Comparison of expression of the fragile site at Xq27 in T and B lymphocytes

Summary We compared the fragile X (fraX) expression in T and B lymphocytes from four hemizygous males with fraX. Blood cultures were stimulated with a T cell mitogen (phytohemagglutinin: PHA) and with a B cell mitogen (pokeweed mitogen: PWM). A significant decrease in fraX expression was observed in cultures stimulated with PWM when compared to PHA-stimulated ones.     

Probe ultrasonification of egg yolk plasma forms low-density lipoprotein nanoparticles that efficiently protect canine semen during cryofreezing

Around the world, many couples have turned to in vitro fertilization as a viable solution to fertility issues. Low-density lipoprotein (LDL) is a protein best known for transporting fat molecules throughout the body, but it has also been shown to protect sperm cells during cryopreservation due to its micellar structure. In the present study, we aimed to evaluate different protocols for the preparation of nanoparticles from egg yolk plasma (EYP) containing LDL to improve the viability of cryopreserved canine semen. EYP was subjected to three distinct treatments: ultrasonification in an ultrasound bath at 40 kHz for 30 min (LDL-B); ultrasonification via an ultrasound probe at 50% amplitude for 30 min (LDL-P); and high-pressure homogenization at 10,000 PSI for six cycles (LDL-H). Sperm quality was assessed after thawing using computer-assisted sperm analysis and flow cytometry. The results revealed that compared to the EYP control, the LDL-P formulation presented significantly higher efficiency (p < 0.05) in maintaining total and progressive sperm motility, sperm membrane integrity and fluidity, and levels of intracellular reactive oxygen species. The LDL-P nanoparticles had an average size of approximately 250 nm, a PDI value of 0.3, and −1.15 mV of zeta potential, which are very important because it is an indicator of the stability of a colloidal dispersion. Therefore, we conclude that ultrasonication of EYP using a probe is an efficient method for the preparation of LDL nanoparticles that would enhance the cryoprotection of semen during freezing.

Nanoscale structures exhibit functional properties that are unique and not observed at the macroscale (1). This is mainly due to the high surface area at the nano-level, which allows an intense interaction of the nanoparticles with the matrix they are introduced in (2). In animal reproduction, certain metal-conjugated nanoparticles (3), natural extracts (4), and vitamin E nanoemulsions (5) with antioxidant properties are reported to be among the most promising candidates for application in sperm cryopreservation (6).Low-density lipoproteins (LDLs) at a concentration of 8% have been applied in canine semen freezing with encouraging results (2). However, the traditional process of LDL extraction was multi-step, and mistakes were inevitable during the procedure. Therefore, in 2016, studies on simplified LDL extraction processes were published, and it were also reported that egg yolk plasma (EYP) exhibited similar properties for canine semen cryopreservation (7, 8). EYP offers several advantages, including easier procurement, rapid and inexpensive extraction directly from the entire yolk, and the possibility of sterilization and upscaling to the industrial level (9).Belala and Delay (10) experimentally demonstrated the equivalence of LDL and liposomes in the cryoprotection of dog sperm. However, in another study on the biomimetic membrane, differences were observed between the interaction of liposomes and that of LDL with the sperm membrane and prostatic fluid, which emphasized the unique functional properties exhibited by LDL lipids during cryopreservation.LDL comprises a spherical molecule, with the size of its monomers ranging from 8 to 1200 nm and a lipid core of triglycerides and cholesterol esters surrounded by a film of phospholipids and proteins (11, 12, 13, 14). When producing particles at nanometric scales, the methodologies used for the synthesis influence the homogenization, reduction, dispersion, and emulsification of the synthesized compounds (15). Ultrasonic generators and high-pressure homogenizers are the mechanical methods of energy formation that may be used for synthesizing nanostructures composed of lipids (16). Therefore, it is possible to manipulate the nanoforms of LDL micelles derived from EYP, with possible increases in the efficiency of interaction and absorption during cryopreservation. In this context, the present study aimed to evaluate different protocols for the preparation of LDL nanoparticles from EYP containing 8% LDL for the maintenance of the viability of cryopreserved canine semen.     

Intermittent Anoxia Reduces Oxygen Free Radicals Formation During Reoxygenation in Rat Hepatocytes

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.     

Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803^T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803^T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids.     

Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease

Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung's disease (HSCR) or colonic aganglionosis. In 20-30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.     

Antitumor activity and COMPARE analysis of bis-indole derivatives

This paper reports the synthesis of new derivatives (formed by two indole systems separated by a central moiety) analogous of potent antitumor agents previously described. The activity of the bis-indoles bearing a pyridine core confirms the good result described in the previous paper and compound 4c was chosen for the first in vivo experiment (Hollow Fiber Assay). COMPARE analysis and structure–activity relationships were also considered. Contrary to data reported by other Authors, no correlations were found between antitumor activity and NQO1 induction.     

A low-protein diet during pregnancy prevents modifications in intercellular communication proteins in rat islets

Background

Gap junctions between β-cells participate in the precise regulation of insulin secretion. Adherens junctions and their associated proteins are required for the formation, function and structural maintenance of gap junctions. Increases in the number of the gap junctions between β-cells and enhanced glucose-stimulated insulin secretion are observed during pregnancy. In contrast, protein restriction produces structural and functional alterations that result in poor insulin secretion in response to glucose. We investigated whether protein restriction during pregnancy affects the expression of mRNA and proteins involved in gap and adherens junctions in pancreatic islets. An isoenergetic low-protein diet (6% protein) was fed to non-pregnant or pregnant rats from day 1–15 of pregnancy, and rats fed an isocaloric normal-protein diet (17% protein) were used as controls.

Results

The low-protein diet reduced the levels of connexin 36 and β-catenin protein in pancreatic islets. In rats fed the control diet, pregnancy increased the levels of phospho-[Ser^279/282]-connexin 43, and it decreased the levels of connexin 36, β-catenin and beta-actin mRNA as well as the levels of connexin 36 and β-catenin protein in islets. The low-protein diet during pregnancy did not alter these mRNA and protein levels, but avoided the increase of levels of phospho-[Ser^279/282]-connexin 43 in islets. Insulin secretion in response to 8.3 mmol/L glucose was higher in pregnant rats than in non-pregnant rats, independently of the nutritional status.

Conclusion

Short-term protein restriction during pregnancy prevented the Cx43 phosphorylation, but this event did not interfer in the insulin secretion.     

Post-Activation Brain Warming: A 1-H MRS Thermometry Study

PurposeTemperature plays a fundamental role for the proper functioning of the brain. However, there are only fragmentary data on brain temperature (T_br) and its regulation under different physiological conditions.MethodsWe studied T_br in the visual cortex of 20 normal subjects serially with a wide temporal window under different states including rest, activation and recovery by a visual stimulation-Magnetic Resonance Spectroscopy Thermometry combined approach. We also studied T_br in a control region, the centrum semiovale, under the same conditions.ResultsVisual cortex mean baseline T_br was higher than mean body temperature (37.38 vs 36.60, P<0.001). During activation T_br remained unchanged at first and then showed a small decrease (-0.20 C°) around the baseline value. After the end of activation T_br increased consistently (+0.60 C°) and then returned to baseline values after some minutes. Centrum semiovale T_br remained unchanged through rest, visual stimulation and recovery.ConclusionThese findings have several implications, among them that neuronal firing itself is not a major source of heat release in the brain and that there is an aftermath of brain activation that lasts minutes before returning to baseline conditions.