Identification of valid reference genes during the differentiation of human myoblasts

<p>Abstract</p> <p>Background</p> <p>Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.</p> <p>Results</p> <p>Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.</p> <p>Conclusion</p> <p>RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.</p

Lower hypoxic ventilatory response in smokers compared to non-smokers during abstinence from cigarettes

Background Carotid body O2-chemosensitivity determines the hypoxic ventilatory response (HVR) as part of crucial regulatory reflex within oxygen homeostasis. Nicotine has been suggested to attenuate HVR in neonates of smoking mothers. However, whether smoking affects HVR in adulthood has remained unclear and probably blurred by acute ventilatory stimulation through cigarette smoke. We hypothesized that HVR is substantially reduced in smokers when studied after an overnight abstinence from cigarettes i.e. after nicotine elimination. Methods We therefore determined the isocapnic HVR of 23 healthy male smokers (age 33.9 ± 2.0 years, BMI 24.2 ± 0.5 kg m−2, mean ± SEM) with a smoking history of >8 years after 12 h of abstinence and compared it to that of 23 healthy male non-smokers matched for age and BMI. Results Smokers and non-smokers were comparable with regard to factors known to affect isocapnic HVR such as plasma levels of glucose and thiols as well as intracellular levels of glutathione in blood mononuclear cells. As a new finding, abstinent smokers had a significantly lower isocapnic HVR (0.024 ± 0.002 vs. 0.037 ± 0.003 l min−1 %−1BMI−1, P = 0.002) compared to non-smokers. However, upon re-exposure to cigarettes the smokers’ HVR increased immediately to the non-smokers’ level. Conclusions This is the first report of a substantial HVR reduction in abstinent adult smokers which appears to be masked by daily smoking routine and may therefore have been previously overlooked. A low HVR may be suggested as a novel link between smoking and aggravated hypoxemia during sleep especially in relevant clinical conditions such as COPD

The Effect of Obstructive Sleep Apnea and Continuous Positive Airway Pressure Therapy on Skeletal Muscle Lipid Content in Obese and Nonobese Men.

Obstructive sleep apnea (OSA), independently of obesity (OBS), predisposes to insulin resistance (IR) for largely unknown reasons. Because OSA-related intermittent hypoxia triggers lipolysis, overnight increases in circulating free fatty acids (FFAs) including palmitic acid (PA) may lead to ectopic intramuscular lipid accumulation potentially contributing to IR. Using 3-T-1H-magnetic resonance spectroscopy, we therefore compared intramyocellular and extramyocellular lipid (IMCL and EMCL) in the vastus lateralis muscle at approximately 7 am between 26 male patients with moderate-to-severe OSA (17 obese, 9 nonobese) and 23 healthy male controls (12 obese, 11 nonobese). Fiber type composition was evaluated by muscle biopsies. Moreover, we measured fasted FFAs including PA, glycated hemoglobin A1c, thigh subcutaneous fat volume (ScFAT, 1.5-T magnetic resonance tomography), and maximal oxygen uptake (VO2max). Fourteen patients were reassessed after continuous positive airway pressure (CPAP) therapy. Total FFAs and PA were significantly (by 178% and 166%) higher in OSA patients vs controls and correlated with the apnea-hypopnea index (AHI) (r ≥ 0.45, P < .01). Moreover, IMCL and EMCL were 55% (P < .05) and 40% (P < .05) higher in OSA patients, that is, 114% and 103% in nonobese, 24.4% and 8.4% in obese participants (with higher control levels). Overall, PA, FFAs (minus PA), and ScFAT significantly contributed to IMCL (multiple r = 0.568, P = .002). CPAP significantly decreased EMCL (-26%) and, by trend only, IMCL, total FFAs, and PA. Muscle fiber composition was unaffected by OSA or CPAP. Increases in IMCL and EMCL are detectable at approximately 7 am in OSA patients and are partly attributable to overnight FFA excesses and high ScFAT or body mass index. CPAP decreases FFAs and IMCL by trend but significantly reduces EMCL

Cellular Imaging of Human Atherosclerotic Lesions by Intravascular Electric Impedance Spectroscopy

Background: Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) is able to provide information about the cellular composition of biological tissue. The present study was performed to determine the influence of inflammatory processes in type Va (lipid core, thick fibrous cap) and Vc (abundant fibrous connective tissue while lipid is minimal or even absent) human atherosclerotic lesions on the electrical impedance of these lesions measured by EIS. Methods and Results: EIS was performed on 1 aortic and 3 femoral human arteries at 25 spots with visually heavy plaque burden. Severely calcified lesions were excluded from analysis. A highly flexible micro-electrode mounted onto a balloon catheter was placed on marked regions to measure impedance values at 100 kHz. After paraffin embedding, visible marked cross sections (n = 21) were processed. Assessment of lesion types was performed by Movats staining. Immunostaining for CD31 (marker of neovascularisation), CD36 (scavenger cells) and MMP-3 (matrix metalloproteinase-3) was performed. The amount of positive cells was assessed semi-quantitatively. 15 type Va lesions and 6 type Vc lesions were identified. Lesions containing abundant CD36-, CD31- and MMP-3-positive staining revealed significantly higher impedance values compared to lesions with marginal or without positive staining (CD36+455650 V vs. CD36- 346653 V, p = 0.001; CD31+436643 V vs. CD31- 340655 V, p = 0.001; MMP-3+ 400668 V vs. MMP-3- 323633 V, p = 0.03)

Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations

Einfluss von PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide) beziehungsweise des PACAP-Rezeptors PAC1 auf die Morphologie der Nierenrinde bei cholesterin- und normalgefütterten ApoE-/- Mäusen

Hintergrund: Krankheiten des Herz-Kreislaufsystems waren im Jahr 2015 mit 39% die häufigste Todesursache in Deutschland und sind weltweit ein großes gesundheitliches Problem. Die Atherosklerose nimmt in diesen Pathologien einen nicht zu vernachlässigenden Stellenwert ein. Im entzündlichen Entstehungsprozess der chronischen Gefäßerkrankung spielen Lipidstoffwechsel und Makrophagen eine wichtige Rolle. Das ApoE-/- Mausmodell ist etabliert, um Atherosklerose und dessen Einflussfaktoren sowie Nierenerkrankungen zu untersuchen. Das Peptid PACAP (Pituitary Adenylate Cyclase-Activating Protein) gehört zur Glucagon-PACAP-VIP Protein-Superfamilie. Es wirkt antiinflammatorisch, antiapoptotisch und antioxidativ und zeigt diese Eigenschaften auch in der Niere in einem nephroprotektiven Effekt. In dieser Studie wurde erstmals untersucht, inwieweit die Defizienz von PACAP bzw. dessen Rezeptor PAC1 im ApoE-/- Mausmodell inflammatorische Reaktionen und morphologische Veränderungen der Niere hervorrufen und/oder beeinflussen. Zusätzlich wurde der Einfluss der Verabreichung eines cholesterinangereicherten Futters auf diese Parameter untersucht. Material und Methoden: Für die Untersuchung standen folgende Maus-Genotypen zur Verfügung: WT, ApoE-/-, PACAP-/-/ApoE-/- bzw. PAC1-/-/ApoE-/-. Den jeweils zehn Wochen alten Mäusen wurde für 20 Wochen entweder Normalfutter (NF) oder cholesterinangereicherte Nahrung (CED) verabreicht. Es ergaben sich insgesamt sieben Mausgruppen, vom WT gab es nur eine CED Gruppe. Körpergewicht und Plasma-Cholesterin- sowie Triglyceridspiegel wurden gemessen. Nach Tötung der Tiere erfolgte die Organentnahme. Die Nieren wurden kryofixiert. 6μm dünne Kryoschnitte der Niere wurden in einer Azan- und ORO-Färbung histologisch, histomorphometrisch und immunhistologisch mittels der Antikörper MOMA-2 (Monozyten und Makrophagen), F4/80 (vorwiegend residente Makrophagen), TNFα und IL-6 (proinflammatorische Zytokine) gefärbt und analysiert. Anhand der Azanfärbung wurden die Nierenschnitte bezüglich Anzahl, Durchmesser und Fläche der Glomeruli sowie ihres Bindegewebsanteils untersucht und vermessen. Ergebnisse: Die verabreichte CED führte innerhalb der PACAP-/-/ApoE-/- Mäuse zu signifikant schwereren Tieren als unter NF. Die PACAP-/-/ApoE-/- Mäuse waren nach CED signifikant schwerer als die ApoE-/- Mäuse (Kontrolle). Die PACAP-/-/ApoE-/- Mäuse waren unter NF jedoch leichter als die ApoE-/- Mäuse. Hier besteht eine Wechselwirkung zwischen Gewicht und Genotyp. Die Plasma-Cholesterinspiegel waren nach CED bei ApoE-/-, PACAP-/-/ApoE-/- und PAC1-/-/ApoE-/- Mäusen signifikant höher als nach NF. Nach CED waren die Plasma-Triglyceridspiegel der PACAP-/-/ApoE-/- Mäuse signifikant höher als die der ApoE-/- Mäuse. Bei der morphologischen Untersuchung ergab sich nach CED eine signifikant erhöhte Anzahl an Nierenkörperchen bei den PACAP-/-/ApoE-/- Mäusen im Vergleich zu den ApoE-/- Mäusen. Die Fläche der Nierenkörperchen war im Vergleich zu den ApoE-/- Mäusen nach CED bei den PAC1-/-/ApoE-/- Mäusen signifikant um 22%, bei den WT Mäusen um 21% niedriger. Auch bzgl. des Durchmessers der Nierenkörperchen waren diejenigen der PAC1-/-/ApoE-/- Mäuse bei NF und CED kleiner als bei den ApoE-/- Mäusen. Bezüglich des renalen Bindegewebsanteils bzw. einer möglichen Fibrosierung konnten wir keine signifikanten Unterschiede zwischen Futtergruppen und Genotypen nachweisen. Die ApoE-/- Mäuse zeigten nach CED eine doppelt so hohe Dichte an MOMA-2+ Zellen im Nierenkortex wie die WT Mäuse. Die PAC1-/-/ApoE-/- Mäuse zeigten nach CED eine viermal geringere Dichte an F4/80+ Zellen im Nierenkortex als die ApoE-/- Mäuse (Kontrolle). Innerhalb der ApoE-/- Mäuse bewirkte die CED eine um 2,5fach höhere Dichte an F4/80+ Zellen als bei Verabreichung von NF. Der Futterunterschied wirkte sich auch auf die Dichte der IL-6+ Zellen im Nierenkortex aus: Nach CED zeigten PACAP-/-/ApoE-/- Mäuse glomerulär und tubulär eine höhere Dichte an IL-6+ Zellen als nach NF. Bei den ApoE-/- Mäusen war die Dichte an IL-6+ Zellen im Tubulussystem nach CED höher als nach NF. Die ApoE-/- Mäuse zeigten im Tubulussystem nach NF eine höhere Dichte an TNFα+ Zellen als nach CED. Nach NF war in den Tubuli von PACAP-/-/ApoE-/- Mäusen die Dichte an TNFα+ Zellen signifikant geringer als bei den ApoE-/- Mäusen. Schlussfolgerung: Abschließend lässt sich sagen, dass bei den PAC1-/-/ApoE-/- Mäusen nach CED kleinere Glomeruli und eine geringere Dichte residenter Makrophagen als bei ApoE-/- Mäusen (Kontrolle) gefunden wurden. Die CED bewirkte eine signifikant höhere Dichte an IL-6+ Zellen in Glomeruli und Tubuli der PACAP-/-/ApoE-/- Mäuse im Vergleich zu NF