Qiagen Rotor Gene

Background: Lactobacillus reuteri SKKU-OGDONS-01 was isolated from chicken intestines for further development as an antiviral feed additive. This study aimed to investigate probiotic properties of chicken isolates in mice model and in silico analysis.Results: Compared to known probiotics, Lactobacillus paracasei ATCC 334, Lactobacillus reuteri SKKU-OGDONS-01 showed immune-boosting effects despite short persistence in the mice intestine. Especially, the expression levels of IFN-β and IFN-γ were increased 4 and 40 times higher than those of the control mice. In proportion to the immune-boosting effects elicited by chicken isolates, the antiviral efficacy against murine norovirus (MNV) was also remarkable. For the purpose of evaluating the potential for development as feed additives, the expression levels of probiotic markers such as long-term acid adaptation, stress response, and adhesion-related proteins were investigated using in silico method, and the results showed that these proteins were expressed at high levels in chicken isolate. Conclusion: Our study demonstrated that chicken isolate, Lactobacillus reuteri SKKU-OGDONS-01 can also elicit high probiotic properties in mice even though it originated in chicken. We expect that this chicken isolate will be able to induce much higher probiotic activity in chickens to develop feed additives for poultry.Keywords: Lactobacillus reuteri SKKU-OGDONS-01, probiotics, antiviral efficacy, probiotic marker, cytokine

Dongjun Kim, Mun-ju Cho, Yongjun Lee, Seungchan Cho, Eui-Joon Kil, Sung June Byun, Sukchan Lee. 2020.

Background Malaria transmission from humans to mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However accurate quantification of gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available. Methods qRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes ( pfs25 and pfMGET respectively) using synthetic cRNA standards and in-vitro cultured gametocytes. Assay were validated and assay performance was investigated using blood samples of clinical trial participants (ClinicalTrials.gov reference number NCT02431637 and NCT02431650) using these standards and compared to absolute quantification by droplet digital PCR (ddPCR). Results The number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5 - 307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6 - 14.9) for the male-specific transcript pfMGET . These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71x10 6 to 5.71 gametocytes/mL for pfs25, and 1.73x10 7 to 5.59 for pfMGET. The limit of detection was 3.9 (95% CI 2.5-8.2) gametocytes/mL for pfs25, and 26.9 (95% CI 19.3 - 51.7) gametocytes/mL for PfMGET . Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated high level agreement (ICC=0.998 for pfs25 and 0.995 for pfMGET ). Conclusions We developed and validated qRT-PCR assays that are able to accurately quantify levels of female and male P. falciparum gametocytes at submicroscopic densities. The assays showed excellent reproducibility, sensitivity, precision, specificity and accuracy. The methodology will enable the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.

Claire YT Wang, Emma Ballard, Stacey Llewellyn, Louise Marquart, Teun Bousema, James S. McCarthy, Katharine A Collins. 2020.

BackgroundThe spread of virus via the blood stream has been suggested to contribute to extra pulmonary organ failure in Coronavirus disease 2019 (COVID-19). We assessed SARS-CoV-2 RNAemia (RNAemia) and the association between RNAemia and inflammation, organ failure and mortality in critically ill COVID-19 patients.MethodsIn a tertiary center we included all patients with PCR verified COVID-19 and consent admitted to ICU. SARS-CoV-2 RNA copies above 1000/ml measured by PCR in plasma was defined as RNAemia and used as surrogate for viremia.ResultsOf the 92 patients RNAemia was found in 31 (34%) and RNA in plasma was detected in 63 (58%). Hypertension and corticosteroid treatment was more common in patients with RNAemia . RNAemia levels were higher in patients with RAAS-inhibitor treatment or corticosteroid treatment than those without. Extra-pulmonary organ failure biomarkers and the extent of organ failure were similar in patients with and without RNAemia, but the former group had more renal replacement therapy and higher mortality (26 vs 16%; 35 vs 16%, respectively, p=0.04). RNAemia was not an independent predictor of death at 30 days after adjustment for age. ConclusionSARS-CoV2 RNA copies in plasma is a common finding in ICU patients with COVID-19. Although viremia was not associated with extra pulmonary organ failure it was more common in patients who did not survive to 30 days after ICU admission.Trial registrationClinicalTrials NCT04316884

Josef D. Järhult, Michael Hultström, Anders Bergqvist, Robert Frithiof, Miklos Lipcsey. 2020.

Background Autologous fat grafting is becoming increasingly common worldly. However, the long-term retention of fat grafting is still unpredictable due to the inevitable fibrosis that arises during tissue repair. Fibrosis may be regulated by T-cell immune responses that are influenced by adipose-derived stem cells (ASCs). Accordingly, we hypothesized that overly abundant ASCs might promote fibrosis by promoting T-cell immune responses to adipose tissue. Methods We performed 0.3 ml fat grafts with 10⁴/ml, 10⁶/ml and 10⁸/ml ASCs and control group in C57 BL/6 mice in vivo. We observed retention, fibrosis, T-cell immunity, and macrophage infiltration over 12 weeks. In addition, CD4 + T-helper 1 (Th1) cells and T-helper 2 (Th2) cells were co-cultured with ASCs or ASCs conditioned media (CM) in vitro. We detected the ratio of Th2%/Th1% after 24 and 48 hours. Results In vivo, the retention rate was higher in the 10⁴ group, while even lower in the 10⁸ group with significantly increased inflammation and fibrosis than the control group at week 12. There was no significance between control group and the 10⁶ group. Also, the 10⁸ group increased infiltration of M2 macrophages, CD4 + T-cells and Th2/Th1 ratio. In vitro, the ratio of Th2%/Th1% induced by the ASCs-transwell group was higher than the ASCs-CM group and showed concentration-dependent. Conclusions High concentrations of ASCs in adipose tissue can promote Th1–Th2 shifting, and the excess of Th2 cells might promote the persistence of M2 macrophages and increase the level of fibrosis which lead to a decrease in the long-term retention of fat grafts. In addition, we found that ASCs promoted Th1–Th2 shifting in vitro.

Xinyao Chen, Yunzi Chen, Zijue Wang, Ziqing Dong, Yao Yao, Ye Li, Jing Xia, Jingyan Guan, Xinhui Wang, Rongcun Sun, Feng Lu, Lijun Hao, Sai Luo. 2020.

Background: Acute myocardial injury (AMI), which is induced by renal ischemia-reperfusion (IR), is a significant cause of acute kidney injury (AKI)-related associated death. Obesity increases the severity and frequency of AMI and AKI. Tanshinone IIA (TIIA) combined with cyclosporine A (CsA) pretreatment was used to alleviate myocardial cell apoptosis induced by renal IR, and to determine whether TIIA combined with CsA would attenuate myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats. Methods: Male rates were fed a high fat diet for 8 weeks to generate obesity. AKI was induced by 30 min of kidney ischemia followed 24 h of reperfusion. Obese rats were given TIIA (10 mg/kg·d) for 2 weeks and CsA (5 mg/kg) 30 min before renal IR. Related indicators were examined.Results: TIIA combined with CsA alleviated the pathohistological injury and apoptosis induced by renal IR in myocardial cells. In addition, TIIA combined with CsA improved cardiac function and decreased the serum myocardial enzyme spectrum in obese rats after renal IR. At the same time, TIIA combined with CsA improved mitochondrial function. Abnormal function of mitochondria was supported by decreases in the respiration controlling rate (RCR), intracellular adenosine triphosphate (ATP), oxygen consumption rate, and mitochondrial membrane potential (MMP), and increases in mitochondrial reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore (mPTP), mitochondrial DNA damage, and mitochondrial respiratory chain complex enzymes (Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ). The injury of mitochondrial dynamic function was assessed by a decrease in Drp1, and increases in Mfn1 and Mfn2, and mitochondrial biogenesis injury was assessed by decreases in PGC-1, NRF1, and TFam. TIIA combined with CsA can attenuate apoptosis through modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.Conclusion: We used isolated mitochondria from rat myocardial tissues to demonstrate that myocardial mitochondrial dysfunction occurred along with renal IR to induce myocardial cell apoptosis; obesity aggravated apoptosis. TIIA combined with CsA attenuated myocardial cell apoptosis by modulating mitochondrial function through the PI3K/Akt/Bad pathway in obese rats.

He Tai, Xiao-lin Jiang, Yue Li, Liang Kong, Si-cheng Yao, Nan Song, Mei-jun Lv, Jin Wu, Ping Yang, Guan-lin Yang, Jin-song Kuang, Zhi-ming Lan, Lian-qun Jia. 2020.

The induction of autophagy, the catabolic pathway by which damaged or unnecessary cellular components are subjected to lysosome-mediated degradation and recycling, is impaired in Collagen VI (COL6) null mice and COL6-related myopathies. This autophagic impairment causes an accumulation of dysfunctional mitochondria, which in turn leads to myofiber degeneration. Our previous work showed that reactivation of autophagy in COL6-related myopathies is beneficial for muscle structure and function both in the animal model and in patients. Here we show that pterostilbene (Pt)—a non-toxic polyphenol, chemically similar to resveratrol but with a higher bioavailability and metabolic stability—strongly promotes in vivo autophagic flux in the skeletal muscle of both wild-type and COL6 null mice. Reactivation of autophagy in COL6-deficient muscles was also paralleled by several beneficial effects, including significantly decreased incidence of spontaneous apoptosis, recovery of ultrastructural defects and muscle remodeling. These findings point at Pt as an effective autophagy-inducing nutraceutical for skeletal muscle with great potential in counteracting the major pathogenic hallmarks of COL6-related myopathies, a valuable feature that may be also beneficial in other muscle pathologies characterized by defective regulation of the autophagic machinery.

Samuele Metti, Lisa Gambarotto, Martina Chrisam, Martina La Spina, Martina Baraldo, Paola Braghetta, Bert Blaauw, Paolo Bonaldo. Frontiers in Cell and Developmental Biology, 8, 2020.

Mycobacterium avium, an opportunistic intracellular pathogen, is a member of the non-tuberculous mycobacteria species. M. avium causes respiratory disease in immunosuppressed individuals and a wide range of animals, including companion dogs and cats. In particular, the number of infected companion dogs has increased, although the underlying mechanism of M. avium pathogenesis in dogs has not been studied. Therefore, in the present study, the host immune response against M. avium in dogs was investigated by transcriptome analysis of canine peripheral blood mononuclear cells. M. avium was shown to induce different immune responses in canine peripheral blood mononuclear cells at different time points after infection. The expression of Th1-associated genes occurred early during M. avium infection, while that of Th17-associated genes increased after 12 h. In addition, the expression of apoptosis-related genes decreased and the abundance of intracellular M. avium increased in monocyte-derived macrophages after infection for 24 h. These results reveal the M. avium induces Th17 immune response and avoids apoptosis in infected canine cells. As the number of M. avium infection cases increases, the results of the present study will contribute to a better understanding of host immune responses to M. avium infection in companion dogs.

Suji Kim, Hyun-Eui Park, Woo Bin Park, Seo Yihl Kim, Hong-Tae Park, Han Sang Yoo. Frontiers in Cellular and Infection Microbiology, 10, 2021.

The NETO2 gene (neuropilin and tolloid-like 2) encodes a protein that acts as an accessory subunit of kainate receptors and is predominantly expressed in the brain. Upregulation of NETO2 has been observed in several tumors; however, its role in tumorigenesis remains unclear. In this study, we investigated NETO2 expression in breast, prostate, and colorectal cancer using quantitative PCR (qPCR), as well as the effect of shRNA-mediated NETO2 silencing on transcriptome changes in colorectal cancer cells. In the investigated tumors, we observed both increased and decreased NETO2 mRNA levels, presenting no correlation with the main clinicopathological characteristics. In HCT116 cells, NETO2 knockdown resulted in the differential expression of 17 genes and 2 long non-coding RNAs (lncRNAs), associated with the upregulation of circadian rhythm and downregulation of several cancer-associated pathways, including Wnt, transforming growth factor (TGF)-β, Janus kinase (JAK)-signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways. Furthermore, we demonstrated the possibility to utilize a novel model organism, short-lived fish Nothobranchius furzeri, for evaluating NETO2 functions. The ortholog neto2b in N. furzeri demonstrated a high similarity in nucleotide and amino acid sequences with human NETO2, as well as was characterized by stable expression in various fish tissues. Collectively, our findings demonstrate the deregulation of NETO2 in the breast, prostate, and colorectal cancer and its participation in the tumor development primarily through cellular signaling.

Maria S. Fedorova, Anastasiya V. Snezhkina, Anastasiya V. Lipatova, Vladislav S. Pavlov, Anastasiya A. Kobelyatskaya, Zulfiya G. Guvatova, Elena A. Pudova, Maria V. Savvateeva, Irina A. Ishina, Tatiana B. Demidova, Nadezhda N. Volchenko, Dmitry Y. Trofimov, Gennady T. Sukhikh, George S. Krasnov, Anna V. Kudryavtseva. Frontiers in Genetics, 11, 2020.

Numerous researches have focused on the genetic variations affecting SARS-CoV-2 infection, whereas the epigenetic effects are inadequately described. In this report, for the first time, we have identified potential candidate genes that might be regulated via SARS-CoV-2 induced DNA methylation changes in COVID-19 infection. At first, in silico transcriptomic data of COVID-19 lung autopsies were used to identify the top differentially expressed genes containing CpG Islands in their promoter region. Similar gene regulations were also observed in an in vitro model of SARS-CoV-2 infected lung epithelial cells (NHBE and A549). SARS-CoV-2 infection significantly decreased the levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in lung epithelial cells. Out of 14 candidate genes identified, the expression of 12 genes was upregulated suggesting promoter hypomethylation, while only two genes were downregulated suggesting promoter hypermethylation in COVID-19. Among those 12 upregulated genes, only HSPA1L and ULBP2 were found to be upregulated in AZA-treated lung epithelial cells and immune cells, suggesting their epigenetic regulation. To confirm the hypomethylation of these two genes during SARS-CoV-2 infection, their promoter methylation and mRNA expression levels were determined in the genomic DNA/RNA obtained from whole blood samples of asymptomatic, severe COVID-19 patients and equally matched healthy controls. The methylation level of HSPA1L was significantly decreased and the mRNA expression was increased in both asymptomatic and severe COVID-19 blood samples suggesting its epigenetic regulation by SARS-CoV-2 infection. Functionally, HSPA1L is known to facilitate host viral replication and has been proposed as a potential target for antiviral prophylaxis and treatment.

Jibran Sualeh Muhammad, Narjes Saheb Sharif-Askari, Zheng-Guo Cui, Mawieh Hamad, Rabih Halwani. Frontiers in Genetics, 12, 2021.

Fusarium is a filamentous fungus commonly found in the environment and is the major cause of fungal keratitis. We report a case of keratomycosis caused by Fusarium solani in a patient using disposable soft contact lenses. A delay in diagnosis led to the initiation of an empirical antifungal treatment with the subsequent deterioration of the patient's clinical condition. The use of the real-time quantitative PCR assay confirmed keratitis from F. solani providing a result in &lt;48 h and therefore giving the possibility of quickly starting targeted antifungal therapy. The patient had an improvement in eye condition after the diagnosis of keratitis by F. solani and the rapid change to targeted antifungal treatment. For the rapid identification of corneal fungal pathogens, we believe that PCR may be added for the diagnosis of mycotic keratitis pending the isolation in culture that is necessary for in vitro susceptibility testing.

Laura Trovato, Antonio Marino, Giovanni Pizzo, Salvatore Oliveri. Frontiers in Medicine, 8, 2021.

Exosomes have been introduced as a new alternative delivery system for the transmission of small molecules. Tumor-derived exosomes (TEXs) not only contain tumor-associated antigens to stimulate antitumor immune responses but also act as natural carriers of microRNAs. The aim of the current study was to evaluate the efficacy of miR-124-3p-enriched TEX (TEXomiR) as cell-free vaccine in the induction of antitumor immune responses in a mouse model of colorectal cancer. Briefly, the exosomes were isolated from cultured CT-26 cell line, and modified calcium chloride method was used to deliver miR-124-3p mimic into the exosomes. We used a CT-26-induced BALB/c mouse model of colorectal cancer and analyzed the effect of TEXomiR on survival, tumor size, spleen and tumor-infiltrated lymphocytes, and splenocyte proliferation. Furthermore, intra-tumor regulatory T cells, cytotoxic activity of the splenocytes, and cytokine secretion was also evaluated to describe the anti-tumor immune response. When the tumor size reached 100 mm³, the mice were injected with TEXomiR, TEX, and/or phosphate-buffered saline (PBS) subcutaneously three times with 3-day interval, and then tumor size was monitored every 2 days. The in vitro results indicated that TEXs could efficiently deliver functional miR-124-3p mimic. The in vivo evaluation in tumor-bearing mice showed that treatment with TEXomiR can elicit a stronger anti-tumor immune response than unloaded TEX and PBS. Significant tumor growth inhibition and increased median survival time was achieved in tumor-bearing mice treated with TEXomiR. A significant decrease in CD4/CD8 and Treg/CD8 ratio in tumor tissue was demonstrated. Moreover, increased cytotoxicity and proliferation of splenocytes in the TEXomiR group compared to the TEX and PBS groups were identified. Taken together, our data demonstrated that tumor-derived exosomes efficiently deliver miR-124-3p mimic, and TEXomiR promotes anti-tumor immune responses.

Ramazan Rezaei, Kaveh Baghaei, Seyed Mahmoud Hashemi, Mohammad Reza Zali, Hossein Ghanbarian, Davar Amani. Frontiers in Medicine, 8, 2021.

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeonHaloferax volcaniito identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase inH. volcaniigrowth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5′ UTR of the gene HVO_1309.In vitroprocessing assays showed that the BHB sites in the 5′ UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.

Thandi S. Schwarz, Sarah J. Berkemer, Stephan H. Bernhart, Matthias Weiß, Sébastien Ferreira-Cerca, Peter F. Stadler, Anita Marchfelder. Frontiers in Microbiology, 11, 2020.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Felix Grünberger, Robert Reichelt, Ingrid Waege, Verena Ned, Korbinian Bronner, Marcell Kaljanac, Nina Weber, Zubeir El Ahmad, Lena Knauss, M. Gregor Madej, Christine Ziegler, Dina Grohmann, Winfried Hausner. Frontiers in Microbiology, 11, 2021.