bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II (Ang II) in the paraventricular nucleus (PVN) stimulates elevations in PVN superoxide levels in the rat
Authors: Knupp Lauren C , Haywood Joseph R , Northcott Carrie A
Published: June 2021

Blood‐borne Ang II has been suggested to stimulate angiotensinergic brain pathways through the PVN to increase mean arterial pressure (MAP). In addition, Ang II in the brain, as well as in the vasculature, has been shown to elevate superoxide (O2−). However, it has yet to have been shown that PVN specific Ang II increases PVN O2− levels in vivo. Therefore it was hypothesized that Ang II microinjected in the PVN in vivo would generate O2− in the PVN. PVN directed bilateral microinjections of Ang II (100 nl; 3x10−9mol/L) or aCSF in anesthetized sprague dawley rats resulted in no change in MAP. Immediately following the microinjection the animals were euthanized and the PVN was isolated for chemiluminescent lucigenin O2− measurements. There was a significant increase in PVN O2− in Ang II microinjected animals compared to aCSF controls [Ang II: 1.834 + 0.2887 nmol/min*mg vs. aCSF: 1.000 + 0.2590 relative superoxide (nmol/min*mg); p<0.05]. The change in PVN O2− levels was similar in animals that were given intravenous infusions of Ang II (30 ng/kg/min; 15 ul/min; 60 minutes) [Ang II: 1.486 + 0.1316 vs. Saline: 1.024 + 0.1097 relative O2− (nmol/min*mg); p<0.05]. In conclusion, both blood borne Ang II and PVN Ang II result in elevations in the formation of O2− in the PVN. These data provide further evidence that blood‐borne Ang II may stimulate angiotensinergic brain pathways through the PVN to increase O2− in the PVN.
Increased expression of Ndufa10, a subunit of mitochondrial complex 1 in the paraventricular nucleus of the SHR
Authors: Jun Joo Yun , Yuan Lihui , Raizada Mohan K
Published: June 2021

PVN integrates signals from circulating angiotensin II (AngII), transmits them to the RVLM and increases sympathetic nerve activity and blood pressure (BP). Although oxidative stress (OS) in the PVN has been implicated in this process, little is known regarding as mechanism. Our proteomic studies have established that Ndufa10, a subcomplex of mitochondrial NADH dehydrogenas involved in ROS and OS is increased in the PVN of the SHR. This observation, coupled with central role of mitochondria in OS, led us to propose that Ndufa10 is the key protein whose expression is critical in the establishment of neurogenic hypertension. Thus, we compared levels of Ndufa10 in the PVN of WKY rats and SHR, and determined if its expression is linked to the genetic aspects of hypertension. PVN from the SHR showed a ~1.8‐fold increase in the mRNA and protein levels of Ndufa10 compared to WKY rat PVN. Neuronal cells in primary culture from prehypertensive SHR brain were used next to determine if this increase was genetically linked or was a result of chronic exposure of the rat to high BP. Both mRNA and protein levels of Ndufa10 were 1.3‐to 2‐fold higher in neuronal culture of the SHR compared to the WKY rat. Together, these observations suggest that an increase in the expression of Ndufa10 is linked to the genetic aspects of hypertension and may be responsible for increased OS in the PVN of the SHR.
Aldosterone and Salt Induces Hypertension Associated with Increased Oxidative Stress in the Brain of Apolipoprotein E Knockout Mice
Authors: Araki Shuichiro , Hirooka Yoshitaka , Kishi Takuya , Ito Koji , Koga Yasuaki , Konno Satomi , Yasukawa Keiji , Utsumi Hideo , Sunagawa Kenji
Published: June 2021

Background

Oxidative stress in brain plays a crucial role in the neural mechanism of hypertension. Apolipoprotein E‐knockout mice (apoEKO) reveal increased oxidative stress in the vasculature, heart, and kidney. Aldosterone (ALDO)‐salt load promotes hypertension and organ damage. We hypothesized that apoEKO was susceptible to ALDO‐salt thereby causing hypertension associated with the increased oxidative stress in the brain using an in vivo electron spin resonance (ESR) method.

Methods and Results

Eight‐week‐old apoEKO and their wild type (WT) were treated with either ALDO (6 microg/mouse/d, s.c. osmotic minipump) and 8% NaCl‐containing chow, or vehicle and standard chow (Veh). After 6‐week treatments, differences in SBP and the in vivo ESR/spin probe decay rates in brain, an index of oxidative stress in brain, between ALDO‐salt and Veh treatments were compared between apoEKO and WT. SBP was increased significantly in apoEKO (117.8±2.1 vs 105.2±1.8 mm Hg, n=6 each, P<0.05), but not in WT (111.8±2.1 vs 104.8±1.8 mm Hg, n=6 each, ns). In vivo ESR decay rate was increased significantly in apoEKO (0.297±0.009 vs 0.243±0.008 min−1, P<0.01), but not in WT (0.248±0.011 vs 0.232±0.012, min−1, ns). Oxidative stress evaluated by dihydroethidium staining in the rostral ventrolateral medulla was increased in ALDO‐salt‐treated apoEKO compared with Veh‐treated apoEKO.

Conclusion

These results suggest that ALDO‐salt induces hypertension associated with the increased oxidative stress in the brain in ApoEKO.
PARTICIPATION OF NITRIC OXIDE AND PROSTAGLANDINS IN THE CARDIOVASCULAR PARAMETERS OF MSG OBESE RAT
Authors: Martins‐Pinge Marli Cardoso , da Cunha Natália Veronez , de Abreu Samantha Bagolan , Grassiolli Sabrina , Pinge‐Filho Phileno
Published: June 2021

The circulating levels of the cytokines, interleukin and of inflammatory mediators such as prostaglandin, amongst others, are elevated in obese individuals. Also, clinical and experimental data support a link between endothelial dysfunction and inflammation. The aim of this study was to evaluate the effects of 30 days treatment with prostaglandins and nitric oxide inhibitors in the development of obesity induced by neonatal Monosodium Glutamate (MSG) administration upon blood pressure and heart rate in conscious animals. Control or msg obese 90‐day‐old male Wistar rats were anesthetized to femoral artery and vein catheterization for recording mean arterial pressure (MAP) and heart rate (HR) in a conscious state preparation. 24 hours later MAP and HR parameters were analysed by one hour. Obese rats presented hypertension (MAP= 150±1 mmHg) and tachycardia (HR=432±15 bpm) compared with control animals (MAP=116±6 mmHg; HR=334±17 bpm). After treatment with celecoxib, the hypertension was attenuated (MAP=128±2 mmHg; HR=393±21 bpm). The treatment with L‐NAME did not modify MAP parameter (MAP=143±7 mmHg). Our data showed that msg obese rat is also a model of hypertension; which cause may involve at least in part the participation of prostaglandins.
EVIDENCE FOR A HEART RATE MECHANISM ASSOCIATED WITH TOLERANCE TO PROGRESSIVE CENTRAL HYPOVOLEMIA
Authors: Convertino Victor A , Rickards Caroline A , Ryan Kathy L
Published: June 2021

The tachycardia that accompanies reduced blood volume (e.g., hemorrhage) is associated with elevated sympathetic nerve activity and vagal withdrawal. Subjects with high tolerance (HT) to central hypovolemia display higher heart rates (HR) than low tolerant (LT) subjects. We tested the hypothesis that HT to central hypovolemia would be associated with higher HR, sympathetic activity and vagal withdrawal. Subjects were instrumented to record R‐R intervals (RRI) from ECG and beat‐by‐beat systolic arterial pressure (SAP) from a Finometer® during a progressive LBNP protocol taken to presyncope. Subjects with tolerance = 60 mmHg LBNP were classified as LT (n=31) while subjects who tolerated LBNP levels > 60 mmHg were classified as HT (n=76). The following calculations were performed: power spectral density in the high frequency for RRI (RRIHF; a surrogate of vagal activity that is inversely correlated with sympathetic activity) via fast Fourier transform and spontaneous baroreflex sensitivity (BRS) via RRI‐SAP down‐down sequences. At presyncope, higher (P < 0.001) HR in the HT group (120 ± 2 bpm) than the LT group (99 ± 3 bpm) was associated with lower BRS (HT = 1.7 ± 0.3 vs. LT = 5.7 ± 0.9 ms/mmHg; P = 0.006), and similar (P = 0.377) reductions in RRIHF. Our data support the hypothesis that higher HR associated with HT to progressive central hypovolemia appears to be mediated by sympathetic rather than vagal mechanisms. Similar RRIHF between groups in the presence of a higher BRS in the LT subjects suggests that a sympathetic mechanism for HT may reflect increased cardiac adrenoreceptor responsiveness rather than efferent nerve activity.
T1α/Podoplanin through regulation of RhoA activation directs human lymphatic endothelial cell migration
Authors: Navarro Angels , Perez Ricardo E , Rezaiekhaligh MO , Mabry Sherry M , Ekekezie Ikechukwu I
Published: June 2021

We have recently shown that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression in human lung lymphatic microvascular endothelial cells HMVEC‐LLy (Am J Physiol Lung Cell Mol Physiol 295: L543‐L551, 2008). We have also reported that podoplanin is required for a full migratory response to VEGF in these cells and that complete reorientation of the microtubule‐organizing center (MTOC) in response to VEGF in scratch‐wound assays, necessary for directional migration, is achieved only if the cells express podoplanin (FASEB J 2008; 22:1178.20). In the present study our objective was to determine if podoplanin regulates the migratory response of HMVEC‐LLy to VEGF via modulation of RhoA activation.

RESULTS

HMVEC‐LLy treated with CT04, a Rho inhibitor, achieve 60% wound closure by 48 hr in the presence of VEGF vs 100% in controls. Also, MTOC polarization in treated vs control cells is only 40% vs 80% as assesed at 3 hr after wounding. Furthermore, in transfection experiments, cells expressing a dominant negative mutant RhoA achieve only 20% wound closure vs 60% in control cells by 48 hr in the presence of VEGF.

CONCLUSION

Taken together our findings indicate that the migratory response of HMVEC‐LLy to VEGF stimulation is dependent on precise regulation of RhoA activity, a process dependent on podoplanin.
Protein Kinase C (PKC) Mediates Purinergic Receptor Induced Contraction in MC3T3‐E1 Osteoblasts
Authors: Timothee Patricia , Fomin Victor , Czymmek Kirk , Duncan Randall
Published: June 2021

P2X7 receptor activation mediates the load‐induced increase in bone formation, in vivo. We find that activation of this receptor produces a rapid contraction of the osteoblast that we hypothesize is modulated by two distinct pathways; RhoA GTPase and PKC. To test this hypothesis, we measured MC3T3‐E1 preosteoblast contractions using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor in the presence or absence of specific inhibitors of RhoA GTPase and PKC pathways. BzATP, a known agonist of the P2X7 receptor, was added to the cells and changes in cell area following BzATP stimulation were quantified using Differential Interphase Contrast (DIC) microscopy. Addition of 0.5mM BzATP to MC3T3‐E1 cells resulted in a 34.14% reduction in cell area. Similar results were seen using a PKC activator, PMA. Non‐specific inhibition of PKC with, GF109203X, significantly attenuated the BzATP‐induced contraction. Specific inhibition of PKCá, a Ca2+ dependent isoform of PKC, amplified the contraction in response to BzATP, suggesting that PKCá is not responsible for the P2X7 induced contraction. Inhibition of myosin light chain kinase and Rho kinase (ROCK) also failed to block BzATP‐induced contractions. These studies suggest that PKC mediates the response of P2X7 receptor activation that may be important in skeletal remodeling. (Supported by INBRE2 P20 RR016472‐08 and NIH/NIDDK R01 DK058246)
Evidence that biotin status is impaired in human pregnancy
Authors: Stratton Shawna L , Matthews Nell I , Bogusiewicz Anna , Zhu Shan , Mock Donald M
Published: June 2021

Marginal biotin deficiency is teratogenic in some mammals, raising concern that biotin deficiency can cause human birth defects. Our previous studies have shown that about one‐half of pregnant women become deficient, but indicators of biotin status depended on renal function, which is altered by pregnancy per se. We have validated lymphocyte activity of the biotin‐dependent enzyme propionyl‐CoA carboxylase (PCC) as a measure of biotin status. We used PCC activity to test whether women develop marginal biotin deficiency during pregnancy. Both early and late in pregnancy, PCC activity was less (p < 0.0002) than non‐pregnant controls. In a pilot study, PCC response to biotin supplementation at 10x RDA (300 µg/d for 2 wks) was measured in 8 pregnant women with low PCC activities. Activity of PCC was determined before and at the end of the supplement period. PCC activity increased for each woman; the mean± SD increase was 103% ± 59% (p < 0.001). These results provide evidence that marginal biotin deficiency is common in pregnancy and suggests that supplementation effectively repletes biotin status. Supported by AR tobacco settlement funds, NIH R37 DK 36823, and UAMS CRC M01RR14288.
The Mechanism for Type II Collagen in Modulating Osteochondral Differentiation of Mesenchymal Progenitor Cells
Authors: Chiu Li‐Hsuan , Yang Charng‐Bin , Fang Chia‐Lang , Lai Wen‐Fu T , Wu Yu‐Wei , Tsai Yu‐Hui
Published: June 2021

Type II collagen is mainly presented in the cartilage as well as in the developing bone, and is considered as a cartilaginous ECM. It is known that human mesenchymal progenitor cells (hMPCs) have a great affinity for Type I collagen which enhances osteogenesis; however, no report has addressed on the modulating role of type II collagen during MPC osteochondral differentiation. In our work, CD90+, SH2+, SH3+, CD34‐, CD14‐ hMPCs were isolated, and the mRNA expression pattern in the presence of beta‐glyceralphosphate and BMP‐2 were analyzed. The data reveal a transient osteochondral stage which expresses type II collagen during the osteogenic induction. At this stage, an up‐regulated VLA2 complex expression which is considered as a potential type II collagen receptor and an increasing type II collagen expression were observed. The differentiating MPCs showed an elevation in FAK and JNK but not ERK and P38 phosphorylation levels upon their attachment to type II collagen coated surface. Furthermore, in osteogenic medium, MPCs cultured on type II collagen‐coated surface showed an accelerated calcification of the cells. Consequently, type II collagen may facilitate osteogenesis through a VLA2‐FAK‐JNK signaling axis during the early differentiation stage of MPCs. This work was supported by the National Science Council grant NSC 97‐3112‐B‐038‐003, Taiwan.
Unstirred layers in measurements of CO2 permeability of the red cell membrane
Authors: Gros Gerolf , Endeward Volker
Published: June 2021

We have used the mass spectrometric 18O technique to measure at 37°C CO2 permeability, PCO2, of human red blood cells (RBC) in saline, to which dextran of mol.wt. 60 kDa at concentrations between 0 and10 g% had been added. Apparent PCO2 was found to fall with increasing [dextran] down to 1/3, which was interpreted as an effect of unstirred layers around the RBC, USLe, that increase in thickness d with rising [dextran]. The viscosity of the dextran solutions was determined in an Ubbelohde viscometer. Plotting 1/PCO2 vs. solution viscosity gave a straight line. As theoretical hydrodynamics predict that USLe disappear at 0 viscosity, we extrapolated the straight line to a viscosity of 0, giving a "true" PCO2 of 0.16 cm/s, ~ 30% higher than the 0.12 cm/s measured in saline. We interpret the difference of these figures using the relation of diffusion resistances 1/PCO2,apparent ‐ 1/PCO2,true = d / DCO2, solution, where DCO2, solution is the CO2 diffusion coefficient in the saline of the unstirred layer. We obtain from this a d in saline of 0.5 µm. This figure is compatible with previous estimates of δfor RBCs in well‐stirred solutions. We conclude that USLe cause a moderate underestimation of PCO2. We also present a theoretical model showing that the intracellular unstirred layer (USLi) in RBC causes an additional underestimation of PCO2 by 30%, leading to a PCO2 in the absence of both USLe and USLi of ~ 0.2 cm/s.
Reactive oxygen species‐dependent down regulation of protein phosphatase contributes to tyrosine hydroxylase activation by intermittent hypoxia
Authors: Raghuraman Gayatri , Rai Vandana , Peng Ying‐Je , Prabhakar Nanduri R , Kumar Ganesh K
Published: June 2021

In a previous study we showed that intermittent hypoxia (IH) activates tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis via increased phosphorylation of Ser‐40 without altering total TH protein (Kumar et al., J. Appl. Physiol. 95: 536‐544, 2003). In the present study, we examined the role of protein phosphatase(s) in IH‐evoked TH activation in the brainstem of rats. IH increased TH activity in the medullary regions of the brainstem associated with regulation of cardio‐respiratory systems. This increase TH activity was associated with elevation of dopamine (DA) levels, and increase in TH phosphorylation at Ser‐31 and Ser‐40. IH also resulted in the down‐regulation of protein phosphatase 2A (PP2A) expression and activity. Reactive oxygen species (ROS) were elevated in the brainstem regions in IH exposed rats. Anti‐oxidants prevented IH‐induced down‐regulation of PP2A as well as increases in serine phosphorylation of TH with a concomitant reduction of TH activity and DA to normoxic control levels. These findings demonstrate that ROS‐dependent down regulation of protein phosphatase 2A plays a critical role in IH‐induced activation of TH and increase in DA synthesis which may in part contribute to altered catecholaminergic neurotransmission in the brainstem during IH (Supported by grants from NIH HL‐89616 and HL‐90554).
Role of MicroRNAs in homocysteine induced oxidative stress
Authors: Mishra Paras K , Tyagi Neetu , Kumar Munish , Kundu Soumi , Givvimani Shrikanth , Sen Utpal , Tyagi Suresh C
Published: June 2021

MicroRNAs (miRs) are a novel class of short, non‐coding, endogenous RNA molecules that negatively modulate gene expression primarily through base pairing at 3' untranslated region of target RNA causing mRNA cleavage or translational repression. Homocysteine (Hcy) is a thiol‐containing amino acid and elevated level (hyperhomocysteinemia‐HHcy) causes oxidative stress, one of the central causes of cardiovascular disease. We hypothesize that disregulation of miRs causes oxidative stress in HHcy. To test this hypothesis, we cultured HL1 cardiomyocytes and treated them with different doses of Hcy. Message from oxido‐redox enzymes were determined by RT‐ PCR and Western Blot. MicroRNA array was used to assess the differential expression of miRs in HHcy cardiomyocytes which were further confirmed by individual miR assay. The results suggest that in HHcy cardiomyocytes miR‐188 (‐3p and ‐5p) were dramatically down regulated. Individual miR assay also confirmed it. MMP‐9 which is a signature of extracellular matrix (ECM) remodeling was increased in HHcy cardiomyocytes. These findings suggest the role of miR‐188 in Hcy induced oxidative stress leading to ECM remodeling.
Subcellular localization of the metal‐ion transporters Zip14 and DMT1 in human hepatoma (HepG2) cells
Authors: Zhao Ningning , Knutson Mitchell
Published: June 2021

The liver is the main storage site for body iron. This organ normally takes up plasma transferrin‐bound iron (TBI), but can also acquire non‐transferrin‐bound iron (NTBI), which appears in plasma in iron overload. Uptake of TBI involves binding to the transferrin‐receptor at the cell surface, followed by internalization into recycling endosomes. Upon endosomal acidification, iron is released from transferrin and translocated into the cytosol by a transmembrane protein. We used confocal laser‐scanning microscopy to examine the subcellular distribution of the transmembrane metal‐ion transporters DMT1 (Divalent Metal Transporter 1) and Zip14 in HepG2 hepatoma cells. For the localization studies, cells were transfected to express green fluorescent protein (GFP) fusion proteins. We found that Zip14‐GFP was readily detectable at the hepatocyte plasma membrane whereas DMT1‐GFP was not. Both fusion proteins displayed abundant punctuate intracellular staining that partially colocalized with internalized Texas‐red labeled transferrin, a marker of recycling endosomes. Localization of Zip14‐GFP to the plasma membrane is consistent with its postulated role in the uptake of NTBI at the cell surface. Detection of Zip14‐GFP in recycling endosomes further implicates this protein in the assimilation of TBI. We conclude that Zip14 may therefore play a role in normal uptake of iron by the liver. Supported by NIH grant R01DK080706 (MK).
Potential abnormalities in iron metabolism in hyperlipidemia patient fibroblasts
Authors: Morrison Chevaun , Sauble Eric N , Nguyen Annie , La Alice , Bach Gideon , Linder Maria C.
Published: June 2021

Mucolipidosis type IV (MLIV) is an autosomal recessive neurodegenerative disorder that results from a mutation in mucolipin 1, a 580 amino acid non‐selective cation channel present on lysosomal membranes. This mutation disrupts sorting, transport and/or fusion of endosomes and lysosomes, and subjects suffer from iron deficiency anemia. To establish whether lysosomal turnover of endogenous ferritin was disrupted, fibroblasts from normal and MLIV subjects were pretreated with 59Fe‐labeled ferric ammonium citrate (180 uM) for 24h to produce ferritin (Ft) and then with desferioxamine to induce Ft turnover in lysosomes (Kidane et al, Am J Physiol 291: C445, 2006). No defects in Ft turnover and Fe release were observed, as demonstrated with iodixanol density gradients separating cytoplasmic and lysosomes/endosomal Ft and its iron. However, when exogenous (cationized) horse spleen Ft was administered, turnover of this iron was slowed and more was held in lysosomes and endosomes in the case of cells from diseased subjects. Accumulation of cytoplasmic Ft protein (measured by ELISA) was also reduced. We conclude that a reduced rate of processing of iron entering by endocytosis may slow recycling of red cell iron and contribute to the development an iron deficiency‐like anemia. Supported in part by PHS Grant No. RO1 HD 46949.
Obesity and poor physical function contribute to food insecurity in older adults in Georgia senior centers
Authors: Penn Dawn M , Hausman Dorothy B , Fischer Joan G , Reddy Sudha , Johnson Mary Ann
Published: June 2021

It is unclear why obesity is sometimes associated with food insecurity (FIS). The hypothesis that poor physical function (PF) may be related to this paradox was tested in a convenience sample of older adults receiving nutrition services from senior centers in Georgia (N =759, mean age 75, 81% female, 36% black, 63% white). PF was measured using the short physical performance battery (Guralnik et al, 1994) and FIS defined as a "no" response to "Do you always have enough money to buy the food you need?" The prevalence of FIS was 18%, obesity (BMI > 35) was 18%, and poor (0‐5), moderate (6‐9) and good physical function (10‐12) was 23.3%, 51.8%, and 25.0%. Compared to food secure, the FIS were younger (73 vs 76, p < 0.01), likely to be black (69.0% vs 31.0%, p < 0.01), had higher BMI (31.4 ± 7.6 vs 29.0 ± 6.3, p < 0.02), and lower PF (6.8 ± 2.8 vs 7.6 ± 2.8, p < 0.03). In logistic regression analyses the risk of being FIS vs food secure was significantly increased by being <80 years, being black, obese (OR: 1.65 [1.01, 2.68]), and having poorer PF (OR: 1.78 [1.02, 3.09]) after controlling for confounders. Thus, impaired PF and obesity independently contributed to FIS and may indicate an enhanced need for nutrition assistance. Further efforts are warranted to understand the relationships among obesity, poor PF, and FIS and ways to address them in nutrition assistance programs targeted to older adults. Funding: GA‐AES, GA Division of Aging Services.
Resolution of experimental liver fibrosis in mice by targeted delivery of connective tissue growth factor siRNA
Authors: Brigstock David , Lawrencia Carmel
Published: June 2021

Resolution of experimental liver fibrosis in mice by targeted delivery of connective tissue growth factor siRNA

Connective tissue growth factor (CTGF) promotes liver fibrosis by stimulating collagen production in activated hepatic stellate cells (HSC). Here, we investigated the curative properties of CTGF siRNA delivered to activated HSC in acute or chronic models of CCl4‐induced liver fibrosis. Modified liposomes containing CTGF siRNA were coated with a collagen Type VI receptor‐binding peptide. Balb/c mice received daily injections of CCl4 or oil control for either 3 or 5 weeks. During the last 2 weeks of these regimens, some mice also received concurrent daily injections of CTGF siRNA in targeted liposomes ("CTGF siRNA‐TL") or scrambled CTGF siRNA in targeted liposomes ("CTGF SsiRNA‐TL"). Administration of CCl4 for 3 or 5 weeks resulted in high levels of α‐SMA‐positive cells and deposition of collagen. Whereas this response was unaffected by administration of CTGF SsiRNA‐TL, neither α‐SMA‐positive cells nor collagen deposits were present in CTGF siRNA‐TL‐treated livers. Real‐time PCR showed that the CCl4‐induced mRNA expression of α‐SMA, collagen Type I α1, TGF‐β, or CTGF was restored to baseline values by CTGF siRNA‐TL (p< 0.01) but not by CTGF SsiRNA‐TL. Thus, this targeted liposomal delivery system designed to deliver CTGF siRNA to activated HSC was effective in either preventing or reversing fibrosis even in the continued presence of the fibrotic insult.
Potential role for beta‐catenin in oval cell differentiation during liver injury repair
Authors: Thompson Michael D , Monga Satdarshan S
Published: June 2021

Atypical ductular proliferation (ADP) occurs in the liver following DDC‐induced liver injury. We previously noted a blunted ADP in mice liver lacking β‐catenin after short‐term DDC exposure. To examine whether the presence of active‐β ‐catenin would provide a growth or differentiation advantage to atypical ductular cells in mice, liver‐specific β‐catenin over expressing mice (TG) with a stabilizing mutation at ser45 were fed the DDC diet. No significant differences in appearance and numbers of A6‐positive cells were observed, but we did observe increased numbers of A6‐positive cells with hepatocyte‐like morphology in TG mice as early as 2 weeks and a dramatic difference at 5 months. These cells may indicate an increased potential for proliferating cells to differentiate in the setting of increased stable β‐catenin. We hypothesized that such a differentiation advantage would allow the liver to recover more quickly following a change from DDC back to a normal diet. Indeed, TG mice exposed to DDC diet for 4 weeks followed by 2 days of normal chow had significantly less A6‐positive cells and had recovered close to normal liver histology compared to what is observed in WT. In sum, these findings indicate diverse roles Wnt/β ‐catenin signaling during oval cell response in mice including a role in the differentiation process during liver repair.
The clinical application of cultured cells – are they permanent or just an effective temporary dressing?
Authors: James S. Elizabeth
Published: June 2021

Cultured epithelial cells from stem cell niches have been used to heal wounds and to reconstruct damaged corneal surfaces with varying degrees of success over the years. In collaborative work with the Queen Victoria Hospital and The Blond McIndoe Centre in East Grinstead, UK, a range of patients have been treated with cultured epithelial cells. Clinical cases of burns patients, eye patients and soft tissue reconstruction patients will be shown, demonstrating some successes together with longer term follow up. The question is raised as to whether the cultured cells have performed as permanent transplants or merely good biological bandages with short term effects. DNA fingerprinting studies to follow the fate of cultured donor cells, which have led to successful reconstruction of patient corneal surfaces, will shed some light upon the survival of allogeneic cells. The question of survival of autologous cultured epithelial cells is harder to answer. The outcomes for three cases of soft tissue reconstruction (full thickness skin replacement and facial cheek replacement) using autologous cells will go some way to answering this question
DYAD PEDAGOGY: AN IDEAL GAME LEARNING METHOD FOR USE IN STATE‐ OF‐ THE‐ART GROSS ANATOMY LABORATORY
Authors: Márquez Samuel , Sherman Lloyd R
Published: June 2021

A new gross anatomy laboratory facility at SUNY Downstate Medical Center emerged like the phoenix from the former pre‐war laboratory. The facility opened Fall 2008 with the college of medicine class of 2012. The laboratory is equipped with 20 36inch hi‐definition monitors, mobile VCR, and tableside computers which faculty orchestrate to meet the diverse learning needs of their students. Dyad pedagogy, which arranges students in pairs, was used to optimize learning of gross anatomy in the high technology enabled laboratory. Dyads selected key dissection themes set by the instructor and were asked to create a 3 minute video‐log of their dissection. The video‐logs presented by dyads to peers and faculty though the new monitors. Each presentation demonstrated that students had a command of detailed knowledge and understanding of the anatomy, far greater than what is ascertained from paper and pencil assessments, including conventional laboratory practical examinations. The experience was rated at the highest level by faculty and students for laboratory skill, knowledge and understanding of anatomy, and dyad pedagogy effectiveness. The use of dyad pedagogy in a high‐tech laboratory environment breaks new ground in anatomical education as students learn beyond the iteration of facts ‐ learning creative problem solving, collaboration, and communication skills that reinforce and expand anatomical knowledge.
Cortex rules: The neural mechanisms differentiating multisensory integration and unisensory integration in the midbrain
Authors: Stanford Terrence R. , Alvarado JuanCarlos , Vaughan J. William , Rowland Benjamin , Stein Barry E.
Published: June 2021

Multisensory neurons in the superior colliculus (SC) integrate cues from different senses and the responses to multisensory stimuli often equal or exceed the summed influences of the modality‐specific stimulus components. This capacity is unique to multisensory information as we have shown that such rarely occurs for pairs of within‐modal stimuli (e.g., two visual stimuli)(Alvarado et al., 2007). That the same neuron integrates excitatory inputs differently depending on their source is fortuitous from a functional perspective but the architecture underlying this dual capacity is unclear. A likely candidate is a projection to the SC from the anterior ectosylvian cortex (AES)(Wallace and Stein, 1994; Jiang et al., 2001; Alvarado et al., 2007). We examined the degree to which the AES‐derived cortico‐collicular projection is multisensory‐specific by evaluating the impact of cortical inactivation on both unisensory and multisensory integration in the same multisensory neurons. Cortical inactivation nearly abolished multisensory enhancement but had no impact on unisensory integration. These findings suggest that this cortico‐collicular circuit has evolved for the purpose of combining information across multiple senses and highlights an essential distinction between brain mechanisms for integrating stimuli within and across senses. This research was supported by NIH grants EY016716 and NS36916.
Behavior of nascent polypeptide chains in and on the ribosome
Authors: Beckmann Roland
Published: June 2021

The earliest environment a nascent polypeptide chain will encounter is the ribosomal tunnel through the large ribosomal subunit. In all kingdoms of life this tunnel has the same dimensions with a length of ca 100 Angstroms and a width ranging from 10 to 20 Angtsroms. After the first third of the tunnel a constriction is present and the last third adopts a funnel like widening towards the exit. In most cases the nascent polypeptide is thought to move through the tunnel without engaging in specific interactions with the tunnel wall. However, certain nascent chains can interact with the tunnel wall in order to stall the translational machinery. Other nascent chains may adopt secondary structure already inside the tunnel in order to specifically initiate folding before emerging from the tunnel. Moreover, it has been proposed that specific nascent chains such as hydrophobic transmembrane domains can trigger allosteric regulation of ribosomal ligands bound at the tunnel exit. Here, we present cryo‐electron microscopic (cryo‐EM) structures of several ribosome‐nascent chain complexes at sub‐nanometer resolution. We observe distinct conformations and interaction patterns for stalling nascent chains and, in addition, secondary structure formation in the tunnel.
Influence of the protein source on rat adiposity during energy restriction.
Authors: Chevalier Laure , Quignard‐Boulange Annie , Bos Cecile , Fromentin Gilles , Tome Daniel , Gaudichon Claire
Published: June 2021

The aim of this study was to assess modifications of body composition after a high protein, energy‐restrained diet in overweight rats, with emphasis on the effect of the protein source. 56 rats received an obesity induction diet (OI) for 5 weeks. A control group (n=8) remained on the same diet whereas other rats were restricted (‐40% of energy intake) for 3 weeks with either the same diet (n=8) or with a 33 % protein diet (HP) formulated with casein, whey, soy or mixes of casein:whey (50:50 or 80:20). Control OI rats exhibited a higher adiposity than restricted rats (+29%, P<0.0001). Whatever the diet composition, the energy restriction elicited the same body weight loss characterized by a decrease of fat mass without lean mass change. Compared to control OI rats, the epididymal adipocyte sizes were lower in rats consuming the OI restricted diet (‐10%, P<0.05), the soy and the casein:whey 80:20 diet (‐20%, P<0.05). Compared to OI‐restricted rats, the effect of restriction was greater in the casein:whey 80:20 group (P<0.01). Our results reveal that, under energy restriction, the nature of the protein does not impact weight loss and body composition within a short period but are likely to generate changes on a longer period. Gene expression measurements in adipose tissue are in progress.
Gestational weight gain is associated with adherence to a Western dietary pattern in low‐income women
Authors: Rahn Maike , Strawderman Myla M , Olson Christine M
Published: June 2021

The association of dietary patterns with gestational weight gain (GWG) has not been investigated. Body size, diet and behavioral factors were assessed in a prospective observational cohort of pregnant women in rural New York. GWG was the difference between weight at first and last prenatal visit, excessive GWG was based on Institute of Medicine criteria (IOM 1990). Dietary patterns were determined with orthogonal factor analysis utilizing food groups created from the Willett FFQ. Computed factor scores of the identified dietary patterns were inserted into regression equations with GWG measures as outcome, adjusting for BMI, income, gestational age, timing of weight and diet assessments, parity, physical activity, smoking and energy intake. Factor analysis (n=503) resulted in a Prudent factor with high loadings for fruits and vegetables and other healthful foods, a Western factor (WF) composed of soda, meats and processed foods, and a Fat factor. The 481 women with complete data had a mean GWG of 30 lbs, with 40% experiencing excessive GWG and 43% low income. In low‐income women, GWG increased by 1.5 lbs with each unit rise in the WF, and the odds of excessive GWG were 2.34 times greater when WF scores ≥ median compared to reference (interaction p's ≤ 0.05).

In low‐income women, a Western dietary pattern is associated with higher GWG and increased risk of excessive GWG.

This research was supported by the NIH Grant No. DK69448.
Evidence that protein requirements in healthy school‐age children are significantly underestimated in current recommendations
Authors: Elango Rajavel , Humayun Mohammad A , Ball Ronald O , Pencharz Paul B
Published: June 2021

Protein (PRO) requirements in healthy school‐age children were determined by the indicator amino acid oxidation (IAAO) method. Seven, healthy children (6 ‐ 11 y) each randomly received a minimum of 6 graded PRO intakes (range= 0.1 ‐ 2.56 g/kg/d) to a total of 47 studies. The diets provided energy at 1.7 X REE and were made isocaloric using carbohydrates. PRO was given as an amino acid mixture based on egg protein pattern, except phenylalanine and tyrosine intakes, which were maintained constant across intakes. Oxidation of L‐[1‐13C]‐phenylalanine to 13CO2 (F13CO2) was measured in breath samples at baseline and isotopic steady state. Linear regression crossover analysis identified a breakpoint (requirement) at minimal F13CO2 in response to different PRO intakes.

Figure 1. Phenylalanine oxidation (F13CO2, µmol/kg/hr) in response to graded protein intakes

The mean and population‐safe PRO requirements were determined to be 1.35 and 1.6 g/kg/d, respectively (Figure 1). These results are significantly higher than the mean and population‐safe PRO requirements of 0.76 and 0.95 g/kg/d, respectively, currently recommended by the Dietary Reference Intakes (DRI 2005) for macronutrients. This study is the first to directly estimate protein requirements in children using stable isotopes and indicates that the current recommendations are severely underestimated. (CIHR supported)
Copper deficiency increases ferroportin expression in rats
Authors: Jenkitkasemwong Supak , Prohaska Joseph R. , Knutson Mitchell D.
Published: June 2021

Ferroportin is a transmembrane iron‐export protein that plays an important role in dietary iron absorption and iron recycling from senescent red blood cells. Ferroportin expression increases with tissue iron loading but decreases in response to hepcidin, the circulating iron‐regulatory hormone that binds to ferroportin, causing its internalization and degradation. Hepcidin expression increases with iron loading but decreases during anemia. We examined ferroportin and hepcidin expression in copper‐deficient (CuD) rats, which have elevated liver iron levels but are anemic. We compared CuD and copper‐adequate (CuA) animals (n=4/group) after one month of treatment postweaning. Hemoglobin levels were lower in CuD than in CuA rats (101 vs. 159 g/L). Western blot analysis revealed that ferroportin levels increased in CuD rats relative to CuA rats by 4‐ and 1.6‐fold in liver and spleen, respectively (P<0.02). Increased ferroportin expression in CuD rat tissues was associated with increased ferroportin transcript abundance, as measured by qRT‐PCR. Copy number of hepatic hepcidin mRNA was more than 50‐fold lower in CuD than CuA rats despite a doubling of liver iron. We conclude that tissue ferroportin levels increase in CuD rats because of diminished hepcidin levels and increased ferroportin mRNA abundance.
Stereoscopic three‐dimensional reconstruction of the female pelvis and pelvic contents for education
Authors: Sergovich Aimee Lynn , Johnson Marjorie , Wilson Timothy D
Published: June 2021

The anatomy of the pelvis is complex, and its three‐dimensional organization is conceptually difficult for students to grasp. The aim of this project is to create an explorable and projectable stereoscopic three‐dimensional (3‐D) model of the female pelvis and pelvic contents for anatomical education. The model is created using cyrosection images obtained from the Visible Human Project in conjunction with a general purpose three‐dimensional segmentation and surface rendering program. Anatomical areas of interest are identified and labeled on consecutive images. They are then reassembled into a three‐dimensional model. Currently the model includes the pelvic girdle, organs of the pelvic cavity, surrounding muscles, the perineum, and large blood vessels. Each structure can be individually controlled (ex. added, subtracted, made transparent) in order to reveal the organization of and relationships between structures. The model can also be manipulated and/or projected stereoscopically in order to visualize structures and relationships from different angles with excellent spatial perception. Due to its ease of use and versatility this model may provide powerful teaching tool for learning in the classroom or in the laboratory. Research support: Ontario Graduate Scholarship.

Grant Funding Source

Internal
Reconstruction of the Cerebral Ventricular System System in Stereoscopy
Authors: Lew Christina , Wilson Timothy D
Published: June 2021

The complexity of the human cerebral ventricular system, coupled with its deep position within the brain, may pose a problem for conceptualizing its anatomy. Cadaveric dissection may be inadequate for learning the anatomy of the cerebral ventricular system as ventricular structures represent spaces rather than physical structures. Three‐dimensional (3D) computer reconstruction of the ventricular system may offer a solution. This study aims to create an accurate 3D computer reconstruction of the ventricular system with surrounding structures from 4T magnetic resonance (MR) scans of a male cadaver. Using 3D segmentation and rendering software, structures of interest were reconstructed through manual segmentation. The finished model can be manipulated by adding or removing structures, altering opacity, or taking slices through the model to highlight inner structures. Complex physiologic concepts, such as cerebrospinal fluid flow, can be shown using the 3D model through a video animation. Stereoscopic projection can be used increase depth perception and emphasize spatial relationships between anatomical structures. This model is suited for both self‐directed learning and classroom teaching of 3D anatomical structure and spatial orientation of the ventricles, their connections, and their relation to adjacent neural and skeletal structures. Research support: Ontario Graduate Scholarship.

Grant Funding Source

Internal
The Role of Fibronectin During Vasculogenesis
Authors: Rupp Paul A. , Kosa Edina , Szabo Andras , Filla Mike B. , Czirok Andras
Published: June 2021

The role of fibronectin in vasculogenesis was studied with time‐lapse deconvolution microscopy in quail embryos and in cultures of endothelial cells invading 3D collagen I gels. We show that fibronectin accumulates very early at the endothelial cell surfaces ‐‐ sometimes earlier than the endothelial cell‐surface epitope QH1 is detectable. By analyzing the displacements of pulse‐labeled fibronectin fibers around the nascent vessels, we show that the fibronectin accumulated is of mesodermal origin, and additional filaments are not assembled de novo during the process. Correlation analysis of endothelial cell movements and the morphology of fibronectin filaments show that fibronectin does not act as a vascular pre‐pattern to guide multicellular sprouts. However, fibronectin remodeling is essential to normal vasculogenic sprout formation ‐‐ perturbed fibronectin assembly alters endothelial cell motility. The work was funded by NIH (R01 HL87136).
The actin cytoskeleton regulates matrix‐metalloproteinase‐2 expression in vascular smooth muscle cells in response to insulin‐like growth factor
Authors: Risinger George Mitchell , Updike Dawn L , Bullen Elizabth C , Tomasek James J , Howard Eric W
Published: June 2021

Vascular smooth muscle cells (VSMCs) can display a variety of phenotypes from a differentiated contractile phenotype to a de‐differentiated migratory one. This plasticity facilitates VSMC participation in vessel remodeling events like atherogenesis. The cytoskeleton can regulate the expression of contractile genes through the actin‐mediated regulation of myocardin‐related transcription factor (MRTF)‐A. We asked if the cytoskeleton was capable of regulating factors associated with the de‐differentiated phenotype, namely, matrix‐metalloproteinase (MMP)‐2. We used insulin‐like growth factor‐1 (IGF‐1) to induce VSMC de‐differentiation, which was characterized by changes in the actin cytoskeleton and a decreased level of the contractile marker smooth muscle α‐actin, and found that IGF‐1 induced up‐regulation of MMP‐2. Alteration of the actin cytoskeleton affected this regulation. Co‐stimulation of VSMCs with IGF‐1 and the F‐actin stabilizing agent jasplakinolide blocked MMP‐2 up‐regulation, while the F‐actin de‐stabilizing agent latrunculin B enhanced up‐regulation. These data suggest that the F‐actin cytoskeleton plays an inhibitory role in the regulation of MMP‐2 in VSMCs. Since MRTF‐A is critical for regulation of the contractile genes in VSMCs, future work will determine the role of MRTF‐A in MMP‐2 regulation. (Funding: NIH R01 GM60651)

Grant Funding Source

NIH
Teaching the limbic system: A historical perspective
Authors: Culberson James L.
Published: June 2021

In 1878 Paul Broca named le grande lobe limbique as one more thing to worry about in thestruggle to understand the forebrain. It was 60 years later beforeJames Papez wired this unnoticed lobe into his insightful 'circuit for emotion'. Again no oneseemed to notice for 20 more years,until P.D. McLean described a muchmore pervasive, functioninglimbic system that assumed real behavioral relevance (and added complexity!). By the time Walle Nauta extended the systemfrom basal forebrain into the midbrain, it was getting too difficult for anyone to figure out!Even as anatomists and students sweated connections, clinicians made clear that this limbus was problematic in many epilepsy patients, and that the'limbic system' was implicated in a wide range of disorders of affect and in dysfunction of many innate survival behaviors. Since both anatomy and function of the limbic system are recognizably important andcomplex, neuroscientists facultieslearned to teach it and students tried to cope.Whilewe struggled together, well intentionedDeans and Curriculum Committeescheerfully choppedaway teaching hours from med neuroscience (where recognizable courses survived!) to accommodate'competencies'. Given these realities, this talk will consider the dilemma: How shall we teach a limbic system that is ever more complex, while instructional time is ever more constrained and the system is almost daily more clinically relevant?
Clinical correlations to limbic structure and function
Authors: Rao Jayaraman
Published: June 2021

Since the original description of the "le grand lobe limbic" by Paul Broca in 1878, several areas of the frontal, temporal, and amygdala regions have been added on to be part of the "limbic lobe". While the debate about what constitutes the limbic lobe is continuing, dysfunctions of several regions of the limbic lobe have been implicated to cause many neuropsychiatric disorders. Recent experimental evidence strongly suggest that the orbitofrontal cortex, dorsolateral and medial prefrontal cortex and subgenual anterior cingulate gyrus may be involved in obsessive compulsive disorder, anxiety disorders, drug dependence, suicidal depression, bipolar and mood disorders. The discussion will expand on the experimental and clinical evidence supporting these evolving concepts. The potential target sites to improve the clinical syndrome of obsessive compulsive behavior and depression with deep brain stimulation technique will be elaborated.