a significant association between the miR-194 decrease and poor CC survival. In essence, ROC curve analysis showed that miR-194-5p and miR-195-5p could serve as valuable markers for the development of cervix dysplasia in individuals who are positive for high-risk HPVs.ConclusionsThis study revealed that hsa-miR-194-5p and hsa-miR-195-5p may possess tumor suppressor capabilities in the context of cervical dysplasia progression. However, it remains uncertain whether these microRNAs are implicated in the transition of patients with high dysplasia to cervical cancer. We also showed the potential capability of candidate miRNAs as novel diagnostic biomarkers related to cervical dysplasia progression.

Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

Bimetallic S. pachycarpa@Ag-Doped ZnO Alloy Nanoparticles Unveil Therapeutic Promise: Revolutionizing Multiple Myeloma Treatment

In recent years, nanoscience has emerged as a prominent research field, focusing on exploring the therapeutic properties of herbal medicine. This research presents the green synthesis of Ag-doped ZnO nanoparticles using the fruit extract of Sophora pachycarpa (S. pachycarpa@Ag-doped ZnO NPs). Various characterization methods, including XRD, EDAX, TEM, and FT-IR, were employed to identify the nanoparticles. The XRD technique confirmed the crystallinity of S. pachycarpa@Ag-doped ZnO NPs, with an average size of 47nm. In this investigation, we explored the potential toxicity of S. pachycarpa fruit extract and S. pachycarpa@Ag-doped ZnO NPs on U266B1 cells. The cells were treated for 24 and 72hours with varying concentrations (0.5-1mg/ml) of S. pachycarpa and S. pachycarpa@Ag-doped ZnO NPs. The percentage of cell viability was determined using the AlamarBlue Assay. Additionally, the expression level of the P53 gene was evaluated by Real-Time PCR, with the β-actin gene serving as an internal reference gene for comparison. Viability results demonstrated that approximately 92% and 62% of cells remained alive after treatment with 1mg/ml dose of S. pachycarpa for 24 and 72hours, respectively, compared to the control. In the case of cells treated with S. pachycarpa@Ag-doped ZnO NPs, an IC50 of 585.664µg/ml was acquired after 24h of treatment. The expression level of P53 in cells treated with S. pachycarpa@Ag-doped ZnO NPs and S. pachycarpa significantly increased in a concentration-dependent manner, reaching 10380 and 1.62 fold expressions in 2mg/ml of S. pachycarpa@Ag-doped ZnO NPs and S. pachycarpa, respectively. Based on the obtained results, it can be concluded that S. pachycarpa@Ag-doped ZnO NPs can be considered a new avenue for Multiple myeloma (MM) treatment. However, further studies are necessary to identify more detailed aspects of the drug's mechanism of action, such as investigating the signaling pathway and protein level via western blotting.

Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues.

Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

ShRNA Targeting Lentiviral Vector Minus-Strand Product Improves the Viral Titer During Viral Packaging.

Lentiviral vector (LVV) has been used as one of the common carriers for gene therapy in clinical trials. LVV-mediated clinical trials have being reported in successfully treating hundreds of β-thalassemia cases. These LVVs bear an inversely placed β-hemoglobin (HBB) gene expression cassette for preserving introns during the viral RNA packaging. Consequently, these LVVs often produce a small amount of negatively orientated transcript driven by its internal gene promoter and would lower the viral titer by the minus-strand complemented with the viral backbone. To overcome this problem, we designed shRNAs specifically target the minus-strand RNA driven by the LVV internal promoter that resulted in a notable increase in the viral titer. This report demonstrates a simple and positive mean for increasing the effectiveness for gene therapy with the LVV system.

Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review.

Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high throughput. The stability of internal reference genes has progressively emerged as a major factor affecting the precision of qRT-PCR results. However, the stability of the expression of the reference genes needs to be determined further in different cells or organs, physiological and experimental conditions. Methods for evaluating these candidate internal reference genes have also evolved from simple single software evaluation to more reliable and accurate internal reference gene evaluation by combining different software tools in a comprehensive analysis. This study intends to provide a definitive reference for upcoming research that will be conducted on fruit trees. The primary focus of this review is to summarize the research progress in recent years regarding the selection and stability analysis of candidate reference genes for different fruit trees.

Screening and validation of optimal miRNA reference genes in different developing stages and tissues of Lilium henryi Baker

Dynamic miRNA detection using the qRT-PCR technique requires appropriate reference genes to ensure data reliability. Previous studies have screened internal reference genes in plants during embryonic development and various stress treatment, involving relatively few tissues and organs. There is no relevant miRNA study in Lilium henryi Baker and limited research on the optimal miRNA reference genes in lilies, such as 5S, 18S, U6 and Actin. Twelve genes were selected as candidate reference genes whose expression stability was analyzed in petals at different developmental stages and other tissues using various algorithms, such as geNorm, NormFinder, BestKeeper, and Delta CT. The results revealed that the optimal combination of reference genes for Lilium henryi Baker petals at different developmental stages was osa-miR166m and osa-miR166a-3p, while that for different tissues of Lilium henryi Baker was osa-miR166g-3p and osa-miR166a-3p.Four important genes related to growth and development regulation, namely, osa-miR156a, osa-miR395b, osa-miR396a-3p, and osa-miR396a-5p, were selected for validation. The findings of the present study could contribute to future investigations onmiRNA expression and the related functions in Lilium henryi Baker while providing important references for the normalization of the miRNA expression in other varieties of lily.