DNA Ratio Research Articles

An optimized approach for processing of frozen lung and lavage samples for microbiome studies.

The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.

Dietary administration of silk microparticles improves the epidermal and dermal regeneration after a skin wounding in gilthead seabream (Sparus aurata L.)

The impact of dietary supplementation with silk fibroin (SF) microparticles on the wound healing process in gilthead seabream (Sparus aurata) skin was studied. A control diet was enriched with different SF levels: 0 (control), 50 (SF50 diet), and 100 (SF100 diet) mg Kg−1 to form three experimental diets and was fed to seabream for 30 days. Experimental wounds were performed and after 7 days post-wounding (dpw) skin mucus immunity, macroscopic wound closure, and skin regeneration were studied at a microscopic and genetic level. Results indicated that fish fed SF100 did not suffer the decreases in protease and IgM levels observed in the skin mucus of wounded fish fed with the control diet. Macroscopic findings illustrated that dietary SF100 significantly improved the wound closure ratio compared to those reared in the control group. At a microscopic level, changes in the shape of keratocyte cells were evident in the wounded fish. In addition, the intercellular spaces present between epidermal cells and their proliferation in the epidermis, as well as the presence of blood vessels in the dermis were significantly statistically higher in the skin of fish fed the SF100 diet and sampled at 7 dpw compared to those observed in the skin of fish fed the control or SF50 diets. Moreover, regarding the RNA: DNA ratio, statistically significant increases and decreases were observed in fish fed the control and SF100 diet, respectively, in non-wounded and wounded fish. Interestingly, dietary SF100 supplementation improved skin cell proliferation, enhanced the inflammatory phase, and increased the expression of important genes involved in tissue repair and extracellular matrix formation. In conclusion, the SF100 diet can be considered as an appropriate feed additive to improve wound healing in gilthead seabream.

Multiplex Droplet Digital PCR Assay for Detection of MET and HER2 Genes Amplification in Non-Small Cell Lung Cancer.

Simple SummaryNon-small-cell lung cancer (NSCLC) remains one of the most common tumors with a high mortality and morbidity rate. Alterations in HER2 and MET could be a target for anti-tumor drugs or lead to resistance to anti-EGFR therapeutics. Existing methods for detecting HER2 and MET amplifications are time and labor-consuming, and alternative methods are needed. We report the first multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR, and optimal ddPCR conditions were selected. The developed ddPCR was validated on artificial samples with various DNA concentrations and MET and HER2 ratios. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed, and, among them, five specimens (1.15%) were MET-positive and six samples (1.38%) were HER2-positive. The multiplex ddPCR assay could be used for screening MET and HER2 amplification in NSCLC samples.Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

Effects of dietary lipid levels on growth, feed utilization, RNA/DNA ratio, digestive tract enzyme activity, non-specific immune response and optimum inclusion in feeds for fingerlings of rohu, Labeo rohita (Hamilton)

The current investigation was conducted to assess the effects of different levels of dietary lipid on growth performance, feed utilization, digestive tract enzyme activity, lipid deposition, and non-specific immune response of fingerling Labeo rohita (2.23 ± 0.06 g). Six isonitrogenous (400 g kg−1) purified diets with increasing levels of lipid (20, 40, 60, 80, 100, 120 g kg−1 diet) were fed to triplicate groups of 30 fish per tank at 08:00, 12:00, and 16:00 h to apparent satiety for 8 weeks. Absolute weight gain (AWG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), protein retention efficiency (PRE) and RNA:DNA ratio improved significantly (P < 0.05) up to 100 g kg−1 lipid in diet. Somatic indices and whole body lipid positively correlated with the increase in dietary lipid. Dietary lipid levels significantly affected hepatic antioxidant activities. Liver superoxide dismutase (SOD) and catalase (CAT) activities improved significantly up to 100 g kg−1 lipid in diet. However, liver thiobarbituric acid reactive substances activity (TBARS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities showed a negative correlation with the increase in levels of lipid in the diets. Highest intestinal protease and lipase activity was also noted in fish received 100 g kg−1 lipid in diet. Second-order polynomial analysis of AWG, FCR, RNA/DNA ratio, SOD and serum lysozyme activity against varying levels of dietary lipid reflected the requirement in the range of 94.77–98.35 g kg−1 dietary lipid for optimum growth of L. rohita.

Anti-cancer Effects of 5-Aminoimidazole-4-Carboxamide-1-β-D-Ribofuranoside (AICAR) on Triple-negative Breast Cancer (TNBC) Cells: Mitochondrial Modulation as an Underlying Mechanism.

Triple-negative breast cancer (TNBC) is known for Warburg effect and defects in the mitochondria. AMP-dependent kinase (AMPK) activates the downstream transcription factors PGC-1α, PGC-1β, or FOXO1, which participate in mitochondrial biogenesis. 5- aminoimidazole-4-carboxamide riboside (AICAR) is an analog of adenosine monophosphate and is a direct activator of AMPK. In the present study, we have made an attempt to understand the influence of AICAR on TNBC cells, MDA-MB-231, and the underlying changes in mitochondrial biogenesis, if any. We investigated AICAR induced changes in cell viability, apoptosis, migratory potential, and changes in the sensitivity of doxorubicin. In response to the treatment of MDA-MB-231 breast cancer cells with 750 μM of AICAR for 72 hours, followed by 48 hours in fresh media without AICAR, we observed a decrease in viability via MTT assay, reduction in cell numbers along with the apoptotic appearance, increased cell death by ELISA, decreased lactate in conditioned medium and decrease in migration by scratch and transwell migration assays. These changes in the cancer phenotype were accompanied by an increase in mitochondrial biogenesis, as observed by increased mitochondrial DNA to nuclear DNA ratio, a decrease in lactic acid concentration, an increase in MitoTracker green and red staining, and increased expression of transcription factors PGC-1α, NRF-1, NRF-2, and TFAM, contributing to mitochondrial biogenesis. Pre-treatment of cells with AICAR for 72 hours followed by 48 hours treatment with 1 μM doxorubicin showed an increased sensitivity to doxorubicin as assessed by the MTT assay. Our results show that AICAR exerts beneficial effects on TNBC cells, possibly via switching off the Warburg effect and switching on the anti-Warburg effect through mitochondrial modulation.

Application of droplet digital PCR method for DNA methylation-based age prediction from saliva.

The recent studies reported that DNA methylation markers show changes with age, and expected that the DNA methylation markers can be effectively used for estimation of age in forensic genetics. In this study, we applied droplet digital PCR (ddPCR) method to investigate the DNA methylation pattern in the CpG sites, and we constructed an age prediction model based on the ddPCR method. The ddPCR is capable of highly sensitive quantitation of nucleic acid and detection of sequence variations in gene by separating the sample into large number of partitions and clonally amplifying nucleic acids in each partition. We extracted DNA from saliva samples collected from several age groups. The DNA was bisulfite converted and subjected to ddPCR using specifically designed primers and probes. The methylation ratio of each sample was calculated and correlation between the methylation ratio and the chronological age was analyzed. In the results, methylated DNA ratio at the 4 CpG sites (cg14361627, cg14361627, cg08928145 and cg07547549) showed strong correlation with chronological age. Percent-methylation values at 4 CpG markers and chronological ages of the 76 individuals were analyzed by multiple regression analysis, and we constructed an age prediction model. We observed a strong correlation (Spearman's rho=0.922) between predicted and chronological ages of 76 individuals with a MAD from chronological age of 3.3years. Collectively, the result in this study showed the potential applicability of ddPCR to predict age from saliva.

Process improvement of adeno-associated virus (AAV) production.

Adeno-associated viruses (AAVs) have been well characterized and used to deliver therapeutic genes for diseases treatment in clinics and basic research. This study used the triple transient transfection of AAV-DJ/8 as a model expression system to develop and optimize the laboratory production of AAV for research and pre-clinical applications. Specifically, various production parameters, including host cell, transfection reagent, cell density, ratio of plasmid DNA and cells, gene size, and production mode, were tested to determine the optimal process. Our results showed that the adherent production using HEK 293AAV with calcium transfection generated the highest volumetric productivity of 7.86x109 gc/mL. The optimal suspensive production using HEK 293F had best AAV productivity of 5.78x109 gc/mL in serum-free medium under transfection conditions of transfection density of 0.4x106 cells/mL, plasmid DNA:cells ratio of 1.6 μg:106 cells and synthesized cationic liposomes as transfection reagent. The similar AAV productivity was confirmed at scales of 30 mL - 450 mL in shaker and/or spinner flasks. The in vitro transfection and in vivo infection efficiency of the harvested AAV-DJ/8 carrying luciferase reporter gene was confirmed using cell line and xenograft mouse model, respectively. The minimal or low purification recovery rate of AAV-DJ/8 in ion-exchange chromatography column and affinity column was observed in this study. In summary, we developed and optimized a scalable suspensive production of AAV to support the large-scale preclinical animal studies in research laboratories.

Associations of Maternal Polyunsaturated Fatty Acids With Telomere Length in the Cord Blood and Placenta in Chinese Population.

Few studies have investigated the correlation between maternal polyunsaturated fatty acids (PUFAs) and telomeres in offspring, and the underlying influential mechanisms. In this study, we assessed the associations of maternal PUFAs with telomere length (TL) and DNA methylation of the telomerase reverse transcriptase (TERT) promoter in the cord blood and the placenta. A total of 274 pregnant women and their newborn babies were enrolled in this study. Maternal blood before delivery, the cord blood, and the placenta at birth were collected. Fatty acids in maternal erythrocytes and cord blood cells were measured by gas chromatography (GC). TL in the cord blood and the placenta was determined using real-time quantitative PCR (qPCR) by calculating the product ratio of telomeric DNA to the single-copy gene β-globin. The TERT promoter methylation was analyzed by DNA bisulfite sequencing. The associations of maternal fatty acids with TL were analyzed by univariate and multivariate regression. We found that low concentrations of docosapentaenoci acid (DPA, C22: 5n-3) and total n-3 PUFAs, adrenic acid (ADA, C22: 4n-6), and osbond acid (OA, C22: 5n-6) and high concentrations of linoleic acid (LA, C18: 2n-6) in maternal erythrocytes were associated with the shortened TL in cord blood cells (estimated difference in univariate analysis −0.36 to −0.46 for extreme quintile compared with middle quintile), and that low concentrations of cord blood docosahexaenoic acid (DHA, C22: 6n-3) were related to the shortened TL in cord blood cells. Differently, high concentrations of α-linolenic acid (LNA, C18: 3n-3), eicosatrienoic acid (EA, C20: 3n-3), DHA, and γ-linoleic acid (GLA, C18:3n-6) in maternal erythrocytes were associated with the shortened TL in the placenta (estimated difference in univariate analysis −0.36 to −0.45 for higher quintiles compared with the middle quintile). Further examination demonstrated that the concentrations of DHA and total n-3 PUFAs in maternal erythrocytes had positive associations with DNA methylation of the TERT promoter in the cord blood instead of the placenta. These data suggest that maternal PUFAs are closely correlated to infant TL and the TERT promoter methylation, which are differently affected by maternal n-3 PUFAs between the cord blood and the placenta. Therefore, keeping higher levels of maternal n-3 PUFAs during pregnancy may help to maintain TL in the offspring, which is beneficial to long-term health.

Pembrolizumab induces HIV latency reversal in people living with HIV and cancer on antiretroviral therapy

In people living with HIV (PLWH) on antiretroviral therapy (ART), virus persists in a latent form where there is minimal transcription or protein expression. Latently infected cells are a major barrier to curing HIV. Increasing HIV transcription and viral production in latently infected cells could facilitate immune recognition and reduce the pool of infected cells that persist on ART. Given that programmed cell death protein 1 (PD-1) expressing CD4+ T cells are preferentially infected with HIV in PLWH on ART, we aimed to determine whether administration of antibodies targeting PD-1 would reverse HIV latency in vivo. We therefore evaluated the impact of intravenous administration of pembrolizumab every 3 weeks on HIV latency in 32 PLWH and cancer on ART. After the first infusion of anti-PD-1, we observed a median 1.32-fold increase in unspliced HIV RNA and 1.61-fold increase in unspliced RNA:DNA ratio in sorted blood CD4+ T cells compared to baseline. We also observed a 1.65-fold increase in plasma HIV RNA. The frequency of CD4+ T cells with inducible virus evaluated using the tat/rev limiting dilution assay was higher after 6 cycles compared to baseline. Phylogenetic analyses of HIV env sequences in a participant who developed low concentrations of HIV viremia after 6 cycles of pembrolizumab did not demonstrate clonal expansion of HIV-infected cells. These data are consistent with anti-PD-1 being able to reverse HIV latency in vivo and support the rationale for combining anti-PD-1 with other interventions to reduce the HIV reservoir.

Artificial light at night (ALAN) causes variable dose-responses in a sandy beach isopod.

Artificial Light at Night (ALAN) is expanding worldwide, and the study of its influence remains limited mainly to documenting impacts, overlooking the variation in key characteristics of the artificial light such as its intensity. The potential dose-response of fitness-related traits to different light intensities has not been assessed in sandy beach organisms. Hence, this study explored dose-responses to ALAN by exposing the intertidal sandy beach isopodTylos spinulosusto a range of light intensities at night: 0 (control), 20, 40, 60, 80 and 100lx. We quantified the response of this species at the molecular (RNA:DNA ratios), physiological (absorption efficiency) and organismal (growth rate) levels. Linear and non-linear regressions were used to explore the relationship between light intensity and the isopod response. The regressions showed thatincreasing light intensity caused an overall ~ threefold decline in RNA:DNA ratios and a ~ threefold increase in absorption efficiency, with strong dose-dependent effects. For both response variables, non-linear regressions also identified likely thresholds at 80lx (RNA:DNA) and 40lx (absorption efficiency). By contrast, isopod growth rates were unrelated (unaltered) by the increase in light intensity at night. We suggest that ALAN is detrimental for the condition of the isopods, likely by reducing the activity and feeding of these nocturnal organisms, and that the isopods compensate this byabsorbing nutrients more efficiently in order to maintain growth levels.

Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation.

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

No protective benefits of low dose acute L-glutamine supplementation on small intestinal permeability, epithelial injury and bacterial translocation biomarkers in response to subclinical exertional-heat stress: A randomized cross-over trial

ABSTRACT Exertional heat stress disrupts gastrointestinal permeability and, through subsequent bacterial translocation, can result in potentially fatal exertional heat stroke. Glutamine supplementation is a potential countermeasure although previously validated doses are not universally well tolerated. Ten males completed two 80-minute subclinical exertional heat stress tests (EHSTs) following either glutamine (0.3 g kg FFM−1) or placebo supplementation. Small intestinal permeability was assessed using the lactulose/rhamnose dual sugar absorption test and small intestinal epithelial injury using Intestinal Fatty-Acid Binding Protein (I-FABP). Bacterial translocation was assessed using the total 16S bacterial DNA and Bacteroides/total 16S DNA ratio. The glutamine bolus was well tolerated, with no participants reporting symptoms of gastrointestinal intolerance. Small intestinal permeability was not influenced by glutamine supplementation (p = 0.06) although a medium effect size favoring the placebo trial was observed (d = 0.73). Both small intestinal epithelial injury (p < 0.01) and Bacteroides/total 16S DNA (p = 0.04) increased following exertional heat stress, but were uninfluenced by glutamine supplementation. Low-dose acute oral glutamine supplementation does not protect gastrointestinal injury, permeability, or bacterial translocation in response to subclinical exertional heat stress.

Multivalent Display of Lipophilic DNA Binders for Dual-Selective Anti-Mycobacterium Peptidomimetics with Binary Mechanism of Action

Multivalent Display of Lipophilic DNA Binders for Dual-Selective Anti-Mycobacterium Peptidomimetics with Binary Mechanism of Action

Mitochondrial DNA copy number as a prognostic marker is age-dependent in adult glioblastoma.

BackgroundGlioblastoma (GBM) is the most common and aggressive form of glioma. GBM frequently displays chromosome (chr) 7 gain, chr 10 loss and/or EGFR amplification (chr7+/chr10-/EGFRamp). Overall survival (OS) is 15 months after treatment. In young adults, IDH1/2 mutations are associated with longer survival. In children, histone H3 mutations portend a dismal prognosis. Novel reliable prognostic markers are needed in GBM. We assessed the prognostic value of mitochondrial DNA (mtDNA) copy number in adult GBM.MethodsmtDNA copy number was assessed using real-time quantitative PCR in 232 primary GBM. Methylation of POLG and TFAM genes, involved in mtDNA replication, was assessed by bisulfite-pyrosequencing in 44 and 51 cases, respectively.ResultsMedian age at diagnosis was 56.6 years-old and median OS, 13.3 months. 153/232 GBM (66 %) displayed chr7+/chr10-/EGFRamp, 23 (9.9 %) IDH1/2 mutation, 3 (1.3 %) H3 mutation and 53 (22.8 %) no key genetic alterations. GBM were divided into two groups, “Low” (n = 116) and “High” (n = 116), according to the median mtDNA/nuclear DNA ratio (237.7). There was no significant difference in OS between the two groups. By dividing the whole cohort according to the median age at diagnosis, OS was longer in the “High” vs “Low” subgroup (27.3 vs 15 months, P = .0203) in young adult GBM (n = 117) and longer in the “Low” vs “High” subgroup (14.5 vs 10.2 months, P = .0116) in older adult GBM (n = 115). POLG was highly methylated, whereas TFAM remained unmethylated.ConclusionmtDNA copy number may be a novel prognostic biomarker in GBM, its impact depending on age.

Cow–calf efficiency of beef cows grazing different herbage allowances of rangelands: hepatic mechanisms related to energy efficiency

Context Improvement in efficiency of energy utilisation of cow–calf systems could benefit beef production profitability and sustainability. Energy efficiency has been associated with mitochondrial function; therefore, hepatic mitochondrial function and oxidative stress could play a key role in energy efficiency of beef cows. Aim We evaluated the effect of two herbage allowances from rangelands (Campos biome) on cow–calf energy efficiency, hepatic mitochondrial density and function, and oxidative stress of purebred (Hereford and Aberdeen Angus) and reciprocal F1 crossbred beef cows. Methods Mature cows (n = 32) were used in a complete randomised block design with a factorial arrangement of herbage allowance (2.5 vs 4 kg dry matter/day; low vs high) and cow genotype (pure vs crossbred) over 3 years. At the end of the third year, cows were slaughtered at 190 ± 10 days postpartum. Liver was dissected and weighed, and samples were collected and snap-frozen pending analysis of mitochondrial density and oxidative stress markers. Estimated cow–calf energy efficiency was calculated by using total cow estimated metabolisable energy intake as input and calf energy retained at weaning as output. Key results Cow–calf energy efficiency was greater (P ≤ 0.07) for high than low herbage allowance and for crossbred than purebred cows. Mitochondrial density biomarkers (hepatic citrate synthase enzyme activity, citrate synthase mRNA, and mitochondrial:nuclear DNA ratio) were greater (P ≤ 0.03) for high than low herbage allowance. Plasma pro-oxidants and plasma antioxidant capacity were greater (P ≤ 0.07) for crossbred than purebred cows. Plasma oxidative stress index and expression of hepatic 4-hydroxynonenal protein adducts were affected (P ≤ 0.06) by herbage allowance × cow genotype interaction. Conclusion Greater cow–calf energy efficiency was associated with greater hepatic mitochondrial density without differences in mitochondrial function. Contrary to expectation, greater efficiency of crossbred than purebred cows was associated with increased hepatic oxidative damage, which probably reflects greater liver metabolic activity in crossbreds. Implications Herbage allowance and cow genotype affect cow–calf efficiency, hepatic mitochondrial function and oxidative stress markers. Greater efficiency of crossbred cows seems associated with increased hepatic oxidative damage.

Anti-cancer effects of sitagliptin, vildagliptin, and exendin-4 on triple-negative breast cancer cells via mitochondrial modulation

Triple-negative breast cancer (TNBC) cell line MDA-MB-231 is known for Warburg metabolism and defects in mitochondria. On the other hand, dipeptidyl peptidase-IV (DPP-IV) inhibitors such as sitagliptin and vildagliptin and GLP-1 agonist exendin-4 are known to improve mitochondrial functions as well as biogenesis, but no study has evaluated the influence of these drugs on mitochondrial biogenesis on metastatic breast cancer cell line. We have recently reported anticancer effects of 5-aminoimidazole-4-carboxamide riboside on MDA-MB-231 cells via activation of AMP-dependent kinase (AMPK), which activates the downstream transcription factors PGC-1α, PGC-1β, or FOXO1 for mitochondrial biogenesis; above-mentioned incretin-based therapies are also known to activate AMPK. This study evaluated the effects of sitagliptin, vildagliptin, and exendin-4 on MDA-MB-231 cells and the underlying changes in mitochondrial biogenesis, were examined. Treatment with sitagliptin (100 µM), vildagliptin (100 µM), and exendin-4 (10 nM) for 72 h to MDA-MB-231 cells led to a decrease in viability indicated by MTT assay, cell migration by scratch, and transwell migration assays, accompanied with marginal reduction in cell numbers along with the apoptotic appearance, the rate of apoptosis, and decreased lactate content in conditioned medium. These changes in the cancer phenotype were accompanied by an increase in the mitochondrial DNA to nuclear DNA ratio, increased MitoTracker green and red staining, and increased expression of transcription factors PGC-1α, NRF-1, NRF-2, TFAM, and HO-1. Pre-treatment of cells with these incretin-based drugs followed by 48 h treatment with 1 µM doxorubicin increased doxorubicin sensitivity as observed by a decrease in viability by MTT assay. Thus, sitagliptin, vildagliptin, and exendin-4 exert their beneficial effects on TNBC cells via an increase in mitochondrial biogenesis that helps to switch Warburg metabolism into anti-Warburg effect. Therapeutic response was in the order of: sitagliptin > vildagliptin > exendin-4.

The intratumoral microbiome: Characterization methods and functional impact.

Live-pathogenic bacteria, which were identified inside tumors hundreds year ago, are key elements in modern cancer research. As they have a relatively accessible genome, they offer a multitude of metabolic engineering opportunities, useful in several clinical fields. Better understanding of the tumor microenvironment and its associated microbiome would help conceptualize new metabolically engineered species, triggering efficient therapeutic responses against cancer. Unfortunately, given the low microbial biomass nature of tumors, characterizing the tumor microbiome remains a challenge. Tumors have a high host versus bacterial DNA ratio, making it extremely complex to identify tumor-associated bacteria. Nevertheless, with the improvements in next-generation analytic tools, recent studies demonstrated the existence of intratumor bacteria inside defined tumors. It is now proven that each cancer subtype has a unique microbiome, characterized by bacterial communities with specific metabolic functions. This review provides a brief overview of the main approaches used to characterize the tumor microbiome, and of the recently proposed functions of intracellular bacteria identified in oncological entities. The therapeutic aspects of live-pathogenic microbes are also discussed, regarding the tumor microenvironment of each cancer type.

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus caryophyllus semperflorens flore pleno) Cultivars

Abstract Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm−3 of 2,4-D and 200 mg·dm−3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm−3 of BA, 0.6 mg·dm−3 of NAA, 5 mg·dm−3 of AgNO3, and 40 mg·dm−3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm−3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Acute L-glutamine supplementation does not improve gastrointestinal permeability, injury or microbial translocation in response to exhaustive high intensity exertional-heat stress

ABSTRACT Purpose: Exertional-heat stress adversely distrupts (GI) barrier integrity and, through subsequent microbial translocation (MT), can result in potentially fatal exertional-heat stroke. Acute glutamine (GLN) supplementation is a potential nutritional countermeasure, although the practical value of current supplementation regimens is questionable. Method: Ten males completed two high-intensity exertional-heat stress tests (EHST) involving running in the heat (40°C and 40% relative humidity) at lactate threshold to volitional exhaustion. Participants ingested GLN (0.3 g kg FFM−1) or a non-calorific placebo (PLA) one hour prior to the EHST. Venous blood was drawn pre-, post- and one-hour post-EHST. GI permeability was assessed using a serum dual-sugar absorption test (DSAT) and small intestinal epithelial injury using plasma Intestinal Fatty-Acid Binding Protein (I-FABP). MT was assessed using the Bacteroides/total 16S DNA ratio. Results: Volitional exhaustion occurred after 22:19 ± 2:22 (minutes: seconds) in both conditions, during which whole-body physiological responses and GI symptoms were not different (p > 0.05). GI permeability (serum DSAT) was greater following GLN (0.043 ± 0.020) than PLA (0.034 ± 0.019) (p = 0.02; d = 0.47), but small intestine epithelial injury (I-FABP) increased comparably (p = 0.22; = 0.16) following the EHST in both trials (GLN Δ = 1.25 ± 0.63 ng ml−1; PLA Δ = 0.92 ± 0.44 ng ml−1). GI MT (Bacteroides/total 16S DNA ratio) was unchanged in either condition following the EHST (p = 0.43). Conclusion: Acute low-dose (0.3 g kg−1 fat free mass) GLN supplementation ingested one hour before high-intesity exertional-heat stress worsened GI permeability, but did not influence either small intestinal epithilial injury or microbial translocation. Abbreviations: ANOVA: Analysis of variance; CV: Coefficient of Variation; DSAT: Dual Sugar Absorption Test; EDTA: Ethylenediaminetetraacetic acid; EHST: Exertional Heat Stress Test; ELISA: Enzyme Linked Immunosorbent Assay; FFM: Fat Free Mass; GI: Gastrointestinal; GFR: Glomerular Filtration Rate; GLN: Glutamine; HPLC: High Performance Liquid Chromatography; HR: Heart Rate; I-FABP: Intestinal Fatty-Acid Binding Protein; ISAK: International Society for the Advancement of Anthropometric Kinanthropometry; L/R: Lactulose-to-Rhamnose; LT: Lactate Threshold; MT: Microbial Translocation; mVAS: Modified Visual Analogue Scale; PBS: Phosphate-Buffered Saline; PLA: Placebo; qPCR: Quantitative Polymerase Chain Reaction; RH: Relative Humidity; RPE: Rate of Perceived Exertion; SD: Standard Deviation; SEM: Sensor Electronics Module; Tcore: Core Body Temperature; Tbody: Mean Body Temperature; Tskin: Mean Skin Temperature; TS: Thermal Sensation; V̇O2max: Maximal Oxygen Uptake. Highlights The pathophysiology of exertional-heat stroke is widely hypothesised to be at least in part attributable to a systemic inflammatory response caused by the leak of gastrointestinal microbes into the circulating blood. Acute high-dose (0.9 g kg FFM−1) L-glutamine supplementation is widely promoted as a practical strategy to protect gastrointestinal barrier integrity during exertional-heat stress. However, previously validated doses are often poorly tolerated and cannot be recommended for widespread implementation. This study examined the efficacy of low-dose (0.30 g kg FFM−1; ∼20 grams) acute L-glutamine supplementation on small intestinal injury, permeability, and microbial translocation in response a high-intensity exertional-heat stress test to exhaustion (20–30 min). This type of exercise accounts for the majority of exertional-heat stroke cases in the military. Despite being universally well-tolerated across all participants, acute low-dose L-glutamine supplementation worsened gastrointestinal permeability, without influencing either small intestinal injury or microbial translocation. These findings do not support the application of low-dose L-glutamine supplementation to help prevent exertional-heat stroke.

IRF7 and RNH1 are modifying factors of HIV-1 reservoirs: a genome-wide association analysis

BackgroundCombination antiretroviral treatment (cART) cannot eradicate HIV-1 from the body due to the establishment of persisting viral reservoirs which are not affected by therapy and reinitiate new rounds of HIV-1 replication after treatment interruption. These HIV-1 reservoirs mainly comprise long-lived resting memory CD4+ T cells and are established early after infection. There is a high variation in the size of these viral reservoirs among virally suppressed individuals. Identification of host factors that contribute to or can explain this observed variation could open avenues for new HIV-1 treatment strategies.MethodsIn this study, we conducted a genome-wide quantitative trait locus (QTL) analysis to probe functionally relevant genetic variants linked to levels of cell-associated (CA) HIV-1 DNA, CA HIV-1 RNA, and RNA:DNA ratio in CD4+ T cells isolated from blood from a cohort of 207 (Caucasian) people living with HIV-1 (PLHIV) on long-term suppressive antiretroviral treatment (median = 6.6 years). CA HIV-1 DNA and CA HIV-1 RNA levels were measured with corresponding droplet digital PCR (ddPCR) assays, and genotype information of 522,455 single-nucleotide variants was retrieved via the Infinium Global Screening array platform.ResultsThe analysis resulted in one significant association with CA HIV-1 DNA (rs2613996, P < 5 × 10−8) and two suggestive associations with RNA:DNA ratio (rs7113204 and rs7817589, P < 5 × 10−7). Then, we prioritized PTDSS2, IRF7, RNH1, and DEAF1 as potential HIV-1 reservoir modifiers and validated that higher expressions of IRF7 and RNH1 were accompanied by rs7113204-G. Moreover, RNA:DNA ratio, indicating relative HIV-1 transcription activity, was lower in PLHIV carrying this variant.ConclusionsThe presented data suggests that the amount of CA HIV-1 DNA and RNA:DNA ratio can be influenced through PTDSS2, RNH1, and IRF7 that were anchored by our genome-wide association analysis. Further, these observations reveal potential host genetic factors affecting the size and transcriptional activity of HIV-1 reservoirs and could indicate new targets for HIV-1 therapeutic strategies.