ResearchPad - rna-extraction Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Genetic algorithm-based personalized models of human cardiac action potential]]> We present a novel modification of genetic algorithm (GA) which determines personalized parameters of cardiomyocyte electrophysiology model based on set of experimental human action potential (AP) recorded at different heart rates. In order to find the steady state solution, the optimized algorithm performs simultaneous search in the parametric and slow variables spaces. We demonstrate that several GA modifications are required for effective convergence. Firstly, we used Cauchy mutation along a random direction in the parametric space. Secondly, relatively large number of elite organisms (6–10% of the population passed on to new generation) was required for effective convergence. Test runs with synthetic AP as input data indicate that algorithm error is low for high amplitude ionic currents (1.6±1.6% for IKr, 3.2±3.5% for IK1, 3.9±3.5% for INa, 8.2±6.3% for ICaL). Experimental signal-to-noise ratio above 28 dB was required for high quality GA performance. GA was validated against optical mapping recordings of human ventricular AP and mRNA expression profile of donor hearts. In particular, GA output parameters were rescaled proportionally to mRNA levels ratio between patients. We have demonstrated that mRNA-based models predict the AP waveform dependence on heart rate with high precision. The latter also provides a novel technique of model personalization that makes it possible to map gene expression profile to cardiac function.

<![CDATA[Growth enhancement of porcine epidemic diarrhea virus (PEDV) in Vero E6 cells expressing PEDV nucleocapsid protein]]>

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.

<![CDATA[A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib]]>

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

<![CDATA[Comparison of the new fully automated extraction platform eMAG to the MagNA PURE 96 and the well-established easyMAG for detection of common human respiratory viruses]]>

Respiratory viral infections constitute the majority of samples tested in the clinical virology laboratory during the winter season, and are mainly diagnosed using molecular assays, namely real-time PCR (qPCR). Therefore, a high-quality extraction process is critical for successful, reliable and sensitive qPCR results. Here we aimed to evaluate the performance of the newly launched eMAG compared to the fully automated MagNA PURE 96 (Roche, Germany) and to the semi-automated easyMAG (bioMerieux, France) extraction platforms. For this analysis, we assessed and compared the analytic and clinical performance of the three platforms, using 262 archived respiratory samples positive or negative to common viruses regularly examined in our laboratory (influenza A, B, H1N1pdm, Respiratory Syncytial Virus (RSV), human Metapneumovirus (hMPV), parainfluenza-3, adenovirus and negative samples). In addition, quantitated virus controls were used to determine the limit of detection of each extraction method.

In all categories tested, eMAG results were comparable to those of the easyMAG and MagNa PURE 96, highly sensitive for all viruses and over 98% clinical specificity and sensitivity for all viruses tested. Together with its high level of automation, the bioMerieux eMAG is a high-quality extraction platform enabling effective molecular analysis and is mostly suitable for medium-sized laboratories.

<![CDATA[Multiplexing polysome profiling experiments to study translation in Escherichia coli]]>

Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.

<![CDATA[Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation]]>

Hbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

<![CDATA[Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription]]>

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.

<![CDATA[Virus load and clinical features during the acute phase of Chikungunya infection in children]]>


Chikungunya virus (CHIKV) infection is a long known mosquito-borne disease that is associated with severe morbidity, characterized by fever, headache, rashes, joint pain, and myalgia. It is believed that virus load has relation with severity of clinical features.


We performed this study to assess the relationship between virus load and clinical features in children during the acute phase of CHIKV infection, in order to draw insights for better-informed treatment.

Study design

Between June 1, 2009, and May 31, 2010, 338 patients with fever and susceptive to CHIKV during first 4 days of illness were prospectively enrolled from Karnataka Institute of Medical Sciences, Hubli in our hospital based cross sectional observational study. Sybr green quantitative reverse transcription polymerase chain reaction was performed to estimate the virus load.


Quantitative RT-PCR was positive for CHIKV in 54 patients. The median copy number of CHIKV was 1.3x 108 copies/ml (1.7x105-9.9x109 copies/ml). Among the observed clinical features, a statistically significant difference in log mean virus load was found between patients with and without myalgia (log mean 7.50 vs 8.34, P = 0.01).


Patients with myalgia had lower virus load and those without myalgia had a higher virus load.

<![CDATA[Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms]]>

Tomato yellow leaf curl virus (TYLCV) and its related begomoviruses cause fast-spreading diseases in tomato worldwide. How this virus induces diseases remains largely unclear. Here we report a noncoding RNA-mediated model to elucidate the molecular mechanisms of TYLCV-tomato interaction and disease development. The circular ssDNA genome of TYLCV contains a noncoding intergenic region (IR), which is known to mediate viral DNA replication and transcription in host cells, but has not been reported to contribute directly to viral disease development. We demonstrate that the IR is transcribed in dual orientations during plant infection and confers abnormal phenotypes in tomato independently of protein-coding regions of the viral genome. We show that the IR sequence has a 25-nt segment that is almost perfectly complementary to a long noncoding RNA (lncRNA, designated as SlLNR1) in TYLCV-susceptible tomato cultivars but not in resistant cultivars which contains a 14-nt deletion in the 25-nt region. Consequently, we show that viral small-interfering RNAs (vsRNAs) derived from the 25-nt IR sequence induces silencing of SlLNR1 in susceptible tomato plants but not resistant plants, and this SlLNR1 downregulation is associated with stunted and curled leaf phenotypes reminiscent of TYLCV symptoms. These results suggest that the lncRNA interacts with the IR-derived vsRNAs to control disease development during TYLCV infection. Consistent with its possible function in virus disease development, over-expression of SlLNR1 in tomato reduces the accumulation of TYLCV. Furthermore, gene silencing of the SlLNR1 in the tomato plants induced TYLCV-like leaf phenotypes without viral infection. Our results uncover a previously unknown interaction between vsRNAs and host lncRNA, and provide a plausible model for TYLCV-induced diseases and host antiviral immunity, which would help to develop effective strategies for the control of this important viral pathogen.

<![CDATA[The bumblebee Bombus terrestris carries a primary inoculum of Tomato brown rugose fruit virus contributing to disease spread in tomatoes]]>

The bumblebee Bombus terrestris is a beneficial pollinator extensively used in tomato production. Our hypothesis was that bumblebee hives collected from a Tomato brown rugose fruit virus (ToBRFV) infected tomato greenhouse, preserve an infectious primary inoculum. Placing a bumblebee hive collected from a ToBRFV contaminated greenhouse, in a glass-/net-house containing only uninfected healthy tomato plants, spread ToBRFV disease. Control uninfected tomato plants grown in a glass-/net-house devoid of any beehive remained uninfected. ToBRFV-contaminated hives carried infectious viral particles as demonstrated in a biological assay on laboratory test plants of virus extracted from hive components. Viral particles isolated from a contaminated hive had a typical tobamovirus morphology observed in transmission electron microscopy. Assembly of ToBRFV genome was achieved by next generation sequencing analysis of RNA adhering to the bumblebee body. Bumblebee dissection showed that ToBRFV was mostly present in the abdomen suggesting viral disease spread via buzz pollination. These results demonstrate that bumblebee hives collected from ToBRFV-contaminated greenhouses carry a primary inoculum that reflects the status of viruses in the growing area. This new mode of ToBRFV spread by pollinators opens an avenue for detection of viruses in a growing area through analysis of the pollinators, as well as emphasizes the need to reevaluate the appropriate disease management protocols.

<![CDATA[Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation]]>

While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds.

<![CDATA[EspL is essential for virulence and stabilizes EspE, EspF and EspH levels in Mycobacterium tuberculosis]]>

The ESX-1, type VII, secretion system represents the major virulence determinant of Mycobacterium tuberculosis, one of the most successful intracellular pathogens. Here, by combining genetic and high-throughput approaches, we show that EspL, a protein of 115 amino acids, is essential for mediating ESX-1-dependent virulence and for stabilization of EspE, EspF and EspH protein levels. Indeed, an espL knock-out mutant was unable to replicate intracellularly, secrete ESX-1 substrates or stimulate innate cytokine production. Moreover, proteomic studies detected greatly reduced amounts of EspE, EspF and EspH in the espL mutant as compared to the wild type strain, suggesting a role for EspL as a chaperone. The latter conclusion was further supported by discovering that EspL interacts with EspD, which was previously demonstrated to stabilize the ESX-1 substrates and effector proteins, EspA and EspC. Loss of EspL also leads to downregulation in M. tuberculosis of WhiB6, a redox-sensitive transcriptional activator of ESX-1 genes. Overall, our data highlight the importance of a so-far overlooked, though conserved, component of the ESX-1 secretion system and begin to delineate the role played by EspE, EspF and EspH in virulence and host-pathogen interaction.

<![CDATA[Methods for detecting Zika virus in feces: A case study in captive squirrel monkeys (Saimiri boliviensis boliviensis)]]>

A strain of Zika virus (ZIKV) of Asian origin associated with birth defects and neurological disorders has emerged and spread through the Americas. ZIKV was first isolated in the blood of nonhuman primates in Africa and has been detected in the blood, saliva, and urine of a few catarrhine species in both Africa and Asia, suggesting that nonhuman primates may serve as both a source and a reservoir of the virus. The recent introduction of ZIKV to human populations in the Americas presents the potential for the virus to spread into nonhuman primate reservoirs. Thus, it is critical to develop efficient and noninvasive detection methods to monitor the spread of the virus in wild nonhuman primate populations. Here, we describe a method for ZIKV detection in noninvasively collected fecal samples of a Neotropical primate. Fecal samples were collected from two captive squirrel monkeys (Saimiri boliviensis boliviensis) that were experimentally infected with ZIKV (Strain Mexico_1_44) and an additional two uninfected squirrel monkeys. Nucleic acids were extracted from these samples, and RT-qPCR was used to assay for the presence of ZIKV using primers flanking a 101 bp region of the NS5 gene. In both ZIKV-inoculated animals, ZIKV was detected 5–11 days post-infection, but was not detected in the uninfected animals. We compare the fecal results to ZIKV detection in serum, saliva, and urine samples from the same individuals. Our results indicate that fecal detection is a cost-effective, noninvasive method for monitoring wild populations of Neotropical primates as possible ZIKV reservoirs.

<![CDATA[A natural antisense lncRNA controls breast cancer progression by promoting tumor suppressor gene mRNA stability]]>

The human genome encodes thousands of long noncoding RNA (lncRNA) genes; the function of majority of them is poorly understood. Aberrant expression of a significant number of lncRNAs is observed in various diseases, including cancer. To gain insights into the role of lncRNAs in breast cancer progression, we performed genome-wide transcriptome analyses in an isogenic, triple negative breast cancer (TNBC/basal-like) progression cell lines using a 3D cell culture model. We identified significantly altered expression of 1853 lncRNAs, including ~500 natural antisense transcript (NATs) lncRNAs. A significant number of breast cancer-deregulated NATs displayed co-regulated expression with oncogenic and tumor suppressor protein-coding genes in cis. Further studies on one such NAT, PDCD4-AS1 lncRNA reveal that it positively regulates the expression and activity of the tumor suppressor PDCD4 in mammary epithelial cells. Both PDCD4-AS1 and PDCD4 show reduced expression in TNBC cell lines and in patients, and depletion of PDCD4-AS1 compromised the cellular levels and activity of PDCD4. Further, tumorigenic properties of PDCD4-AS1-depleted TNBC cells were rescued by exogenous expression of PDCD4, implying that PDCD4-AS1 acts upstream of PDCD4. Mechanistically, PDCD4-AS1 stabilizes PDCD4 RNA by forming RNA duplex and controls the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. Our studies demonstrate crucial roles played by NAT lncRNAs in regulating post-transcriptional gene expression of key oncogenic or tumor suppressor genes, thereby contributing to TNBC progression.

<![CDATA[MicroRNA isolation and quantification in cerebrospinal fluid: A comparative methodical study]]>

Associated with the pathogenesis of many cancers, including brain tumors, microRNAs (miRNAs) present promising diagnostic biomarkers. These molecules have been also studied in cerebrospinal fluid (CSF), showing great potential as a diagnostic tool in patients with brain tumors. Even though there are some biological and technological factors that could affect the results and their biological and clinical interpretation, miRNA analysis in CSF is not fully standardized. This study aims to compare several RNA extraction and miRNA quantification approaches, including high-throughput technologies and individual miRNA detection methods, thereby contributing to the optimization and standardization of quantification of extracellular miRNAs in CSF. Such knowledge is essential for the potential use of miRNAs as diagnostic biomarkers in brain tumors.

<![CDATA[Evaluation of endogenous reference genes in Bactrocera cucurbitae by qPCR under different conditions]]>

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.

<![CDATA[Expression of the sRNAs CrcZ and CrcY modulate the strength of carbon catabolite repression under diazotrophic or non-diazotrophic growing conditions in Azotobacter vinelandii]]>

Azotobacter vinelandii is a nitrogen-fixing bacterium of the Pseudomonadaceae family that prefers the use of organic acids rather than carbohydrates. Thus, in a mixture of acetate-glucose, glucose is consumed only after acetate is exhausted. In a previous work, we investigated the molecular basis of this carbon catabolite repression (CCR) process under diazotrophic conditions. In the presence of acetate, Crc-Hfq inhibited translation of the gluP mRNA, encoding the glucose transporter in A. vinelandii. Herein, we investigated the regulation in the expression of the small non-coding RNAs (sRNAs) crcZ and crcY, which are known to antagonize the repressing activity of Hfq-Crc. Our results indicated higher expression levels of the sRNAs crcZ and crcY under low CCR conditions (i.e. glucose), in relation to the strong one (acetate one). In addition, we also explored the process of CCR in the presence of ammonium. Our results revealed that CCR also occurs under non-diazotrophic conditions as we detected a hierarchy in the utilization of the supplied carbon sources, which was consistent with the higher expression level of the crcZ/Y sRNAs during glucose catabolism. Analysis of the promoters driving transcription of crcZ and crcY confirmed that they were RpoN-dependent but we also detected a processed form of CrcZ (CrcZ*) in the RpoN-deficient strain derived from a cbrB-crcZ co-transcript. CrcZ* was functional and sufficient to allow the assimilation of acetate.

<![CDATA[Aging-associated patterns in the expression of human endogenous retroviruses]]>

Human endogenous retroviruses (HERV) are relics of ancient retroviral infections in our genome. Most of them have lost their coding capacity, but proviral RNA or protein have been observed in several disease states (e.g. in inflammatory and autoimmune diseases and malignancies). However, their clinical significance as well as their mechanisms of action have still remained elusive. As human aging is associated with several biological characteristics of these diseases, we now analyzed the aging-associated expression of the individual proviruses of two HERV families, HERV-K (91 proviruses) and HERV-W (213 proviruses) using genome-wide RNA-sequencing (RNA-seq). RNA was purified from blood cells derived from healthy young individuals (n = 7) and from nonagenarians (n = 7). The data indicated that in the case of HERV-K (HML-2) 33 proviruses had a detectable expression but in only 3 of those the expression levels were significantly different between the young and old individuals. In the HERV-W family expression was observed in 45 loci and only in one case the young/old difference was significant. However, applying hierarchical clustering on the HERV expression data resulted in the formation of two distinct clusters, one containing the young individuals and another the nonagenarians. This suggests, that even though the aging-associated differences in the expression levels of individual proviruses are minor, there seems to be some underlying aging-related pattern. These data indicate that aging does not have a strong effect on the expression of individual HERV proviruses, but instead several proviruses are affected moderately, leading to age-dependent expression profiles.

<![CDATA[NAM gene allelic composition and its relation to grain-filling duration and nitrogen utilisation efficiency of Australian wheat]]>

Optimising nitrogen fertiliser management in combination with using high nitrogen efficient wheat cultivars is the most effective strategy to maximise productivity in a cost-efficient manner. The present study was designed to investigate the associations between nitrogen utilisation efficiency (NUtE) and the allelic composition of the NAM genes in Australian wheat cultivars. As results, the non-functional NAM-B1 allele was more responsive to the nitrogen levels and increased NUtE significantly, leading to a higher grain yield but reduced grain protein content. Nitrogen application at different developmental stages (mid-tillering, booting, and flowering) did not show significant differences in grain yield and protein content. The NAM-A1 allelic variation is significantly associated with the length of the grain-filling period. While the NAM-A1 allele a was associated with a short to moderate grain-filling phase, the alleles c and d were related to moderate to long grain-filling phase. Thus, selection of appropriate combinations of NAM gene alleles can fine-tune the duration of growth phases affecting sink-source relationships which offers an opportunity to develop high NUtE cultivars for target environments.

<![CDATA[Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep]]>

This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how “Vegfaxxxb-like” PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR.