bluuebunny/crossref_metadata_2025 huggingface dataset:
The heat shock protein 70 family members HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle (144.1)
Authors: Khachatoorian Ronik , Ganapathy Ekambaram , Ahmadieh Yasaman , Wheatley Nicole , Sundberg Christopher , Jung Chun‐Ling , Arumugaswami Vaithilingaraja , Raychaudhuri Santanu , Dasgupta Asim , French Samuel
Published: June 2021

We have previously identified HSC70 in complex with viral NS5A along with other heat shock proteins. HSC70 has been reported to modulate HCV infectivity. Here, we further analyze the NS5A/HSC70 interaction biochemically and investigate the role of HSC70 in viral life cycle. We demonstrate that HSC70 knockdown significantly affects virus production with no cytotoxicity. We observed that HSC70 knockdown did not affect viral RNA replication and protein production. In fact, we found a slight, but statistically significant increase in viral RNA and protein levels likely caused by compensatory upregulation of HSP70, which we have previously shown to be required for viral protein production. NS5A‐augmented IRES‐mediated translation was also not inhibited by HSC70 knockdown and, rather, slightly increased. In contrast, we discovered that intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A. Thus, HSC70 and HSP70 play discrete roles in HCV viral life cycle which is further validated by our finding that knockdown of both HSC70 and HSP70 displays synergistic and dramatic reductions in virus production compared with knockdown of each alone. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection.

Grant Funding Source: NIH R01DK090794, SWF; NIH AI084090, AD
HIF‐2α mediates hypoxia‐dependent upregulation of Serpin‐B3, a marker of early liver carcinogenesis (144.7)
Authors: Cannito Stefania , Turato Cristian , Paternostro Claudia , Biasiolo Alessandra , Pontisso Patrizia , Parola Maurizio
Published: June 2021

SERPINB3 (S‐B3), a cysteine‐proteases inhibitor up‐regulated in cirrhosis, dysplastic nodules and hepatocellular carcinoma (HCC), can trigger epithelial‐to‐mesenchymal transition (EMT) and increased invasiveness in hepatic cancer cells. In this study we have investigated whether hypoxia, through involvement of hypoxia inducible factors (HIFs), may affect S‐B3 expression by employing morphological, molecular and cell biology techniques in liver‐derived cancer cell lines, normal murine liver and liver specimens from HCC patients.

Exposure of HepG2 and HUH7 cells to hypoxia resulted in S‐B3 up‐regulation in terms of mRNA, protein synthesis and release in the extracellular medium. The use of specific siRNAs and of ChIP assay revealed that S‐B3 up‐regulation is mediated by HIF‐2α (not HIF‐1α) binding to S‐B3 promoter and that this is a redox‐dependent event. The correlation between hypoxia, HIF‐2α and S‐B3 expression was supported by immunohistochemistry analysis on normal murine liver and on tumor specimens from HCV‐cirrhotic patients carrying HCC. Hypoxia, through HIF‐2α‐dependent and redox sensitive mechanisms, is the first ever described stimulus of pathophysiological relevance able to up‐regulate expression of S‐B3, a protein proposed as marker of early liver carcinogenesis.
Leukocyte cell‐derived chemotaxin 2 (LECT2) is a potential serum biomarker specific for βcatenin mutation in hepatocellular carcinoma (144.8)
Authors: Okabe Hirohisa , Delgado Evan , Min Lee , Singh Sucha , Tsung Allan , Monga Satdarshan
Published: June 2021

Objective: Molecular characteristic of tumor is currently crucial information in terms of the sensitivity to therapeutic agent and the prediction of clinical course. The aim of this study is to identify a biomarker specific for β‐Catenin gene (CTNNB1) mutation seen in 20‐30% of patients with hepatocellular carcinoma (HCC).

Methods: We looked for secreted factors specific for CTNNB1 mutation using microarray analysis previously published, and validated it using stable transfectants with β‐Catenin wild‐type (Hep3BWT) and mutated form (Hep3BS33Y). Serum level of the biomarker and genetic background of primary tumor were examined in mice with liver cancer caused by diethylnitrosamine (DEN) + phenobarbital diet, which is known to induce HCC via CTNNB1 mutation.

Results: Leukocyte cell‐derived chemotaxin 2 (LECT2) is downregulated in β‐Catenin KO livers. Hep3BS33Y secreted more LECT2 and showed higher promoter activity of LECT2 compared to Hep3BWT. Mice with β‐Catenin mutated (S33Y) liver cancer showed significantly higher serum LECT2 level than control or non‐tumor bearing mice. They showed high glutamine synthetase and lect2 gene expression.

Conclusion: Serum LECT2 level can be a predictive biomarker for β‐Catenin activation in HCC.
Comparison of DPP‐4 inhibition versus GLP‐1 analogue supplementation on survival and vascular complications in experimental sepsis (145.2)
Authors: Stamm Paul , Hausding Michael , Kröller‐Schön Swenja , Mader Michael , Agdauletova Saule , Mikhed Yuliya , Pfeffer Amanda , Welschof Philipp , Steven Sebastian , Oelze Matthias , Schulz Eberhard , Klein Thomas , Münzel Thomas , Daiber Andreas
Published: June 2021

Background: Dipeptidyl peptidase [DPP]‐4 inhibitors are a new class of drug for the treatment of hyperglycemia and recent studies revealed anti‐inflammatory effects of these gliptins in experimental atherosclerosis and septic animals. Therefore the aim of the present study was to compare the effect of gliptins and a direct glucagon‐like peptide (GLP)‐1 analogue on survival and complications in septic shock models.

Methods: Mice or rats were treated prophylactically with linagliptin, sitagliptinor liraglutide for 4‐7 days. Lipopolysaccharide (LPS, 10, 17.5 or 20 mg/kg i.p.) was injected on day 3 or 6 of treatment to induce septic shock. Another control group consisted of DPP‐4‐/‐ mice. Survival was monitored over time in response to high dose LPS and vascular function, nitrosyl‐iron hemoglobin in whole blood or oxidative stress were measured 24 hours after the injection of a sub‐lethal dose of LPS. The Gehan‐Breslow‐Wilcoxon‐Test was used for statistical analysis of Kaplan‐Meier curves.

Results: Linagliptin and liraglutide therapy or DPP‐4 deficiency improved the survival of mice in the state of a septic shock. Liraglutide improved vascular dysfunction in septic rats without improvement of inflammatory parameters such as nitrosyl‐iron hemoglobin in whole blood or leukocyte‐dependent oxidative stress. Vascular dysfunction and other septic complications were improved by linagliptin and less efficiently by sitagliptin associated with less inflammation.

Conclusion: The results reported here on the improvement in the survival of septic animals could provide additional evidence for the potential clinical use of linagliptin and liraglutide, not only for the therapy of diabetic but also septic patients.
Monocyte‐derived macrophages as an active HIV‐1 viral reservoir in patients receiving suppressive cART (145.4)
Authors: Jusino Shirley
Published: June 2021

Macrophages play a crucial role in HIV‐1 latency and persistent viremia, especially in the establishment of viral reservoir, even when the patients are receiving suppressive cART. Macrophages are long‐lived cells that are resistant to the cytopathic effects of viral replication. Due to their abundant distribution and capacity to migrate into tissues we hypothesized that they can contribute to the maintenance of known viral reservoirs such as that in CD4+ T cells. We differentiated the infected monocytes of four HIV‐ infected patients taking anti‐retroviral therapy to create macrophages. Infected PBMC (peripheral blood mononuclear cells) were cultured for ten days in presence of macrophage colony stimulating factor. After differentiation, the monocyte‐derived macrophages (MDM) were co‐cultured with phytohemagglutinin (PHA)‐activated CD8+‐depleted PBMC’s from healthy patients for four weeks. Supernatants were harvested and analyzed for viral RNA using a sensitive reverse‐transcriptase PCR assay using HIV‐1 gag gene primers. Sequencing of the C2V3 region of the env gene from each patient revealed a 99.6% match with WT HXB2 using HIV Sequence Database (Los Alamos National Laboratory). Results demonstrate that after differentiation into macrophages the infected MDM can still harbor the proviral DNA and were able to release replicate competent virions in the supernatant. Future directions include performing cell to cell infection assay. These findings will be employed in an ex‐vivo model to further analyze HIV‐1 infection and transmission in order to demonstrate whether tissue macrophages are a true viral reservoir in HIV‐1 patients under suppressive cART.

Grant Funding Source: NIH Grant R25‐096955
GPCR kinase‐6 inhibits neutrophil infiltration but mediates bacterial killing during lung bacterial infection (145.5)
Authors: Parameswaran Narayanan , Packiriswamy Nandakumar , Lee Taehyung
Published: June 2021

GPCR kinases (GRKs) are serine/threonine kinases known to be critical players in desensitization of GPCRs. Recent studies have implicated GRKs in a number of different physiological and pathophysiological processes including biased signaling from GPCRs as well as signaling from receptors other than non‐GPCRs. In this study we examined the role of GRK6 (one of the seven GRKs) in E. Coli‐induced lung infection and inflammation model using wild type and GRK6 deficient mice. Intra‐tracheal instillation of E. Coli led to enhanced infiltration of neutrophils into lung tissue of GRK6 deficient mice compared to wild type mice. Enhanced neutrophil infiltration was associated with enhanced IL‐17 expression in the GRK6 deficient lung following infection. Interestingly, even though there was increased neutrophil infiltration, the bacterial persistence and the subsequent mortality were significantly higher in the GRK6 deficient mice. Intriguingly our studies also reveal that GRK6 deficient neutrophils are inefficient in bacterial killing, suggesting that the observed phenotype of increased bacterial load and mortality is likely due to defective bacterial clearance by GRK6 deficient neutrophils. Our studies reveal a previously unrecognized and novel role of GRK6 in bacterial clearance in lung infection model.

Grant Funding Source: Supported by NIH
Enteropathogen‐induced toxicity of gut‐microbiota in C. elegans: a new model to investigate mechanisms of post‐infectious intestinal disorders (145.7)
Authors: Gerbaba Teklu , Gupta Pratyush , Wang Xin , Rioux Kevin , Hansen Dave , Buret Andre
Published: June 2021

Giardia leads to Irritable Bowel Syndrome via obscure mechanisms. We hypothesized that this may due in part to Giardia‐induced changes in microbiota. Using C. elegans as a model, we found that Giardia and commensal E. coli (E. coli HB101) synergistically paralyze and kill C. elegans, while Giardia or bacteria alone did not. Giardia also induced lethal paralysis when co‐incubated with attenuated C. rodentium (ESPF and MAP mutants), with gut mucosal microbiota from healthy human donors, or with mucosal microbiota from inflamed UC (ulcerative colitis) sites, but not with microbiota from non‐inflamed UC sites. The killing effect of Giardia required direct contact of live E. coli with C. elegans. Upon exposure to Giardia and/or C. elegans, E. coli HB101 exhibited distinct genome‐wide expression patterns. A total of 180 genes, including genes for flagella biosynthesis (FliP, FliL), biofilm formation (BssR, BssS), and hydrogen sulfide biosynthesis (CysJ, CysI, CysD, CysN, CysH, CysC), were differentially expressed (>2 fold). Expression data suggests decreased biofilm formation and increased anti‐stress response in E. coli upon exposure to C. elegans, and decreased sulfate reduction upon exposure to Giardia or Giardia and C. elegans. Deletion of the Cysteine B gene (positive regulator of the hydrogen sulfide biosynthesis genes) in E. coli was sufficient to induce lethal paralysis in C. elegans. In conclusion, Giardia alters gut‐microbiota homeostasis by modulating expression of genes in commensal bacteria.

Grant Funding Source: NSERC
A novel mechanism for protecting the arthritic joint: microparticles deliver Annexin A1 into cartilage (146.8)
Authors: Headland Sarah , Norling Lucy , Patel Hetal , Dalli Jesmond , Kelly Stephen , Pitzalis Costantino , Dell'Accio Francesco , Perretti Mauro
Published: June 2021

Microparticles (MPs), subcellular vesicles released from most eukaryotic cells, play important roles in inflammation. We found abundant endogenous neutrophil MPs present in rheumatoid arthritis (RA) synovial fluid compared to paired plasma analyses. These neutrophil MPs were highly enriched in Annexin A1 (AnxA1), a potent anti‐inflammatory protein. Addition of in vitro generated AnxA1+ve neutrophil MPs to IL‐1‐stimulated 3D chondrocyte cultures, rodent cartilage explants, or injected intra‐articularly in a model of inflammatory arthritis, surprisingly, afforded protection from arthritogenesis. This was evidenced by reduced matrix degradation and inflammatory mediator production and an increase in matrix‐synthesis gene expression, effects mediated primarily by AnxA1 and its receptor FPR2/ALX. Current dogma dictates that cartilage is an impenetrable avascular matrix through which metabolites must diffuse; we present the first evidence that MPs can access chondrocytes through the cartilage in vivo. Adoptive transfer of fluorescently labeled mouse neutrophils i.v. into mice at the peak of arthritis demonstrated abundant presence of MPs within arthritic cartilage (wrists, digits), but not intact (femoral head) cartilage, or in naïve mice. With this data we propose that MPs enriched with AnxA1 could offer therapeutic possibilities in the protection of cartilage in situ in arthritis patients.

Grant Funding Source: Supported by The Oliver Bird Rheumatism Programme, The Nuffield Foundation; The Wellcome Trust
Sequence of vascular patterning and gene transcription in the chick chorioallantoic membrane (15.1)
Authors: Ysasi Alexandra , Belle Janeil , Bennett Robert , Wagner Willi , Chamoto Kenji , Gibney Barry , Ackermann Maximilian , Konerding Moritz , Tsuda Akira , Mentzer Steven
Published: June 2021

Introduction: The chick chorioallantoic membrane (CAM) is a well‐established model of both vasculogenesis and angiogenesis; however, little is known about the genetic control of vascular patterning in the CAM.

Methods: Using recent advances in chicken genomics, we investigated the relative expression of 84 angiogenesis genes during the growth and remodeling of the CAM microcirculatory network. Chick embryos, cultured ex ovo, were studied during embryonic development days (EDD) 8‐14. UV laser microdissection was used to harvest capillary plexus and 1st, 2nd, and 3rd order conducting vessels for qRT‐PCR analysis.

Results: Two transcription peaks were observed between EDD 8 and 14. The first peak occurred prior to EDD 10 and was correlated with maturation of capillary meshwork. The dominant gene expressed during this developmental phase was Epas1. The second peak occurred on EDD 12 and was correlated with growth of the 1st and 2nd order conducting vessels. Dominant transcription peaks were observed in Tgfb3, Timp3, Fn1, F3, Col18a1, Itgb3, Ptk2, Mdk, Sphk1 and Notch1. Nrp2, Mdk, Tgfb3, and Vegfa demonstrated bimodal regulation with peak expression on EDD 9 and 12.

Conclusion: We observed bimodal transcriptional activity of the chick CAM during vascular network development. The peaks correlated with the initial maturation of the capillary meshwork followed by expansion of the 1st and 2nd order conducting vessels. These results provide insights into the genetic control of vascular patterning.
Mitochondrial respiratory capacity is not markedly reduced in end‐stage human heart failure (15.6)
Authors: Holzem Katherine , Vinnakota Kalyan , Ravikumar Vinod , Beard Dan , Efimov Igor
Published: June 2021

Objectives. We aimed to assess mitochondrial (Mt) substrate respiratory capacities in human donor (D) versus failing (F) hearts.

Background. Heart failure (HF) is the end‐stage of cardiovascular disease, in which the heart can no longer sustain systemic metabolic demands. During HF progression, the heart switches energy substrate utilization from preferential use of fatty acids (FA) towards greater glucose consumption. We hypothesized that impaired Mt FA respiratory capacity may contribute to this substrate utilization shift.

Methods. We isolated intact Mt from left ventricular (LV) tissue collected from D (n=7) and end‐stage F (F, n=9) human hearts and measured Mt respiration. Briefly, hearts were recovered in the operating room and cardioplegically arrested. Mt were isolated via differential centrifugation, and respiration measurements were collected with an oxygraph cell using ADP (350 μM) and pyruvate + malate (PM, 10 mM/2 mM) or palmitoylcarnitine (PC, 25 μM) as substrates.

Results. For both substrates, we calculated the apparent capacity for oxidative phosphorylation (State 3) and respiratory control ratios (State 3/4 and State 3/2). In contrast with our hypothesis, Mt capacities for oxidative phosphorylation were not significantly reduced for F compared with D hearts for PM (195.9 ± 19.1 vs 158.8 ± 9.0 nM O2/min/mg, p = 0.12) or PC (122 ± 23.0 vs 91.9 ± 14.4 nM O2/min/mg, p = 0.19). Respiratory control ratios were also not different between D and F heart groups for either substrate. To control for myocardial Mt content, we assessed citrate synthase (CS) specific activity in LV muscle homogenates from the same hearts used for respiration measurements, which were consistent between D and F myocardium (14.5 ± 2.4 vs 14.7 ± 1.8 U/g).

Conclusions. Mt respiratory capacities are not markedly impaired in end stage human HF for either glycolytic or FA substrates. Thus, the substrate utilization switch in HF does not appear to be the result of impaired Mt FA oxidation.

Grant Funding Source: Supported by NHLBI R01HL11439501A1
Entamoeba histolytica induces caspase‐4/11 activation in inflammasome signaling (152.5)
Authors: Quach Jeanie , Mortimer Leanne , Moreau France , Chadee Kris
Published: June 2021

A hallmark of amebiasis is acute intestinal inflammation dominated by secretions of pro‐inflammatory cytokines from macrophages. Entamoeba histolytica (Eh) in contact with macrophages activates caspase‐1 by the inflammasome complex resulting in the maturation of IL‐1β and ‐18. The role of inflammatory caspase‐4, ‐5, ‐12 in humans, and caspase‐11, ‐12 in mice are less understood. Caspase‐11, the murine ortholog of caspase‐4, mediates non‐canonical inflammasome activation, suggesting that caspase‐4 may have similar roles. As caspase‐4 might be an immune sensor in Eh infection the aim of this study was to identify the requirements for Eh‐induced caspase‐4/11 activation. Macrophages were treated with live Eh, soluble Eh proteins and secreted components derived from viable Eh. Only live Eh activated caspase‐1, ‐4, and ‐11 in a contact‐dependent manner. Blockade of Eh Gal‐lectin adhesin inhibited binding to macrophages and caspase‐1, ‐4 and ‐11 secretions. Interestingly, cysteine protease 5, another protein critical for Eh virulence, was required for caspase‐1 inflammasome activation but not for caspase‐4/11 activation. To summarize, caspase‐4/11 activation occurred in a contact‐dependent manner and the requirements for their activation are different from caspase‐1, arguing for distinct, non‐redundant roles. Determining how Eh activates these caspases may lead to new therapeutics to treat amebiasis.

Grant Funding Source: Supported by NSERC
Targeted angiotensin converting enzyme over‐expression in myelomonocytes prevents cognitive decline in Alzheimer’s‐like disease (153.3)
Authors: Bernstein Kenneth , Koronyo Yosef , Shen Xiao , Fuchs Sebastien , Koronyo‐Hamaoui Maya
Published: June 2021

The cognitive decline in Alzheimer's disease (AD) is associated with elevated brain levels of amyloid β‐protein (Aβ), particularly Aβ1‐42. Angiotensin‐converting enzyme (ACE) can enzymatically degrade Aβ1‐42 and its overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE10/10 mice, which over express ACE in myelomonocytic cells, with the double‐transgenic APPSWE/PS1ΔE9 mouse model of AD (AD+). At 7 months, soluble Aβ1‐42 was reduced by 44% (136 vs 245 pg/mg) and Aβ1‐40 was reduced by 32% (21 vs 31 pg/mg). Plaque burden was reduced by as much as 79% (13 vs 62 103mm2) and insoluble Aβ1‐42 by 64% (189 vs 521 pg/mg). There was also a substantial reduction in astrogliosis (49‐57% at 7 months, 50% at 13 months). AD+ACE10/10 mice demonstrated less overall brain infiltrating cells, consistent with less AD pathology, though ACE‐overexpressing monocytes and macrophages were increasingly abundant surrounding and engulfing Aβ plaque. At 11 and 12 months, AD+ACE10/WT and AD+ACE10/10 mice were virtually equivalent to non‐AD mice in cognitive ability as assessed by maze‐based behavioral tests. This study shows that an enhanced immune response, coupled with increased myelomonocytic cell expression of ACE, resulted in essentially complete prevention of the cognitive decline observed in a murine model of AD.

Grant Funding Source: Supported by HL088000, HL110353, and the C.A.R.T fund, UL1TR000124,
Hydrocephalic mouse models of primary ciliary dyskinesia have strain dependent defects in brain morphology and physiology (153.5)
Authors: Finn Rozzy , Lee Lance
Published: June 2021

Congenital hydrocephalus results from accumulation of cerebrospinal fluid (CSF) and causes severe neurological damage, but the underlying causes are not well understood. Hydrocephalus is associated with primary ciliary dyskinesia (PCD), a pediatric syndrome caused by dysfunction of motile cilia that can also result in sinusitis, male infertility, and situs inversus. We have investigated the causes of hydrocephalus in mouse models of PCD lacking the Pcdp1 and Spef2 proteins. Both models develop gross, lethal hydrocephalus on the C57BL/6J (B6) background but not on 129S6/SvEvTac (129). Histological and immunohistochemical analyses demonstrate that both mutants on B6 have ventricular dilatation, sloughing of ciliated ependyma, disrupted axon myelination, and increased gliosis, indicating significant injury to the cerebral cortex. In contrast, both mutants on both the B6 and 129 backgrounds exhibit perturbed CSF flow despite an absence of substantial morphological defects on 129. These findings indicate that abnormal cilia‐driven flow is not the sole determinant of PCD‐associated hydrocephalus and suggest that there are genetic modifiers that generally influence susceptibility to congenital hydrocephalus.

Grant Funding Source: Supported by the NIH (1P20GM103620‐01A1)
Hyperhomocysteinemia inhibits mouse early postnatal neurogenesis, the role of oxidative stress (153.6)
Authors: Zhang Lixiao
Published: June 2021

HHcy is a risk factor of cardiovascular disease and neurodegenerative disease. There is a strong correlation between HHCY and cognitive disorders or Alzheimer's disease. Patients with Cystathionine‐β‐synthase (CBS) deficiency suffer from mental retardation and psychiatric complications, which begins from neonatal period, suggesting a role of HHcy in neural development. In this study, we investigate the effect of HHcy on early postnatal brain development and related molecular mechanisms. We found that severe HHcy markedly reduce body weight, whole brain size, hippocampal volume and neuron number in dentate gyrus of hippocampus in mouse with CBS gene deficiency, starting from postnatal 7 days. Since neurogenesis is critical for early postnatal hippocampus development, we examined BrdU incorporation in hippocampus of CBS‐/‐ mouse and found that severe HHcy decreased BrdU and NeuN (neuron marker) double positive cells in dentate gyrus starting from postnatal 7 days, a time point matching the occurrence of hippocampus developmental defect. Next, we examined neural progenitor cells (NPC) proliferation in cultured NPC isolated from neonatal mouse hippocampus. We found that DL‐Hcy (200umol) decreased neurosphere size, a standard index of NPC proliferation, without affecting NPC cell death (as demonstrated by cell viability assay with 7‐AAD staining). Further, Hcy increases of cytosolic but not mitochondria ROS production in cultured NPC by electron‐spin resonance (ESR) measurement. ROS scavenger, superoxide dismutase (SOD), reverses Hcy‐induced cytosolic ROS generation and inhibition of cultured NPC proliferation. In conclusion, HHcy impairs early postnatal hippocampal development and neurogenesis, at least in part, via cytosolic oxidative stress

Grant Funding Source: NIH
Proteomics approach to study neuronal regeneration events of the sequenced sea star Patiria miniata (153.7)
Authors: Franco Catarina , Sweredoski Michael , Kudtarkar Parul , Cameron R. , Hess Sonja
Published: June 2021

Sea stars have long been recognized for their ability to fully regenerate injured tissues as a consequence of predation or amputation. Indeed the ability to regenerate the nervous system is maintained throughout the lifespan of the animal. Since sea stars are distantly related to the chordates, their neuronal regeneration is of potential interest to biomedical applications in regenerative medicine [1‐3].

With the advent of sea star (Patiria miniata) genome sequence and gene models we can now effectively study protein expression in sea star neurons using high‐resolution mass spectrometry. Previously, the sparse information on sea star protein sequences limited peptide identification to ca. 15%. This bottleneck was overcome with alignment of peptides to P. miniata genomic sequences. At present, we have over 18,000 peptides identified at identification rates of 60%, results commonly observed in sequenced proteomes. The methods we are developing allow greater sensitivity and unprecedented identification of low abundance proteins in the starfish nervous system This level of sensitivity is crucially important for the study for neuronal regeneration events: Sonic hedgehog protein, Neuromodulin and Piwi‐like protein 1 [4] are among the identified proteins and clearly highlight P. miniata as a promising model to understand protein regulation during neuronal regeneration.

Grant Funding Source: Betty and Gordon Moore Foundation, Beckman Institute, Fulbright, PROLAB (ASBMB)
What put the “slink” in a saber cat? Scapular and pelvic adaptations enhance stealthy stalking behavior in Smilodon fatalis (17.2)
Authors: Naples Virginia
Published: June 2021

Mammalian carnivores generally belong either to pursuit or ambush hunting categories. Prey capture requirements for each method differ, and these strategies generally correlate with familial taxonomic levels. Canidae tend toward pursuit, while most Felidae specialize in ambush. The extinct saber‐tooth, Smilodon fatalis, is considered an ambush predator because it is large bodied, robustly muscled and short limbed. These attributes limit this cat’s ability to move sinuously, and therefore act as a constraint on hunting postures. Also, no characters have been identified that enhance Smilodon’s adaptations for concealment while stalking prey. This study of a sample of the typically unassociated limb and limb girdle elements from the asphalt deposits of Rancho La Brea reveals morphological features derived from unique pectoral and pelvic adaptations that improve this cat’s ability to move with great flexibility. Reoriented scapular and truncated ilial shapes give Smilodon a lower crouching profile than is shown by other felids. Usually, in a slinking cat, the posterodorsal aspect of the scapula and the anterodorsal ilial region project above the vertebral spinous processes. In contrast, in Smilodon, with fore and hind limbs flexed to a degree equal to other cats, the shoulder and hip regions fall below the spinous processes, enhancing the cat’s ability to flatten its body to facilitate concealment while stalking prey.
Assessing bone and soft tissue morphology of the scapula region: a cadaver dissection‐based study to improve uncemented glenoid metalback fixation (17.3)
Authors: Mathews Sandra , Burkhard Marco , Link Karl , Franke Ingeborg , Harper Gareth , Quereshi F , Bloch Hans‐Rudolf , Ullrich Oliver , Ruhli Frank , Eppler Elisabeth
Published: June 2021

Shoulder pathologies ‐ esp. degenerative disorders ‐ are of the most common orthopaedic diseases nowadays. The outcome of operative treatment such as Anatomic Total and Reverse Total Shoulder Arthroplasty is highly dependent on the individual anatomical situation. Surgical placement of the superior glenoid baseplate screw e.g. might endanger the suprascapular nerve, which could cause postoperative chronic pain and weakness. Surprisingly, only very few studies investigated e.g. the distance between the screw entry point and the scapular notch. The aim of this study is thus to increase the morphological knowledge on the osseous and neurological structures of this clinically important region. In a cadaver dissection study of 17 cadavers from the institutional body donation programme (8 female, 9 male, average age 78 yrs, range 63‐99 yrs) we documented a.o. the size and inclination angle of the glenoid, the distance from the superior screw entry point to the scapular notch both from outside and through the screw canal and also the distance from the supraglenoid tubercle to the scapular notch. Furthermore, the degree of primary and secondary osteoarthritis (cuff arthropathy) were documented as present in this old age sample and, also, the relationship of the suprascapular nerve to the adjacent tissue including the suprascapular vessels was documented. Data are discussed in light of recommendations for safe screw placement and secure distance to the nerve. Furthermore, the general impact of such morphological studies on clinical and microevolutionary research issues will be highlighted.

Grant Funding Source: Supported by Mäxi Foundation Switzerland
Dissecting through barriers: interprofessional education, problem‐based learning, and gross anatomy (18.3)
Authors: Palombella Andrew , Fernandes Alisha , Iacob Stefan , Salfi Jenn , Wainman Bruce
Published: June 2021

Healthcare delivery is reliant on a team‐based approach. Interprofessional education (IPE) provides a means by which such collaboration skills can be fostered. IPE within healthcare programs has been associated with many benefits, including improvements in patient care and satisfaction, reducing clinical error rates, and diminishing negative professional stereotypes. In recognition of the need for IPE, an IP gross anatomy dissection course was founded at McMaster University in 2009. Data has been collected from 5 cohorts to determine the influence of this IPE format on the attitudes and perceptions of students towards other health professions.

Annually, 28 students from various programs are randomly assigned into IPE teams for 10 weeks. Sessions involve an anatomy and scope‐of‐practice presentation, a small‐group case‐based session, and a dissection. The Interdisciplinary Education Perception Scale (IEPS) and Readiness for Interprofessional Learning Scale (RIPLS) quantitatively measure pre‐ and post‐course attitudes and perceptions towards IPE and other health professions. Weekly surveys and culminating profession‐specific focus groups qualitatively evaluate these variables.

5 year pre‐ and post‐course IEPS and RIPLS scores show significant improvements in positive professional identity, competency and autonomy, role clarity and attitudes toward other health professions. Qualitative results corroborated these findings and made suggestions for the development of similar longitudinal IPE curricula.

The implementation of a 10‐week IPE dissection course provides a unique and effective venue for learning about scope‐of‐practice, fostering positive professional identity, and fostering positive attitudes toward IPE and IP collaboration.
Reprogramming cells and their use in regenerative therapy (19.2)
Authors: Chilian William
Published: June 2021

The growth of the coronary collateral circulation has a beneficial effect on the consequences of ischemic heart disease. Despite this importance, strategies to develop coronary collaterals have not been successful. For example, administration of growth factors has not been successful in stimulating the growth of coronary collaterals in patients. Because of these shortcomings, we adopted new strategies to stimulate coronary collateral vessels using genetically reprogrammed cells. We reprogrammed endothelial cells into a progenitor‐like cell that remains committed to a vascular differentiation program and can become either smooth muscle or endothelium (termed induced vascular progenitor cells [iVPCs]). Administration of iVPCs stimulated the growth of collateral vessels in the heart. Other types of stem cells (mesenchymal stem cells, cardiac stem cells) or native endothelial cells did not have the same stimulatory effect on coronary collateral growth. Although the mechanisms by which iVPCs stimulate collateral growth have not been unequivocally defined, these cells incorporate and engraft in growing blood vessels in the heart. Although the benefit of using reprogrammed cells to stimulate vascular growth in the heart in models of vascular disease is not yet defined, the use of certain types of reprogrammed cells may offer a substantial advance in the treatment of ischemic heart disease.
Development, damage and repair of the mammalian kidney (208.1)
Authors: McMahon Andrew
Published: June 2021

The paired kidneys are vital organs controlling water, salt and pH balance for body fluids, removal of toxic by‐products of metabolism, the pressure and composition of blood, and bone formation. The functional unit of the kidney is the nephron. Depending on the mammalian species, 10’s to hundreds of thousands of nephrons are generated during a lengthy period of fetal life. Low nephron number has emerged as a risk factor for development of kidney disease, and due to an absence of a postnatal nephrogenic program, repair of existing nephrons is the endogenous strategy for restoring function to severely damaged kidneys. A deep understanding of nephron formation, damage and repair, will facilitate and prime the development of knowledge‐based strategies to treat the rising burden of kidney disease. I will discuss our current efforts towards this long‐term goal through the molecular and cellular dissection of nephron stem/progenitor programs during fetal life, and the nephron repair processes initiated on acute kidney injury.
Resting‐state functional connectivity of the brain (21.2)
Authors: Liu Thomas
Published: June 2021

When observed at rest, the brain exhibits spontaneous fluctuations in electrical and metabolic activity. At first glance, these fluctuations appear to occur in a random fashion. However, a deeper investigation shows that the fluctuations carry a surprising amount of information about how the brain is functionally connected. In this talk I will discuss how resting state fMRI, EEG, and MEG measures are being used to probe the functional connectivity of the brain.
Probing tissue microstructures using diffusion MRI (21.3)
Authors: Zhou X. Joe
Published: June 2021

There is mounting evidence indicating that diffusion in biological tissues exhibits a large degree of complexity depending on tissue type, cellular structures, and microenvironment. Measuring water molecular diffusion in vivo using magnetic resonance imaging (MRI) can thus provide a wealth of information on tissue structure and connectivity that are not accessible by other imaging modalities. Given the complexity of human brain structure and connectivity, the extent and accuracy of the information that can be exacted from diffusion MRI depend heavily on specific models. This presentation will review recent advances in a number of emerging diffusion models that relate diffusion MRI data with different aspects of brain tissue structure and connectivity. A specific model, known as the fractional order calculus model, which has been developed using an advanced mathematical tool, will be highlighted with data from the human brain to demonstration its ability to reveal tissue heterogeneity, microenvironment, cellular density, and connectivity. This model, together with others, can provide new insights into linking structure and function towards the connectome.
Inflammation and the regulation of repair responses in developing skin (212.2)
Authors: Wilgus Traci
Published: June 2021

Two general mechanisms exist for healing cutaneous wounds: repair and regeneration. In mature skin, injury initiates a repair process that includes periods of inflammation, cellular proliferation, and scar formation. Ideally, regeneration would occur with complete restoration of normal tissue architecture and function at the site of injury and the absence of a scar. Interestingly, a regenerative process is used to heal injured skin at early stages of development, resulting in scarless healing. However, at later stages of development, the skin heals wounds through a fibrotic repair process that leads to scar formation. Although the mechanisms involved in the switch from scarless to fibrotic healing during fetal skin development are not entirely understood, inflammation is believed to be important. Injury does not incite an inflammatory response in fetal skin early in development, whereas a strong inflammatory response is observed when fetal skin is wounded at late stages of development. We have shown that early events associated with inflammation, including mast cell activation, alarmin release, and pro‐inflammatory mediator production, contribute to the developmental differences in inflammation and healing outcomes in fetal skin. These studies could help identify novel targets for modulating inflammation, limiting scar formation, and promoting regeneration.
Microbiome, host response and wound healing (212.3)
Authors: Hardman Matthew
Published: June 2021

It has long been appreciated that a multitude of bacteria, fungi and viruses colonise our skin (collectively termed the skin microbiome). However, only recently have technological advances allowed comprehensive profiling of the wide diversity of the skin microbiome across homeostasis and disease. A picture is emerging where resident commensal microbes may confer significant beneficial effects on skin health. Conversely pathogenic bacteria are currently being linked to numerous skin diseases. Surprisingly, in the context of wound repair it remains largely unclear how the skin microbiome influences healing outcome, both for normal and pathological healing. Indeed, chronic wound infection remains a clinical diagnosis largely based on the physical appearance of a wound. Here we address the mechanisms whereby a host senses and responds to changes in the wound microbiome. We demonstrate that experimental perturbation of specific aspects of the murine host response directly the skin bacterial profile and lead to delayed wound repair. Our data reveal specific changes to the host innate immune response, including altered antimicrobial peptide production. In fact, numerous factors that influence wound healing also alter the wound microbiome. Finally, we demonstrate clinical relevance with distinct bacterial signatures distinguishing chronic wounds that fail to heal from those that respond to treatment.
Methamphetamine abuse usurps hippocampal neural network operations undergirding memory consolidation (222.2)
Authors: Chirwa Sanika , Aduonum Adwoa , Khoshbouei Habibeh
Published: June 2021

Methamphetamine (METH) abuse impairs verbal and nonverbal memory, recognition, attention, and decision‐making. The exact neural mechanisms that underlie these mnemonic deficits are unknown. We have postulated that METH usurps hippocampal EEG and synaptic plasticity functions resulting in memory deficits. To test this, we utilize multidisciplinary methods and conduct experiments in awake and/or anaesthetized male guinea pigs. Our results show that repeated intraperitoneal injections for 7 days with 1.0 mg/kg METH (comparable to therapeutic doses) produces initial hyperactivity but levels return to baseline over the circadian cycle. By contrast, chronic 10.0 mg/kg METH (similar to abused doses) produce sustained hyperactivity and increases total distances moved in a day, indicating motor sensitization. These animals also exhibit a) impaired novel object recognition memory, and b) decrement in long‐term potentiation (LTP) in vivo. They show significant reductions in delta wave (1‐4 Hz) spectral power following tetanic stimulation to produce LTP; synaptic growth‐associated protein‐43 mRNA and protein are highly up‐regulated in the hippocampus. In related experiments, we found that acute high METH increases baseline synaptic transmission but also decrease LTP in ex‐vivo slices. Our results suggest a causal link between METH‐induced changes in CAI network functions and memory deficits.
Globally monitoring translation one codon at a time through ribosome profiling (231.1)
Authors: Weissman Jonathan
Published: June 2021

The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on ribosome profiling (deep sequencing of ribosome protected fragments) that dramatically expand our ability to follow translation in vivo. I will present recent applications of our ribosome profiling approach including the following: (1) Development of ribosome profiling protocols for a wide variety of eukaryotic and prokaryotic organisms. (2) Uses of ribosome profiling to globally monitor when chaperones, targeting factors or processing enzymes engage nascent chains. (3) Deciphering the driving force and biological consequences underlying the choice of synonymous codons.
Mechanisms coordinating replication and transcription (236.2)
Authors: Niska Joanna , Piccini Daniele , Liberi Giordano , Foiani Marco
Published: June 2021

S‐phase transcription jeopardizes the integrity of replicating chromosomes by hampering replication fork progression. DNA topoisomerases, and the Mec1/ATR checkpoint response contribute to coordinate the clash between replication forks and transcribed genes, counteracting the accumulation of reversed forks, R‐loops and DNA breaks.

Replication termination occurs at specific loci, known as termination zones (TERs). TERs are characterized by the presence of pausing elements, including PolII and PolIII‐mediated transcribed units. We studied the events that mediate replication termination at TERs. We found that specialized DNA helicases mediate replicon fusion during termination and counteract the accumulation of RNA‐DNA hybrids at TERs. The Mec1 checkpoint also participates in the replication termination events. We will discuss the mechanisms controlling replication termination at TERs.
Comparison of NHANES 2007‐2010 methodologies for assessing dietary supplement use, part II (245.3)
Authors: Nicastro Holly , Dodd Kevin
Published: June 2021

In NHANES 2007‐2010 dietary supplement (DS) intake, traditionally measured using a DS questionnaire (DSQ) that assessed usage over the past 30 days, was also measured by up to two 24‐hour dietary recalls (24HR). Estimating total nutrient intake requires combining intake from food sources (on 24HRs) with DS intake. This study describes differences between distributions of total nutrient intake estimated 1) using raw DSQ values and 2) using DSQ values calibrated to the relative frequency on the 24HR. Calibration had little effect on means but significant effects on population percentages with intake above the upper limit (UL) or below the estimated average requirement (EAR). Differences were more pronounced among DS users than non‐users or the overall population. Among females aged 51‐70 who reported using calcium supplements, mean total calcium intake was 1566±27 (estimate±s.e.) before/ 1518±26 mg after calibration (p<0.001). An estimated 8±1% (raw) vs. 7±1% (calibrated) had intake below the EAR (p=0.046), and 23±1% (raw) vs. 18±2% (calibrated) had intake above the UL (p<0.001). These differences were enough to affect the overall distribution as well, suggesting that using the DSQ probability as truth may erroneously extend both the left and right tails of the estimated total nutrient distribution. Combining information from DSQ and 24HR with this method may improve the estimation of total intake distributions.
Comparison of NHANES 2007‐2010 methodologies for assessing dietary supplement use, part I (245.4)
Authors: Dodd Kevin , Nicastro Holly
Published: June 2021

In NHANES 2007‐2010 dietary supplement (DS) intake, traditionally measured using a DS questionnaire (DSQ) that assessed usage over the past 30 days, was also measured by up to two 24‐hour dietary recalls (24HR). This study investigates how choice of DS assessment method affects estimates of total nutrient intake. Among women aged 51‐70 in the 2007‐10 NHANES (n=1740), 65.5±.02% (estimate±s.e.) reported usage of a calcium supplement on the DSQ or on at least one 24HR. Almost 14±1% of all DS reports came from 24HRs alone, and 19±1% came from the DSQ alone. This led to significantly smaller estimated fractions of DS users when only 1 modality was considered: 56.7% for 24HRs and 59.6% for DSQ (p<.001 per test). The intercept and slope of the linear relationship between 24HR relative frequency (RF) of usage and average reported DSQ usage probability were 0.12±0.04 and 0.74±0.04 respectively, suggesting the DSQ under‐reports low usage and over‐reports high usage. Typically, 24HR‐only DS reports appeared on both 24HRs (RF=0.79±0.03), similar to reports with DSQ values of 25‐29/30 days (RF=0.80±0.07). Calibrating DSQ probabilities to 24HR RF led to a 42±14 / 139±36 mg reduction in the mean/85th percentile of usual DS calcium intake, since most (67±1.6%) of DSQ reports were 30/30 days, suggesting that choice of DS assessment may influence estimation of distributions of total nutrient intake for DS users, especially in the upper tails.
Botanical initiative for the Dietary Supplement Ingredient Database: green tea pilot study (245.7)
Authors: Andrews K , Palachuvattil J , Dang P , Gusev P , Savarala S , Pehrsson P , Harnly J , Dwyer J , Betz J , Saldanha L , Bailey R , Costello R , Gahche J , Hardy C , Emenaker N
Published: June 2021

According to 2007‐2010 NHANES, 7.5% of the US population reports taking botanical dietary supplements (DS). One of the botanical ingredients in DS most commonly consumed and studied for its health benefits is green tea. Green tea consumption is associated with decreased risk for mortality, cardiovascular disease and cancer. However, DS labels and label databases provide only partial information about levels of bioactive components in botanicals. In the Dietary Supplement Label Database, 795 products list green tea ingredients in a variety of matrices, both with and without amount/serving data. Complete composition information for botanical DS must be determined analytically so researchers can obtain more accurate estimates of total intake for these bioactives. Green tea DS are being analyzed in a pilot study in order to determine ingredient variability and matrix/methodology limitations as the first step for the DSID Botanical Initiative. Multiple products from >25 green tea DS matrices are being analyzed for total polyphenols (spectrophotometry) and 7 catechins, caffeine and L‐theonine (HPLC/UV or /MS) in 3 labs. National studies for the DSID Botanical Initiative will estimate the level of green tea ingredients and other flavonoids in DS identified with a nationwide sampling plan.

Grant Funding Source: Supported by ARS‐USDA and ODS‐NIH