bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Role of the Metalloprotease FtsH in Biogenesis of the DrrAB Drug Transporter
Authors: Li Wen
Published: June 2021

This study provides the first direct evidence for the dual role of the metalloprotease FtsH in membrane protein biogenesis. Using the physiological substrate DrrAB, it is shown that FtsH is not only responsible for proteolysis of misassembled DrrB but it also plays a much broader role in biogenesis of the DrrAB complex. Previous studies showed that the stable expression of DrrB in the membrane depends on simultaneous expression of DrrA. Here we show that DrrB is proteolyzed by FtsH when it is expressed alone. Moreover, the DrrA and DrrB proteins expressed together in a temp‐sensitive ftsH mutant strain of E. coli were found to be non‐functional due to their incorrect assembly. Simultaneous expression of wild‐type FtsH in trans resulted in normal doxorubicin efflux. Strikingly, doxorubicin efflux could be restored in mutant cells irrespective of whether FtsH was expressed simultaneously with DrrAB or expressed after these proteins had already accumulated in an inactive conformation, thus providing crucial evidence for the ability of FtsH to refold the misassembled proteins. Complementation experiments also showed that the AAA domain of FtsH contains a chaperone‐like activity, however, unlike wild‐type FtsH, it was unable to restore function. Our results therefore show for the first time that FtsH contains the protease as well as refolding functions, and both the AAA and the proteolytic domains of FtsH are required for each of these activities.
The circulating molecular chaperone clusterin interacts with amyloidogenic transthyretin oligomers and modulates amyloid formation
Authors: Greene Michael J , Klimtchuk Elena , Koch Clarissa , Seldin David , Connors Lawreen H
Published: June 2021

Clusterin (CLU) is an abundant, circulating chaperone co‐localizing with amyloid deposits of transthyretin (TTR). The precise interaction of CLU with misfolded, amyloidogenic TTR intermediates has not been defined. In vitro, wild‐type (WT) TTR was unfolded and aggregation induced using NaCl. Cross‐linked aggregation samples from 0–24 h showed a transition from tetramers to oligomers by SDS‐PAGE. Moreover, samples from 0.5 to 4.0 h showed increasing reactivity to the A11 oligomer‐specific antibody. Circular dichroism spectroscopy showed unfolding of native TTR structure, increased beta‐sheet content and aggregate formation. Using surface plasmon resonance (SPR) we investigated the interactions of CLU with surface‐bound native and oligomeric forms of TTR. SPR small molecule binding of Congo red (CR) to oligomers and diflunisal to native tetramers demonstrated the loss of diflunisal binding and increased CR binding to oligomeric TTR. CLU was found to bind to early and late stage oligomers in a concentration dependent manner by SPR. Amyloid formation in the presence of CLU indicated enhanced oligomerization at a 1:1 (TTR:CLU) molar ratio as measured by ThioflavinT binding. Investigation of CLU interactions with oligomeric forms of TTR may provide mechanistic insight for the presence of this chaperone in amyloid deposits. Supported by NIH grant RO1AG031804 (LHC) and the Young Family Amyloid Research Fund.
A search for novel substrate activity in mutants of L‐Alanine Dehydrogenase
Authors: Aldeborgh Hannah , Mundorff Emily
Published: June 2021

The enzyme L‐Alanine dehydrogenase (AlaDH) from Mycobacterium tuberculosis catalyzes the reversible conversion of L‐alanine to pyruvate. AlaDH, however, belongs to the formate‐glycerate dehydrogenase superfamily, whose other members generally reversibly interconvert carbonyls to alcohols, not amines as in the AlaDHs. In a case of convergent evolution, enzymes in the Phe/Glu/Val/Leu amino acid dehydrogenase (AADH) superfamily catalyze the same reaction as AlaDH on larger amino acids, but bare no structural resemblance to AlaDH. Can AlaDH be altered in a way that will allow it to act on different amino acid substrates, or has evolution make it too specific to alanine to alter its activity? Our objective is to find novel activity on various substrates in the highly specific AlaDH. Previously, it has been shown that the replacement of the 94‐phenylalanine binding pocket residue with alanine and serine leads to novel activity on leucine and norleucine in comparison to the wild type, as well as greatly increased activity on norvaline and methionine. Here we present the additional mutation of 133‐methionine to alanine. We successfully express and purify this double mutant, and find additional novel activity.
SP‐101, a novel gefitinib derivative induces apoptosis and tumor inhibition
Authors: Chao Jui‐I , Wang Su‐Pei , Chen Chinpiao , Yang Jinn‐Moon
Published: June 2021

Gefitinib, a tyrosine kinase inhibitor blocks epidermal growth factor receptor (EGFR) activity, has been used in lung cancer therapy. Here, we show a novel gefitinib derivative compound, named as SP‐101, exerts apoptosis and tumor inhibition in human lung cancer. SP‐101 was more effective in inducing the cell death than gefitinib and other derivatives in human lung cancer cell lines, including A549, H1299 and CL3. SP‐101 induced the caspase 3 activation and poly ADP‐ribose polymerase (PARP) protein cleavage. SP‐101 was more sensitive on the inhibition of survivin and cell growth than gefitinib. Transfection with a survivin‐expressed vector reduced the SP‐101‐induced cell death. More importantly, SP‐101 reduced the tumor size and survivin protein levels in the xenografted human lung tumors of nude mice. Besides, a molecular computational docking model shows that SP‐ 101 interacts with the ATP binding site of EGFR kinase domain. These results demonstrate that SP‐101 is a novel gefitinib derivative, which displays apoptosis induction and tumor suppression associated with the blockage of survivin.
p38α and p38δ MAPK are key regulators of p21Cip1 gene expression in human keratinocytes
Authors: Saha Kamalika , Eckert Richard L.
Published: June 2021

PKCδ increases keratinocyte differentiation via a mechanism that involves activation of p38δ MAPK. We recently showed that PKCδ also suppresses cell proliferation by increasing expression of the p21Cip1 cyclin‐dependent kinase inhibitor. However, the downstream effectors that mediate this PKCδ‐dependent regulation are not known. Our present studies suggest that PKCδ activates p38δ which regulates p21Cip1 promoter activity, as exogenously expressed p38δ increases p21Cip1 mRNA and protein level in human keratinocytes. Treatment with SB202190, a p38α/β inhibitor, or dominant negative p38α, leads to a decrease in PKCδ‐dependent p21Cip1 promoter activity, implicating p38α as a second mediator of this regulation. p21Cip1 promoter truncation experiments indicate that p38δ increases the activity via a putative p53 response element. This suggests that p38α and δ may act in conjunction with or through p53 to regulate p21Cip1 gene expression. In addition, we show that p38α and p38δ overexpression increases p53 promoter activity, suggesting that p38α and δ regulate p53 gene expression. We propose that PKCδ activates p38α and p38δ which in turn activates p53 to increase p21Cip1 expression leading to cessation of keratinocyte proliferation.
Paradigm change in clinical anatomical learning: lessons from students’ evaluations
Authors: Jacomo Alfredo Luiz , Akamatsu Flávia Emi , Andrade Mauro
Published: June 2021

Teaching and learning processes are constantly changing as medical knowledge increases exponentially and general students’ behavior demands a continuous improvement of techniques and approaches. The University of São Paulo Medical School has a systematic evaluation of each discipline where students grade 38 different topics ranging to physical conditions to the quality of the classes, allowing the course coordinators to redirect to adjust the course format as needed. We analyzed the changes in the Anatomy course from 1994 to the present and their impact based on the students’ evaluations.

In this study, the different methods to teach Anatomy to 2nd. And 3rd. year students of Medicine employed from 1994 to 2012 were confronted to the students’ evaluations, comparing the general appreciation of the course in different teaching approaches and, in addition, comparing with simultaneous basic disciplines, evaluated by the same questionnaire.

Significant difference in students’ evaluation was observed when the course changed from Problem Based Learning technique to a more formal approach with theoretical classes and practical demonstrations. A still better evaluation was obtained as the practical demonstrations focused aspects showed in theoretical classes given by specialists trained in Anatomy. A similar improvement was not seen in other disciplines.

Continuous feedback from students is essential to adequate teaching strategies to modern challenges to obtain the best results in courses of Clinical Anatomy.
Crystal structures of S6K1 provide insights into the regulation mechanism of S6K1 by the hydrophobic motif
Authors: Ding Jianping , Wang Jianchuan , Zhong Chen , Wang Fang
Published: June 2021

The p70 kDa ribosome protein subunit 6 kinase 1 (S6K1) belonging to the AGC family of kinases is an important downstream effector of the mTOR signaling pathway, and dysfunction of S6K1 has been related to tumorigenesis, metabolic diseases and developmental defects. The kinase activity of S6K1 is stimulated by multiple phosphorylation events, including phosphorylation of T229 in the activation loop and phosphorylation of T389 in the hydrophobic motif. Here we report six crystal structures of S6K1 with or without the hydrophobic motif in various forms, in complexes with a highly specific inhibitor, namely PF‐4708671. The structural and biochemical data reveal in vivo phosphorylation of T389 in the absence of phosphorylation of T229 and demonstrate the functional importance of two conserved residues of the N‐lobe, Q140 on strand β4 and R121 on helix αC, in the establishment of a hydrogen‐bonding network between the N‐lobe and the hydrophobic motif. Phosphorylation of T389 or introduction of a negatively charged group at the corresponding position leads to reinforcement of the network and stabilization of helix αC, particularly via interactions between the negatively charged group and Q140. Furthermore, sequence and structural comparisons of S6K1 with other AGC family kinases suggest that the divergences in both the hydrophobic motif and helix αC confer specificity on the kinase signaling cascades regarding their dependence on PDK1. Together, these results indicate that phosphorylation of the hydrophobic motif in S6K1 is independent of and probably, precedes and promotes phosphorylation of the activation loop by providing a docking site for PDK1.
Identification of intrinsically disordered regions in Angiotensin II receptor AT2R and their correlation with functionality
Authors: Pulakat Lakshmi , Gavini Madhavi , Gavini Nara
Published: June 2021

The Angiotensin II type 2 receptor (AT2R) is G‐protein coupled receptor that is not crystallized. A member of the anti‐inflammatory/vasodialative branch of renin‐angiotensin system (RAS), AT2R‐activation ameliorates cardiovascular diseases, strokes, and attenuates cancers. Developing functional AT2R mimetics is highly relevant to drug development. Emerging evidence suggests that Intrinsically Disordered (ID) regions in proteins facilitate their interaction with multiple protein partners. We hypothesized that loss of function mutations in the AT2R will also disrupt the ID regions of the protein. We employed three in silico ID predictors (PONDR® VLXT, DisEMBLTM and PONDR‐FIT) to identify evolutionarily conserved ID regions of the AT2R. Three conserved ID regions were predicted by these ID predictors that were located at the N‐terminus, 3rd intracellular loop (ICL) and C‐terminal cytoplasmic domain (CCD). This ID‐signature of the AT2R is altered in signaling‐deficient rat and human AT2R mutants. Mutations in the AT2R that do not alter the ID‐signature, do not affect AT2R‐signaling meditated specifically via 3rd ICL and CCD. These observations support the idea that ID‐signature can be unique to a peptide and relates to its functional property and will differ in loss of function mutations. It is conceivable the ID‐signature may serve as a guide for generating AT2R‐mimetic peptides.
Transfection of Primary and iPSC‐derived Cells by Capillary Electroporation
Authors: Pihl Johan , Dalén Jennie Svensson , Aspengren Sara , Karlsson Mattias
Published: June 2021

Primary cells in culture are typically considered to be a highly relevant in vitro models. They can be cultivated and differentiated in vitro, but are typically difficult to transfect using conventional methods. In this work, we've developed a system, Cellaxess ACE, a versatile, single capillary electrode‐based electroporation system that can be used for any adherent cell type. Cellaxess electroporation offers superior transfection quality because of minimal cell processing and low voltages required for electroporation. The focused electrical field delivered by the platform minimizes electrochemical toxicity and joule heating. The platform is truly enabling, e.g. rat mouse & iPSC‐derived human neurons, primary human immune cells and many more cell types with excellent viability and a completely retained morphology. It can be used in any cell culture format, and is an excellent match with high‐content and confocal microscopy readouts. The presented data shows the broad application area for the Cellaxess ACE electroporation system, including cDNA transfection and RNAi experiments on different types of primary cell cultures.
Troponin –“Flipping the Switch”
Authors: Aguirre Jacob , Brinegar Kimberlie , Davidsion Katie , Fournier Ronald , Monteverdr Danielle , William Caitlyn , Galea‐Martinez Sandra A. , Hoffman Ryan
Published: June 2021

According to the American Heart Association,100 young Americans die of sudden cardiac failure annually; some of these deaths can be traced to mutations in the protein troponin. Troponin is located in striated muscle tissues and allows or prevents actin‐myosin interactions, which generate contractile force. Troponin must change structure for muscles to contract, accomplished when calcium ions are released inside the muscle cell. These ions bind to troponin and change its structure. Troponin is comprised of troponin T(TnT), C(TnC), and I(TnI). TnT anchors TnI‐TnC apparatus to tropomyosin, properly orienting it to the actin filament. TnC changes its shape due to calcium‐binding, and removes TnI from the actin surface, ending TnI's inhibition of muscle contraction. The mechanism we studied describes how the protein structures interconvert between their functional states. This may clarify the relationship between troponin mutations and sudden death. The El Capitan High School SMART (Students Modeling a Research Topic) Team partnered a researcher and a group of students using data and 3D printing technology to create a model. Supported jointly by TSRI and UCSD SMART team programs.
A team‐based learning (TBL) experience in gross anatomy laboratory improves physical therapy student perceptions of teamwork
Authors: Killins Anita , Wood Michael , Brooks William S. , Huitt Tiffany W.
Published: June 2021

As healthcare shifts towards increased interdisciplinary patient care, it is essential for students to master group problem solving and develop a positive attitude towards teamwork. Team‐based learning (TBL) fosters high‐level cognitive learning and collaborative group dynamics. The study objective was to determine the effect of TBL on student perceptions of course performance, teamwork, and TBL as an effective learning method in gross anatomy. For this study, we used a Likert scale to survey 2 cohorts of DPT students: one received a traditional cadaveric gross anatomy lab; the other received the TBL hybrid approach wherein students alternated between traditional cadaveric dissections and TBL learning modules. Surveys were administered to traditional format students after they completed gross anatomy and to the TBL format students before and after their gross anatomy course. Our results indicate that all students had positive attitudes towards the TBL method and believed that it would improve their course performance and content mastery. Importantly, perceptions of the necessity for teamwork and collaboration were significantly better for the TBL format students as compared with the traditional format students. Our results, therefore, support the implementation of TBL in the gross anatomy lab as an effective method for developing positive attitudes toward interprofessional communication and teamwork.
PEDF: An Angiogenesis Inhibitor and its role in Glioblastoma multiforme
Authors: Billin Emily , Liesener Hardy , Van Gorden Holly , Yohe Kiera , Mitchell Michael , Fox Colette , Van Keuren Becki , Mirza Shama
Published: June 2021

Glioblastoma multiforme (GBM) is a cancerous brain tumor with almost 100% recurrence rate even after surgery, radiation and chemotherapy. Pigment epithelium‐derived factor (PEDF) has been found in areas where these tumors do not grow as aggressively. PEDF slows the growth of tumors by inhibiting angiogenesis, a physiological process involving growth of new capillaries from pre‐existing blood vessels in the body. Restricting blood flow to the tumor starves it of oxygen and nutrients. The mechanism of PEDF‐mediated inhibition of angiogenesis is unknown. Research has shown that PEDF undergoes posttranslational modifications (PTM), chemical changes to a protein after translation, such as the addition of carbohydrates (glycoslylation) or phosphate groups (phosphorylation), which may occur during various cellular events in tumors. PEDF is phosphorylated at S227, S114 and S24 and glycosylated at N285. Glycosylation may also occur on amino acids within a specific region of the protein (amino acids 371–383). The Wisconsin Virtual Learning SMART (Students Modeling A Research Topic) Team modeled PEDF using 3D printing technology. Identifying the PTMs of PEDF in GBM tumors and plasma samples may further the understanding of angiogenesis inhibition and in turn, may lead to the development of treatments for these lethal cancers. The SMART Team Program is funded by a grant from NIH‐CTSA UL1RR031973.
Modeling the Structure and Function of Argonaute in RNAi
Authors: Clark Philip , Cohen Matthew , Danziger Sunaina , Eisner Lauren , Herz‐Roiphe Rachel , Iwahara Yuri , Kafka Oliver , Kiprovski Aleksandar , Koones Gabrielle , Milich Andrew , Mittal Sonia , Richardson Alia , Rubenstein Jordan , Shapiro Jack , Stern Alexandra , Zhao Yinge , Hackett Jennifer , Joshua‐Tor Leemor
Published: June 2021

RNA Interference (RNAi) regulates gene expression in almost all eukaryotic cells by degrading specific mRNA molecules. A key step in this process is the cleavage of mRNA molecules by the endonuclease Argonaute. The Dalton School SMART Team (Students Modeling A Research Topic) modeled the structure and function of the Argonaute protein using 3D printing technology. RNAi specifically degrades mRNA molecules that are complementary to short interfering RNA (siRNA) molecules. Cells produce siRNAs from double‐stranded RNA molecules that could be made by viruses or that could be transcribed from the cell's own regulatory genes. Argonaute binds an siRNA molecule, forming the RNA‐induced silencing complex (RISC). The siRNA in RISC binds a complementary mRNA, allowing Argonaute to cut the mRNA in the middle, which is then degraded by exonucleases. Argonaute is composed of four domains. Three of the domains form a crescent‐shaped base that includes the active site at the center of the base. A positively‐charged cleft between this base and the fourth domain holds the negatively‐charged siRNA. The siRNA binds to a complementary mRNA, positioning it so that aspartic acid residues in the active site can slice the mRNA into two fragments, thus preventing translation of the mRNA. Strategies that use RNAi to treat disease are currently in development. Supported by a grant from the Camille and Henry Dreyfus Foundation.
Energize the anatomy lab experience with 18 minute tutorials
Authors: Benninger Brion
Published: June 2021

Objective

Classically, anatomy dissection labs consist of 2 or 3 hour sessions with team dissection. The objective of this study was to investigate whether short tutorials would improve scores and diminish fatigue within lab.

Methods

Literature search was conducted on educational literature regarding adult attention span. 6 pre‐organized 18 minute tutorials were integrated into each lab session. Two cohorts, X & Y, were given identical lectures and objectives; cohort Y received integrated tutorial sessions, while cohort X did not. Cohort Y students were subdivided into two overall groups (A & B) and further subdivided into “tutorial” groups of 6–9; group A dissected while B received tutorials, the two then switched the next day.

Results

Literature search revealed multiple studies stating attention span of 15 – 20 minutes. Lab Exam Scores: X: 67.26, 77.65, 69.83, 73.83, Y: 85.64, 88.72, 86.01, 89.3.

Discussion

2 to 3 hours of working on the same task, as a team, can be fatiguing. Several studies have been done on student attention span and find a common denominator of 15 to 20 minutes before attention declines. However, variation and abrupt change can re‐engage students for another 15 – 20 minutes before decline.

Conclusion

This study suggests that 18 minute tutorials were integrated successfully, by means of their scores, because dissection and non‐dissection teams remained engaged throughout lab time.
Indispensible Replication Restart Helicase PriA Aids Bacterial Survival: A SMART Team Story
Authors: Havlena Zoë , Hua Amy , Luo Thomas , Vorperian Sevahn , Bhattacharyya Basudeb , Keck James L.
Published: June 2021

Approximately one disruption in DNA replication occur every cell cycle in bacteria leading to partially duplicated chromosomes. Since unfinished replication can result in genome instability and cell death, bacteria need a mechanism to reload the replication machinery onto the genome. Known as the replication restart primosome (RRP), several proteins function to reload the replicative helicase onto abandoned replication forks, restarting DNA replication. PriA is the most conserved member of the RRP, initiating the dominant replication restart pathway. A helicase, PriA remodels collapsed forks and serves as a platform for binding of other primosomal proteins. The Madison West High School Students Modeling a Research Topic (SMART) Team modeled PriA using 3D printing technology. PriA is a multi‐domain protein and residues important for DNA binding, ATP hydrolysis, and helicase activity are modeled. Since DNA replication restart pathways are essential in preserving genomic integrity and cell viability in bacteria, studies of PriA offer an approach to developing novel antibacterial compounds. Supported by a grant from NIH‐CTSA.
Crystal structure and inhibition studies of isocitrate lyase
Authors: Park Yangshin , Rhee Sangkee
Published: June 2021

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle; it was initially discovered in microorganisms and plays a fundamental role in nature by providing the means for microorganisms to grow on acetate, ethanol, or fatty acids. Several lines of evidence suggest that pathogenic microorganisms use primarily fatty acids, rather than carbohydrates, as carbon substrates during infection. . Data from recent studies indicate that M. grisea isocitrate lyase (ICL), a key enzyme in the glyoxylate cycle, is highly expressed during appressorium‐mediated plant infection. We determined the structure of the ICL at 2.7 Å resolution by X‐ray crystallography. More research is needed to identify inhibitors that selectively target enzymes in the glyoxylate cycle. Recently, we have carried out further studies on ICL using putative inhibitor. Hopefully, recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.
Speedball alters the Vpr‐mediated apoptosis in neurons
Authors: Santiago Ismael Dean , Torres Lillith , Noel Richard
Published: June 2021

HIV infection leads to Associated Neurocognitive Disorders (HAND) in nearly 50% of patients. HIV infects microglia and astrocytes in the brain. Infected astrocytes normally do not support viral replication but do express viral proteins. The HIV‐1 viral protein R (Vpr) is an important factor in viral replication and contributes to HAND by eliciting apoptosis in neurons. HIV positive drug abusers show higher neurotoxicity than their nonabusing counterparts. Speedball is a commonly abused drug mixture of cocaine and heroin. We hypothesize that speedball enhances the Vpr‐mediated neuronal apoptosis. We co‐cultured SH‐SY5Y neurons with Vpr‐transfected SVGA astrocytes in the presence of different concentrations of speedball. We then measured markers of cell apoptosis, cell viability and toxicity using a fluorescent assay. Vpr caused apoptosis and cytotoxicity in neurons. Speedball effects were dependent on dose with higher doses reducing Vpr toxicity. Viability measurements were unchanged under the conditions we tested. These data suggest that the combination of Vpr and high dose speedball induced a protective response in neurons. Supported by NIH: DA026722, MD007579 and GM096955.
Tomato lycopene supplementation modulates leptin, resistin and IL‐6 levels in diet‐induced obese rats
Authors: Ferreira Ana Lucia A , Luvizotto Renata AM , Nascimento Andre F , Imaizumi Erika , Pierine Damiana T , Conde Sandro J , Presti Paula T , Correa Camila R , Yeum Kyung‐Jin
Published: June 2021

To determine if lycopene supplementation can regulate adipokine expression in obesity, male Wistar rats were randomly assigned for receiving control diet (C,n=6) or hypercaloric diet (DIO,n=12) for 6 wks. After this period, DIO animals were randomized into two groups: DIO(n=6) or DIO supplemented with lycopene (DIO+L,n=6). They received corn oil (DIO) or lycopene (DIO+L) by gavage for 6 wks. The lycopene used was tomato oleoresin (Lyc‐O‐Mato, Lycored, Israel) that was mixed with corn oil (10 mg/Kg body wt/day). Animals were killed by decapitation and blood samples and adipose tissue were collected for hormonal determinations and gene expression evaluation (IL‐6, MCP‐1, TNF‐α, leptin and resistin). There was no detectable lycopene in plasma of C and DIO groups, however, mean lycopene plasma concentration was 24 nmol/L in DIO+L. Lycopene supplementation did not affect body weight or adiposity, however significantly decreased leptin, resistin and IL‐6 gene expression and plasma concentrations. It reduced gene expression of MCP‐1 in adipose tissue. Lycopene affects adipokines, reducing leptin, resistin and IL‐6 plasma levels, indicating a potential protective effect. These data suggest that tomato lycopene may be an effective strategy to reduce the inflammation in obesity. Financial support: FAPESP(#10/06100–9, #10/19746–4, #11/19847–8, #11/22786–0, #11/19738–4)
Characterization and distribution of the zinc transporter, Zip14, along the murine GI tract
Authors: Guthrie Gregory J , Cousins Robert J
Published: June 2021

ZIP14 is a member of the SLC39 family of transmembrane zinc transporters, which increase zinc content in the cytoplasmic compartment of the cell either from extracellular influx or organellar efflux of zinc. Within the intestines, zinc transport also displays segment‐specific uptake with higher transport of zinc in the duodenum and jejunum. Intestinal zinc transporters, such as ZIP4, also display dynamic localization to the plasma membrane under specific cellular conditions. ZIP14 is responsive to endotoxin, yet changes in localization due to endotoxin are unknown. ZIP14 localization and its function in both cytoplasmic zinc exchange and site‐specific transport are the aims of this study. Isolated segments of the G.I. tract were used to determine protein distribution of ZIP14. ZIP14 protein is most highly distributed along the duodenum followed by jejunum. For intestinal localization, mice were injected with either PBS, or lipopolysaccharide (LPS, 2 mg/kg). Cytoplasmic and membrane fractions were isolated and protein was used to determine ZIP14 localization. The basolateral membrane shows the greatest expression of ZIP14. Intestinal uptake studies using 65Zn (3μCi, subcutaneous) were performed with either PBS, or LPS (2mg/kg, intraperitoneal). 65Zn uptake in the intestine is higher in the ZIP14−/− compared to ZIP14+/+ genotype. This research is supported by NIH Grant DK94244
Neural crest specific ablation of Jagged1 results in aberrant osteoblast differentiation and maxillary bone development
Authors: Hill Cynthia Renae , Yuasa Masato , Schoenecker Jonathan , Goudy Steven
Published: June 2021

Maxillary hypoplasia results from insufficient growth of the upper jaw bone which affects mastication, respiration and communication. This results from either extrinsic restriction due to premature cranial base suture fusion (eg Apert Syndrome) or from intrinsic growth deficiency of the maxilla (eg Alagille Syndrome). The maxillary bone is formed through intramembranous ossification of neural crest cell derived mesenchyme. Conditional deletion of Jagged1 in the cranial neural crest cells (CNC) using Wnt1‐Cre (Jag1CKO) recapitulated the maxillary hypoplasia phenotype in Alagille Syndrome patients, who have JAGGED1 mutations. Jag1CKO mice die by P21 due to starvation secondary to severe maxillary hypoplasia. Analysis of the maxilla using qPCR and immunohistochemistry on Jag1CKO mice from e14 to e18 revealed reduced expression of early and late markers of osteoblast differentiation. In vitro bone mineralization cultures demonstrated that Jag1CKO maxillary mesenchymal cells have decreased mineralization potential and a diminished induction of osteoblast differentiation markers. In addition, lack of Jagged1 signaling in the maxilla resulted in disregulated BMP and TGFβ signaling pathways. These data suggest that Jagged1 signaling in CNC is required during osteoblast differentiation and that down‐stream targets of Jagged1 signaling include the BMP and TGFβ pathways.
Energy restricted ketogenic diet (ERKD) treatment for advanced glioblastoma multiforme (GBM): Case report
Authors: Nikolai Michele , Noel Mary , Schwartz Kenneth , Kurniali Peter , Chang Howard , Olson Lawrence Karl
Published: June 2021

This study aimed to evaluate a 12 week ERKD protocol for advanced GBM.

A 55 year old white male with progressive GBM was hospitalized to establish ketosis and initiate ERKD. A therapeutic range was established for serum betahydroxybutyrate (BHB) of 3–6 mmol/L and serum glucose of 50–70 mg/dl. Feeding began when patient achieved BHB of ≥3 mmol/L. While he was on ERKD his BHB and glucose were measured twice daily as fasting morning (AM) and two hours post‐prandial in the evening (PM). Initial diet consisted of Ketocal® 3:1 formula, 20–25 kcals/kg. The 3:1 fat: protein + carbohydrate ratio met protein needs of 0.6–0.8 grams protein/kg. The patient and wife received instructions from a dietitian on ketogenic meal preparation.

The BHB and glucose were in therapeutic range initially. As feedings advanced, AM BHB levels decreased to < 3 mmol/L and glucose ≥ 80 mg/dl. Ketosis deepened daily and PM measurements >; 3 mmol/L. BHB and glucose levels did not always correlate. Patient found Ketocal® unpalatable and changed to a food only ketogenic diet. Diet manipulation alleviated his hunger and increased AM BHB. His weight decreased by 6%. Patient withdrew from the study when GBM progressed ERKD protocols can be administered with a trained team to adults with GBM. Study is needed to determine the optimal diet to maintain therapeutic glucose and ketones. Supported by Michigan State University Clinical and Translational Sciences Institute.
Protective effects of anthocyanins against oxidative stress induced by acrylamide in human MDA‐MB‐231 cells
Authors: Song Jian , Chen Fang
Published: June 2021

Acrylamide (AA) occurrs in many cooked starchy foods and has caused widespread concern as a possible carcinogen. Anthocyanins are the most abundant flavonoid constituents in fruits and vegetables. The aim of this study is to investigate whether cyanidin‐3‐glucoside (Cy‐3‐glu) and malvidin‐3‐glucoside (Mv‐3‐glu) possess protective effects on oxidative stress induced by in human MDA‐MB‐231 (adenocarcinoma) cells. Cells were incubated with 0–100 μM Cy‐3‐glu or Mv‐3‐glu for 4 h at 37°C, 5% CO2, followed by incubation with 5.0 mM AA for 20 h, and cytotoxicity assays, determination of antioxidant enzymes activities and immunoblot analysis were performed. Compared to cells treated with AA alone, Cy‐3‐glu and Mv‐3‐glu significantly (p < 0.05) reduced ROS generation, recovered glutathione (GSH) depletion (15%–34%) and decreased the activities of glutathione peroxidase (GPx) (98%–189%) and glutathione S‐transferase (GST) (94%–166%). Moreover, the expression levels of GST and gamma‐glutamyl cysteine synthase (γ‐GCS) were enhanced, and cytochrome P450 2E1 (CYP2E1) expression activated by AA was suppressed. Cy‐3‐glu and Mv‐3‐glu present the protective role against oxidative stress induced by AA in MDA‐MB‐231 cells. The consumption of anthocyanins could mitigate health risk caused by the dietary intake of AA. Funding: National Natural Science Foundation of China (NSFC, 81102152).
Effects of a DHA rich n‐3 fatty acid supplementation on fatty acid composition in cerebrospinal fluid in Alzheimer disease. The OmegAD study
Authors: Vedin Inger , Levi Yvonne Freund , Cederholm Tommy , Basun Hans , Hjorth Erik , Irving Gerd Faxén , Schultzberg Marianne , Eriksdotter Maria , Vessby Bengt , Wahlund Lars‐Olof , Salem Norman , Palmblad Jan
Published: June 2021

Background

Little is known about the transfer of essential fatty acids (FA) across the human blood‐brain barrier (BBB) in adulthood.

Objective

Will oral supplementation for 6 mo with omega‐3 fatty acids (n‐3 FA), high in DHA, change the FA profile in cerebrospinal fluid (CSF) in patients with Alzheimer's disease (AD)?

Methods

FA in CSF were analyzed by gas chromatography and mass spectrometry in 33 patients; 18 received n‐3 FA and 15 placebo. Participants were part of the OmegAD Study, where 204 patients with mild AD received 2.3 g n‐3 FA daily or placebo for 6 mo in a randomized fashion. Correlation analyses were made with plasma FA, CSF markers of AD and of inflammation.

Results

At 6 mo a significant increase in CSF (and plasma) EPA, DHA and total n‐3 levels were noted in the n‐3 FA group, whereas no changes were observed in the placebo group. Changes of CSF DHA levels correlated significantly to changes of total and phosphorylated Tau and soluble IL‐1RII in CSF, i.e. the more the FA increased, the more the AD biomarkers decreased.

N‐3 FA supplementation conferred discrete changes of n‐3 FA in CSF, suggesting transfer over the adult BBB. Significant correlations between CSF n‐3 FA and CSF AD markers suggest possible relationships.
Lean Mass and Handgrip Strength May Be Associated With Dietary Intake
Authors: Navaei Negin , Johnson Sarah A. , Figueroa Arturo , Wong Alexei , Kalfon Roy , Elam Marcus , Payton Mark , Arjmandi Bahram H.
Published: June 2021

Diet and nutrition have been shown to influence muscle mass and function. While certain dietary components, e.g. protein, are known to positively influence muscle mass and function, there is a paucity of studies examining the role that other dietary components, e.g. micronutrients and antioxidants, play in muscle health. The objective of this cross‐sectional study was to investigate the degree to which dietary components are associated with lean body mass and handgrip strength in 46 postmenopausal women. Lean mass and fat mass were measured using dual‐energy X‐ray absorptiometry (DXA). Handgrip strength was evaluated using an isometric handgrip exercise. Diet was analyzed using the Food Processor SQL Nutrition and Fitness Program (ESHA Research, Salem, OR). Body mass index (BMI) was calculated as kg/m2. Pearson correlation coefficients were used to determine the relationship between variables of interest. Percent lean mass was negatively correlated with alcohol intake (r = − 0.38, P = 0.04). Handgrip strength tended to be negatively correlated with trans fatty acid intake (r = − 0.33, P = 0.08) and was negatively correlated with disaccharide intake (r = − 0.38, P = 0.04). All other dietary components were not correlated with percent lean mass or handgrip strength. Further studies are needed to look at the relationship between lifestyle factors, lean mass, and handgrip strength.
Inhibitory effects of ethylacetate fraction from Orostachys japonicus on the induction of apoptosis in human lung cancer cells
Authors: Kwon Ji‐Hye , Ryu Deok‐Seon , Kim Seon‐Hee , Lee Dong‐Seok
Published: June 2021

The aim of this study was to evaluate the anti‐cancer activities of biologically active substances in ethylacetate (EtOAc) fraction derived from a potential herbal plant, Orostachys japonicus in A549 human lung cancer cells. O. japonicus, a perennial herbaceous plant belonging to the family Crassulaceae, is traditionally used as anti‐inflammatory, anti‐febrile, hemostatic, anti‐dote, and anti‐cancer agent. The anti‐cancer effect of EtOAc fraction on A549 cells was investigated via MTS and Western blotting assays. The MTS assay showed that O. japonicus dose‐and time‐dependently decreased cell viability and reduced the number of apoptotic cells. Western blot analysis showed that p53 protein was up‐regulated and bcl‐2 protein was down‐regulated, but bax protein was not regulated in response to treatment of A549 cells with the EtOAc fraction. These results suggest that bioactive EtOAc fraction from O. japonicus has anti‐cancer activities in human lung cancer cells.
Oxidative stress is a key regulator of ectopic calcifications in beta‐thalassemic patients
Authors: Quaglino Daniela , Boraldi Federica , Annovi Giulia , Cianciulli Paolo
Published: June 2021

A surprisingly high percentage of clinical complications affecting beta‐thalassemic (beta‐thal) patients is due to ectopic calcifications. Although this abnormal mineralization is similar to that described in pseudoxanthoma elasticum (PXE), ABCC6 gene mutations have been excluded from the pathogenetic mechanisms. Cultured dermal fibroblasts from beta‐thal patients with and without PXE‐like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated to soft connective tissue mineralization.

Results demonstrate that, in beta‐thal patients, elastic fiber calcification is associated to accumulation of anion superoxide and to increased levels of oxidized proteins and lipids. Possibly, as a consequence of the oxidized environment, carboxylation of the calcification inhibitor, MGP, is impaired.

Data suggest that, independently from the primary gene defect, common pathogenetic pathways are associated to ectopic calcifications in PXE and in a number of beta‐thal patients. Research supported by PXE International.
Diet‐derived Xeno‐autoantigen Sialic acid Promotes Inflammation ‐ Evidence for “Xenosialitis”
Authors: Samraj Annie , Crittenden Alyssa , Banda Kalyan , Gregg Christopher John , Assar Siavash , Diaz Sandra L , Varki Nissi , Varki Ajit
Published: June 2021

Sialic acids are monosaccharides with a 9‐carbon backbone and are terminating units of glycoconjugates that coat the vertebrate cell surface. The two major types are N‐acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N‐glycolylneuraminic acid (Neu5Gc). Although Neu5Gc is deficient in humans, it is incorporated into tissues from diet, the first example of a “xeno‐autoantigen”. Given that occurs in the face of circulating anti‐Neu5Gc antibodies in humans, we hypothesized that this generates an antibody‐antigen reaction and inflammation known as “xenosialitis”. Anti‐Neu5Gc antibodies were generated by immunizing human‐like Cmah null mice with chimpanzee red blood cell ghosts and then passively transferred into Cmah null mice that were on a diet either with or without Neu5Gc. We showed incorporation of Neu5Gc into tissues by immunofluorescence and showed that mice fed with Neu5Gc and injected with anti‐Neu5Gc antibodies showed elevated levels of IL‐6 in the peritoneal fluid and higher levels of acute phase proteins in the serum as compared to controls. We also developed a new DMB‐HPLC method to quantitate the free and bound Neu5Gc in common foods and confirmed previous data that Neu5Gc is indeed enriched in red meats. In humans this possibly contributes to chronic inflammatory processes in diseases epidemiologically associated with red meat consumption, such as atherosclerosis and carcinomas.
The effect of mesenchymal stem cells in a PEGylated fibrin gel seeded on an extracellular matrix on skeletal muscle recovery in a volumetric muscle loss model
Authors: Merscham Melissa , Hammers David , Hseih Pei‐Ling , Da'Costa Adriana , Farrar Roger , Suggs Laura
Published: June 2021

This study investigated the effect of mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into an acellular extracellular matrix (ECM) on recovery of skeletal muscle following volumetric muscle loss (VML) injury. Approximately 1/3 of the mass of the lateral gastrocnemius (LGAS) was removed from 6–9 month male Sprague‐Dawley rats, and immediately replaced with an ECM of the same dimensions. One week later, animals were injected with: saline (SAL), MSCs, PEG, or MSCs in PEG (PEG+MSC). Fifty‐six days after injury, LGAS maximal isometric tetanic tension (Po) of the PEG+MSC treated group was significantly higher compared to all other treatments due to greater mass. The specific tension (N/cm2) however, was only significantly higher compared to SAL and PEG groups. Myofiber cross‐sectional area (CSA) was evaluated throughout the implanted scaffold. Cell‐treated groups trended towards a greater percentage of larger myofibers compared to SAL and PEG groups. These findings suggest that MSCs provide the main regenerative stimulus when infused at 7 days, and demonstrates their importance in regeneration in VML injury models. Functional assessments via neural stimulation demonstrated neural regeneration, however long–term viability is yet to be determined.
Activation of P2X4 Receptors on Glia is Necessary for Opioid or HIV‐1 Associated Neurodegeneration
Authors: Sorrell Mary Ellen , Zou Shipping , Knapp Pamela , Hauser Kurt
Published: June 2021

Individuals who abuse μ opioid receptor agonists are at an increased incidence of HIV‐1‐associated neurocognitive disorders (HAND) with more severe symptoms. Microglia play a major role in HIV neuropathogenesis by harboring the infection and producing inflammatory molecules that lead to neuronal injury/death. Extracellular purines also increase microglial activation and neuronal injury. Morphine reportedly increases microglial motility through P2X4 signaling. To test whether HIV‐1 and opioid‐induced neurotoxicity are mediated via purinergic signaling, cocultures of primary neurons and mixed glia were treated with HIV‐1 Tat, morphine, and/or TNP‐ATP, a non‐selective P2X antagonist. Individual neurons were tracked and neuron survival vs. time was assessed. Tat and morphine neurotoxicity was preventable by treatment with TNP‐ATP. Next we investigated neuronal injury by measuring dendritic pruning and excessive increases in intracellular Ca2+ concentration ([Ca2+]i). In both assays, TNP‐ATP blocked Tat/morphine‐induced injury. To investigate P2X receptor involvement, selective antagonists against the P2X1, P2X3, and P2X7 receptor subtypes were screened. These subtypes were not involved in Tat + morphine neurotoxicity. Lastly, cells from P2X4 null mice were used and confirmed that P2X4 receptors on glia are necessary for opiate/Tat neurotoxicity. Support: NIH DA018633, DA028741, DA007027.
Ozone inhalation induces central oxidative stress, cognitive deficits, depressogenic‐ and anxiogenic‐like behavior in stress‐sensitive rats
Authors: Mokoena Mmalebuso Lynnette , Brink Christiaan B , Harvey Brian H , Oliver Douglas W
Published: June 2021

Oxidative stress is suggested to play a role in the etiology of major depression. Incidentally ozone (O3) inhalation induces central oxidative stress, whereas antidepressants have been associated with neuroprotective properties. Flinders Sensitive Line (FSL – stress‐sensitive) rats inhaled 0 or 0.3 parts per million (ppm) ozone 4 hours daily for 15 days, while simultaneously treated with vehicle, desipramine (15 mg/kg/i.p); escitalopram (15 mg/kg/i.p.) or the antioxidant melatonin (50 mg/kg/i.p). On treatment day 15 we measured cognition (Novel Object Recognition Test), anxiety‐like behaviour (Elevated Plus Maze) and depressive‐like behavior (Forced‐Swim Test), and catalase and superoxide dismutase (SOD) activities from dissected hippocampi and cortexes. Data suggest that O3 significantly impairs memory, displays anxiogenic and depressogenic effects and reduces catalase and SOD activities, all of which are attenuated differentially by serotonin‐ and noradrenalin‐selective antidepressants and melatonin. Ozone inhalation may be a useful model to further study the role of oxidative stress in depression. Project funded by the National Research Foundation (NRF) of South Africa
Pramipexole enhances acquisition of responding with a remifentanil‐associated conditioned reinforcer
Authors: Bertz Jeremiah William , Woods James Henry
Published: June 2021

Dopamine D3 receptor (D3R) preferring agonists and antagonists can alter rats’ drug self‐administration behaviors; however, the effects of D3R‐preferring ligands on the conditioned reinforcing effects of drug‐paired stimuli, specifically, have not been extensively evaluated. Therefore, the present experiments assessed the effects of the D3R‐preferring agonist, pramipexole (PRAM), on rats’ acquisition of a new response with a remifentanil‐paired conditioned reinforcer. First, rats received response‐independent IV injections of remifentanil and presentations of a light‐noise stimulus. In “paired” groups, injections and stimuli co‐occurred. In “random” control groups, injections and stimuli occurred independently. Then, in instrumental acquisition (ACQ) test sessions, all rats could respond in an active nose‐poke that produced the stimulus alone or in an inactive nose‐poke with no scheduled consequences. PRAM (0.0–1.0 mg/kg, SC) was given before ACQ sessions. In “paired” groups, but not “random” groups, rats acquired active nose‐poke responding, and PRAM selectively increased active responding. The effects of PRAM were attenuated by the D2 receptor (D2R) preferring antagonist, L‐741,626, but not by the D3R‐preferring antagonist, SB277011A. D2R activity may, thus, be particularly important for conditioned reinforcement. Supported by NIDA grants DA 020669, DA 024897, and DA 032943.