bluuebunny/crossref_metadata_2025 huggingface dataset:
NOVEL TREATMENT OF BLADDER CANCER WITH CELL THERAPY
Authors: LURIA AYALA R , Jeong Christina , Nolta Jan , Kurzrock Eric
Published: June 2021

Bladder cancer is a common malignant disease in the United States, with an estimated incidence of 71,000 cases in 2009. The very high rate of recurrence and potential for progression is a characteristic of bladder cancer that necessitates close follow‐up and effective management. In this study, we present a new therapeutic approach for preventing recurrent superficial disease and possibly progression to invasive disease. At first, cancerous bladder urothelial cells are removed by an enzymatic treatment using trypsin. Removal of the cancerous urothelial cells is followed by a replacement with ‘healthy’ autologous urothelium that is free of oncogenic mutations. This innovative concept has become feasible with current technologies in stem cell science. Our hypothesis is that pre‐cancerous bladder epithelium and tumor can be removed through de‐epithelialization, and then regenerated with non‐mutagenic cells either from urothelial cell culture or by urothelial differentiated stem cells. Clearly, this technique offers an effective management of patients at risk for developing invasive bladder cancer, which would obviate radical surgery without sacrificing cancer control and survival.
MicroRNA‐610: A novel regulator of gastric cancer migration
Authors: Wang Jing , Zhang Jingwei , Wu Junzhu , Luo Daji , Su Ke , Shi Wentao , Tian Yihao , Wei Lei
Published: June 2021

The main cause of gastric cancer‐related death is more often due to local relapse and long‐distance metastasis. Our previous studies demonstrated that miR‐610, abundantly expressed in normal gastric tissue, showed a significant down‐regulation in SGC‐7901 gastric cancer cells. Conversely, a predicted miR‐610 target, VASP, was over‐expressed in gastric cancer tissue samples. We hypothesized a reciprocal interaction between miR‐610 and VASP. To show this, gastric cancer cells were transfected with a miR‐610 precursor vector to increase endogenous miR‐610 expression. Cell migration was measured by wound healing assay and tumor cell invasion was measured using transwell assay. The interaction between miR‐610 and VASP was ascertained using qPCR, Western Blot and luciferase reporter assays. We demonstrated that ectopic miR‐610 expression reduced the migratory and invasive phenotype of gastric cancer cells. A direct effect of miR‐610 on VASP expression levels was demonstrated at the protein levels via miR‐610 targeting the VASP mRNA 3′‐UTR. In summary, we show that miR‐610 functions as a tumour suppressor miRNA being downregulated in gastric cancer which results in overexpression of VASP. Delivery of miR‐610 into gastric cancer may represent as a novel therapeutic approach to limit gastric cancer metastasis.(The work was supported by the Fundamental Research Funds for the Central Universities No3081006.)
Hypoxic environments cause differential facial shape variation in zebrafish
Authors: Parsons Trish Elizabeth , Weinberg Seth M , Tsang Michael , Vieira Alexandre R
Published: June 2021

High altitude populations exhibit increased risk for craniofacial defects including cleft lip, microtia and branchial arch anomaly complex. A hypoxic maternal environment is thought to be the mechanism responsible for these malformations. In this study, zebrafish were used to test the hypothesis that different hypoxic levels will variably affect facial shape. Three sets of zebrafish were raised in uniform conditions with the exception of dissolved oxygen level. One group was raised at normal conditions (controls, n=7), the second at 30% hypoxia (30H) for 24 hours at 24 hours post fertilization (hpf) and the third at 50% hypoxia (50H) for 24 hours at 24 hpf. Larvae from each group were collected at 120 hpf and stained for cartilage. Dorsoventral images were taken of each specimen and then landmarked to capture viscerocranial morphology. A 2D morphometric analysis was performed on the landmark coordinates. The results show that the mean shape of each group was significantly different and the first two principal components showed a clear separation of the three groups with controls at one end of the shape spectrum, the 50H at the other end and 30H spanning the variation in between. This result indicates that low oxygen levels can cause differential variation in facial shape, which has implications not only for high altitude fetal health, but also other environments, behaviors and genes that affect fetal oxygen delivery.

This project is supported by the University of Pittsburgh Central Research Development Fund (CRDF).

Grant Funding Source : University of Pittsburgh Central
Identification of a proteoglycan‐associated regulatory region active during central nervous system development
Authors: Van Hong Annie , Feenstra Jennifer M , Pira Charmaine U , Oberg Kerby C
Published: June 2021

Lmx1b is a homeodomain transcription factor necessary for limb, kidney, eye and central nervous system (CNS) development. Lmx1b KO mice show reduced cerebellum and tectum formation, loss of dopaminergic/serotonergic neurons, ocular malformations, and distal ventral‐ventral limb symmetry. Direct downstream targets of Lmx1b in the brain and limb remain unknown. Recent microarray studies have implicated the proteoglycans Keratocan, Lumican, and Decorin as possible downstream targets of Lmx1b. Interestingly, these proteoglycans cluster to a locus in the genome (KLD). Using in silico analysis, we identified a conserved non‐coding region with a known Lmx1b binding site (CNR Peak 3). We hypothesize that Lmx1b directly regulates KLD via CNR Peak 3. We isolated CNR Peak 3 and generated a green fluorescent protein (GFP) reporter construct. This construct was electroporated into the chick embryo at Hamburger‐Hamilton (HH) 4 and HH10. GFP was visualized under fluorescent microscopy. GFP expression was detected in the chick HH10 neural tube and HH19 surrounding the developing midbrain and cerebellum. At HH19, Lmx1b and KLD mRNA expression overlap GFP expression. The data suggests CNR Peak 3 plays a role in the regulation of KLD during CNS development. We suspect that Lmx1b mediates KLD regulation through the Lmx1b binding site in CNR Peak 3. Further studies are underway investigating Peak 3 CNR in other Lmx1b patterned tissues.

Grant Funding Source: National Institutes of Child Health and Human Development
Functional defects in mice somatotropes caused by ablation of leptin receptors is reversed, in vitro, by stimulation with Ghrelin
Authors: Syed Mohsin M , Cozart Michael A , Crane Christopher , Haney Anessa C , Akhter Noor , Odle Angela K , Syed Farhan M , Allensworth Melody , Childs Gwen V
Published: June 2021

Deletion mutant mice without leptin receptors on somatotropes (GH cells) have fewer immunolabeled GH cells. This study determined if stimulation will restore GH cells, in vitro. Freshly dissociated pituitaries from male mice were stimulated for 3 h with 0.1–30 nM growth hormone releasing hormone (GHRH) either alone or with ghrelin (10nM). Media were immunoassayed for GH (Millipore, Inc). GHRH (0.3 nM) increased % of immunolabeled GH cells from 33±0.05% (vehicle) to 43±1.8% (p<0.002) in control cells, which secreted 1.79–4X more GH in response to 0.3—10 nM GHRH. Deletion mutant pituitaries contained 16.7±0.4% immunolabeled GH cells, which correlated well with their lower basal GH secretion (8 ng/ml in controls vs 4.9 ng/ml in the mutants p<0.03). GHRH (1–10 nM) restored the immunolabeled GH cells to 30–31% of pituitary cells (30% below levels in stimulated controls) Ghrelin alone stimulated more GH cells in mutants (23%) and enhanced the actions of 0.1—10 nM GHRH resulting in % of GH cells similar to those in the stimulated controls. Ghrelin enhanced secretory actions of 0.3–10 nM of GHRH, resulting in GH levels from mutant cultures equal to that seen in control cultures. These findings indicated that without leptin receptors, somatotropes are quiescent and less responsive to GHRH, suggesting leptin's importance in somatotrope function. This functional deficiency can be reversed in vitro by stimulation with ghrelin.
Effects of laminar shear stress on expression of MT1‐MMP in rat bone marrow‐derived mesenchymal stem cells
Authors: Qin Wang Han
Published: June 2021

Objective

To investigate the effect of laminar shear stress on the expression of membrane type‐1 matrix metalloproteinase (MT1‐MMP) in rat bone marrow‐derived mesenchymal stem cells (rMSCs).

Methods

rMSCs were isolated from SD rat lower extremities and loaded by laminar shear stress with the parallel‐plate flow chamber system, and then the expression of MT1‐MMP mRNA of rMSCs was examined by real‐time PCR.

Results

Shear stress with different magnitude of 5, 15, 30 dynes/cm2 all induced MT1‐MMP mRNA expression, especially high shear stress, 30 dynes/cm2, which was time‐dependent. The expression of MT1‐MMP mRNA was inhibited by SB202190, an inhibitor of p38, but enhanced by wortmannin, an inhibitor of PI3K/Akt.

Conclusion

Shear stress can activate the expression of MT1‐MMP. The amount of MT1‐MMP expression is closely related to stimulating time and the strengths of shear stress, and p38MAPK and PI3K/Akt signal pathway may play a critical role during the process.
Cisplatin induces differentiation of breast cancer cells
Authors: Prabhakaran Praseetha , Hassiotou Foteini , Filgueira Luis
Published: June 2021

Breast tumours are heterogeneous, including cells with cancer stem cell properties and more differentiated tumour cells. As breast cancer stem cells are often resistant to chemotherapy, recent efforts have been focusing on treatments that may shift them towards a more differentiated phenotype, making them more susceptible to treatment. Here, we examined whether t cisplatin induces cell differentiation in four breast cancer cell lines that represent different types of breast tumours (BT549, MDA‐MB‐468, MDA‐MB‐231, MCF7). Cisplatin treatment of 20 μM reduced cell viability from 100% to 50–80%, depending on the cell line. Flow cytometric quantification showed that treatment with cisplatin induced a 18–65% decrease of stem cell marker expression (CD49f, SSEA4) and a 4–97% increase in expression of differentiation markers (CK18, Tubulin). It is concluded that in addition to its effects on cell survival, cisplatin shifts breast cancer cell lines towards a more differentiated phenotype.

Research Support: Malaysian Ministry of Higher Education Postgraduate Scholarship
Abnormal Proliferation and Defective Multilineage differentiation Potential of Adipose‐Derived Stem Cells Isolated from Dystrophin and Utrophin Knockout Mice
Authors: Sohn Jihee , Kozemchak Adam , Oyster Nick , Thompson Seth Davis , Lu Aiping , Gharaibeh Burhan , Tang Ying , Wang Bing , Huard Johnny
Published: June 2021

Adipose derived stem cells (ADSCs) hold potential in cell therapy for a variety of musculoskeletal injuries and diseases including Duchenne Muscular Dystrophy (DMD). DMD is a genetic disease characterized by progressive weakening of the skeletal, cardiac, and diaphragmatic muscles. Using double knock‐out mice (dKO) for dystrophin and utrophin which is a DMD model that better emulates the human phenotype of the disease in mice, we determine if ADSCs from dKO mice are affected in their proliferation and differentiation capacities compared to wild type (WT) ADSCs. ADSCs were isolated from fat tissue of 6 week old dKO‐homo (dys−/−utro−/−), dKO‐hetero (dys−/−utro+/−) and C57BL WT mice and flow cytometry was used to confirm positive expressions of Sca‐1, CD34, CD44 and CD29. Cell proliferation and apoptotic rates investigated using a Live Cell Imaging system showed dKO ADSCs have a higher proliferation rate than WT cells. This may explain the gradual muscle weakness and pseudohypertrophy observed in DMD. Myogenesis, adipogenesis and chondrogenesis are impaired in dKO ADSCs. Impaired myogenesis observed with dKO cells may be a secondary effect of the increased proliferation observed with those cells. These results suggest a dysfunction in the dKO ADSCs and that blocking the exhaustion of ADSCs and stem cell therapy could be used to modulate adipose tissue formation in the muscles of DMD patients to restore structure and function.

Grant Funding Source: NIH
Metabolic signals that drive cell growth and proliferation
Authors: Tu Benjamin , Cai Ling
Published: June 2021

The decision by a cell to enter a round of growth and division must be intimately coordinated with nutrient availability and its metabolic state. These metabolic and nutritional requirements, and the mechanisms by which they induce cell growth and proliferation, remain poorly understood. We discuss recent efforts to understand how cell growth and proliferation are coordinated with metabolism using the budding yeast Saccharomyces cerevisiae.
A Semi‐Purified Soy Protein Diet Preserves Skilled Ladder Rung Walking Performance after Stroke in Rats
Authors: Cheatwood Joseph L , Stout Jessica M , Köppen Jenny R , Wallace Douglas G , Butteiger Dustie N , Banz William J
Published: June 2021

Dietary interventions represent potential strategies for reducing the impact of strokes in people identified as being at enhanced risk. Herein, we examined the effects of a soy protein isolate‐based diet on post‐stroke performance of the skilled ladder rung walking task – a clinically relevant measure of skilled limb function. Adult male LE Hooded rats were fed a semi‐purified diet containing either soy protein isolate or sodium caseinate as the sole protein source. After two weeks on the appropriate diet, baseline performance was recorded on the skilled ladder rung walking task. Rats then underwent a permanent unilateral middle cerebral artery occlusion (MCAO) to induce a stroke lesion. On day 3 post‐stroke, rats were recorded again on the ladder rung walking task to gauge post‐stroke limb use. Rats were then euthanized and brains were removed for assessment of lesion volume and immunohistochemistry. All recordings were analyzed by two blinded reviewers, and errors were recorded as slight or deep slips. At three days after stroke, rats fed the soy protein‐based diet exhibited fewer deep forelimb slips than rats fed the caseinate‐based diet (p<0.05). This represents a significant reduction in the degree of forelimb impairment in the soy protein‐based diet group compared to the caseinate‐based diet group. Comparisons of microglial activation and lesion volume are ongoing. Support: SIUSOM, SIU CIRCNS
Neuroprotective role of Alpha lipoic acid (ALA) in arsenic (As) induced developmental neurotoxicity in rat pups
Authors: Dhar Pushpa , Kaushal Parul , Mehra Raj D
Published: June 2021

Aim

To determine the role of exogenous ALA on arsenic induced morphological features and synaptophysin (SYP) expression in cerebellum of Wistar rat pups.

Material and methods

The experimental group of animals received either NaAsO2 alone (1.5 or 2.5mg/kg bw) or NaAsO2 + ALA (70 mg/kg bw) from postnatal day (PND) 1 to 21 by intraperitoneal (i.p) route whereas the controls received distilled water by the same route. Behavioral tests for evaluation of the arousal, cognition, vestibular and motor coordination ability (cliff avoidance, tail hung, negative geotaxis and midair righting) were carried out on PND 7, 9, 10 and 15 respectively. On PND 22, cerebellum obtained from perfusion fixed animals was processed for paraffin embedding and sectioning. Immunohistochemical localization (SYS) was carried out on cryostat sections.

Results and conclusion

During the experimental period, an apparent decrease (dose dependent) was noted in the performance of NaAsO2 alone exposed pups as compared to the controls. The animals receiving ALA with NaAsO2 showed an apparent improvement in these parameters. Substantial recovery was also evident in As induced disrupted cerebellar cytoarchitecture in animals receiving ALA with NaAsO2. SYP expression, localized mainly in the molecular and granular layers showed an apparent decrease in the NaAsO2 alone exposed animals as compared to control and ALA supplemented group, supporting the hypothesis of the important role of As induced oxidative stress in developmental neurotoxicity and the amelioration of these effects by exogenous ALA.
Expression and Characterization of the Cyr61 Gene in Medakafish
Authors: Monzon Ricardo , Varley Lisa
Published: June 2021

Cyr61 is a member of the CCN family of matricellular proteins, modulating a diverse set of functions in vertebrates with putative roles in development and human disease states. Recent studies have examined CCN function in fish model systems, such as zebrafish (Danio rerio) and medakafish (Oryzias latipes), which provide powerful genetic analysis and accessible visualization of embryonic structures. Analysis of expressed sequence tag (EST) sequences isolated from medakafish provided targeting primer sequences for polymerase chain reaction (PCR), which were used to isolate Cyr61 genetic sequences from both isolated medakafish DNA and cDNA. Amplified PCR products representing overlapping regions in the medakafish genome were sequenced and used in the construction of the medakafish Cyr61 genetic map. Subcloned PCR products from the medakafish Cyr61 gene were then used in the construction of DNA probes for Southern analysis and in the construction of RNA probes for examining the expression of Cyr61 transcripts during medakafish development and in adult tissues. Analysis of the Cyr61 gene in the medakafish reveals comparative expression patterns in embryonic development and adult stages, illustrating the utility of the medakafish as a useful model system for examining Cyr61 function, while providing insights into the development of CCN proteins between different vertebrate species.
Identification of novel stem cell‐specific accessible chromatin domains
Authors: Okino Steven T , Kong Michelle , Wang Yan
Published: June 2021

When permeabilized cells are treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin, can be readily isolated. These small DNA fragments, when analyzed by next‐gen sequencing, provide a global map of accessible chromatin. Here we report the application of this technology in identifying chromatin domains involved in stem cell behavior using NTera2 cells as a model system. By comparing differentiated and undifferentiated NTera2 cells we identified more than 800 genomic regions that are accessible only in undifferentiated cells. We then used these results, together with ENCODE data analyzing human embryonic stem cells and human cancer cells, to identify genomic regions that exhibit stem cell‐specific chromatin accessibility. Importantly, many of these regions are proximal to genes that are associated with cell “stemness” (NANOG and LIN28 are examples). In addition, several stem‐cell specific accessible chromatin domains adjacent to previously uncharacterized, stem‐cell specific non‐coding RNA transcript regions were identified. These findings demonstrate that this approach can serve as a discovery tool to identify chromatin regions that control stem cell behavior. In addition, chromatin regions associated with other biological or pathological states could also be identified using this approach.
Establishment of an NIH‐3T3 Transiently Transfected Ikaros System to Study its Effects on Gene Expression
Authors: Meyer Megan , Van der Steen Travis , Dorsam Glenn
Published: June 2021

This research is focused on improving the treatment and diagnostic therapies of human leukemia. The long term goal of the proposed research is to gain a clearer insight into the molecular mechanisms that cause acute lymphoblastic leukemia (ALL). A recent study showed that a significant population of human ALL patients with a mutation in the tumor suppressor gene, Ikaros (IK), showed an 80% increased rate of relapse despite modern chemotherapy regimens. IK mutated ALL cells have elevated resistance to chemotherapy and are associated with a poor prognosis. The vasoactive intestinal peptide receptor 1 (VPAC1) gene contains numerous high‐affinity IK binding sites, is downregulated during T‐cell activation and is silenced in the majority of human, mouse and rat T‐cell leukemic lines. VPAC1 will be a molecular tool to probe whether ectopically expressed IK protein affects its expression in NIH3T3 cells. Currently, we have conducted a study utilizing bicistronic vectors in an effort to select successfully transfected cells, and observed a decrease in VPAC1 mRNA levels. Future studies will allow for the analysis of IK phosphomimetic mutants and their differential effects on VPAC1 expression. Funding was provided by NCRR/NIH (P20 RR016741).
The ELL component of the SEC complex facilitates RNA polymerase II pause site entry and release
Authors: Byun Jung S , Gardner Kevin
Published: June 2021

This study characterizes the differential mechanistic roles of ELL in intact cells at endogenous genes during mitogen induced transcription. We analyze the stimulus induced spatial loading and distribution of ELL, the PIC complex and members of the super elongation complex (SEC) including AFF4 and P‐TEFb at endogenous genes in intact cells by high resolution chromatin immunoprecipitation (ChIP) and reveal that ELL has multiple roles in the transcription cycle prior to its assembly into the SEC complex. These roles include stabilization of the PIC complex and the licensing of elongation and pause site entry prior to its assembly into and recruitment of the SEC complex. These findings are supported by global gene expression analysis; profiling of endogenous Pol II promoter clearance and pause site entry; and characterization of biochemical protein interactions in cells with and without depletion of endogenous ELL. Moreover, we show that these influences are distinct for ELL as AFF4 gene depletion does not have similar effect. Finally, we use live cell imaging of the kinetic loading of ELL and AFF4 at an endogenous gene locus in intact cells to show that ELL and AFF4 exist in dynamic and kinetically distinct complexes and not exclusively in a single static large molecular complex.
Identification of the telomerase RNA in thermotolerant yeast Hansenula polymorpha
Authors: Smekalova Elena M. , Zvereva Maria I.
Published: June 2021

Telomerase is a ribonucleoprotein which maintains eukaryotic genome integrity via replication of the chromosomal ends. The core enzyme comprises two main components – protein TERT (telomerase reverse transcriptase) which is conserved among all the species and RNA subunit, which in contrary varies a lot in size and sequence which makes it particularly difficult to identify the one from the novel organism. Here we report identification of the telomerase RNA from thermotolerant yeast H. polymorpha. It is one of the smallest among yeast species and consists of 824 nt. Mutational analysis of its template and investigation of the H. polymorpha telomeres revealed that the mechanism of the H. polymorpha telomerase action differs significantly in vivo and in vitro. Telomerase, isolated in vitro efficiently incorporates to the newly synthesized repeat additional noncognate nucleotide which is not present in telomeres. We propose that this action corresponds to nonprocessive state of telomerase and reflects regulation between active and inactive form of the enzyme. This work was supported by: Russian Ministry of Education and Science (P800, P1390 No 02.740.11.07.06, No 16.512.11.2108) and Russian Foundation for Basic Research (Grants 11‐04‐01310‐a and 11‐04‐12051‐ofi‐m‐2011; PNR 5.13).
Whi3p Localizes To Cytoplasmic Foci During Slow Growth At High OD In Saccharyomyces cerevisiae
Authors: Dégallier Joshua Paul , Tembreull Jarrod , Theede Kelly , Segal Scott P.
Published: June 2021

Regulation of cell cycle progression is necessary to ensure that cells do not undergo uncontrolled division, resulting in reduced cell volume. The cell cycle is regulated by several checkpoints, the most important of which is the G1/S checkpoint. In yeast, the G1/S checkpoint is sensitive to the levels of the mammalian cyclin D homolog Cln3p. To ensure that the cell passes through the G1/S checkpoint at the appropriate time, Cln3p levels throughout G1 phase are, in part, controlled by local translation of the CLN3 mRNA by Whi3p, an ER associated RNA binding protein.

Previously, Whi3p was found to genetically interact with SBP1, which encodes a protein involved in general translational repression. This implies that Sbp1p may work in conjunction with Whi3p to control local translation of the CLN3 mRNA. In support, we found that Whi3p is found in cytoplasmic foci at high OD after nutrient exhaustion, where growth is reduced, but are not found during log phase growth. Future studies will determine whether these foci are P‐bodies or stress granules. Furthermore, strains lacking Whi3p have no change in P‐body formation at log phase. Our working model is that Sbp1p represses translation of CLN3 mRNA as it localizes to the endoplasmic reticulum. To ensure repression as the CLN3 mRNA enters the cytoplasm, Sbp1p may associate with the mRNA in the nucleus, as Sbp1p contains a 2‐3‐1 Nuclear Export Sequence.
The regulation of Aurora Kinase B by O‐GlcNAcylation
Authors: Tan Ee Phie , Potnis Anish , Slawson Chad
Published: June 2021

Post‐translational modifications are essential in regulating the function, stability, and localization of proteins. The O‐GlcNAc modification is the attachment of a single N‐acetylglucosamine moiety to serine and threonine residues on nuclear and cytoplasmic proteins. This dynamic modification is processed by the enzymes O‐GlcNAc transferase (O‐GlcNAc addition) or O‐GlcNAcase (O‐GlcNAc removal). The O‐GlcNAc signaling system interacts with and alters other signaling cascades, such as phosphorylation. Previously, we have demonstrated that O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) interact with Aurora Kinase B, a kinase essential in regulating mitosis. Our objective in this study is to determine how Aurora Kinase B is regulated by the O‐GlcNAc signaling system. We employed HeLa cells with either OGT or OGA gain or loss of function and discovered that there is a reduction in the protein levels of Aurora Kinase B and its’ binding partner INCENP. Next, we measured a downstream substrate of Aurora Kinase B, phosphorylation of serine 10 on histone H3, and saw a marked decrease in phosphorylation under all cellular conditions with altered O‐GlcNAcylation. These findings suggest that O‐GlcNAc signaling system plays a role in regulating the activity of Aurora Kinase B through multiple cellular processes, and that aberrant O‐GlcNAcylation can alter Aurora Kinase B signaling and mitotic progression.
Novel telomere‐anchored PCR approach for studying sexual stage telomeres in Aspergillus nidulans
Authors: Kirk Karen E , Wang Nengding , Rizvydeen Saajidha , Kuprys Paulius , Hauer Tyler , Vahedi Mithaq , Davis Shaun , Mirabito Peter
Published: June 2021

Telomeres, the protective caps of linear chromosomes, have been shown to vary in length between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. In the filamentous fungus Aspergillus nidulans, which has several different cell types, the telomere lengths in vegetative hyphae and spores are invariant. No study has determined the telomere length of the sexual ascospores. We report the development of a PCR technique to measure the telomere length in A. nidulans, including the meiotic cells. This novel technique that we call “telomere‐anchored PCR,” measures telomere length using a fraction of the DNA required for terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. Furthermore, an increase in telomere length is not necessary for meiosis in this organism. When the hyphal telomeres were measured in a telomerase reverse transcriptase knockout strain, decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types, is sensitive enough to measure them when exceptionally short, and might be utilized in other organisms.
Translation initiation of non‐LTR retrotransposons by HDVlike self‐cleaving ribozymes
Authors: Ruminski Dana Jannel , Luptak Andrej
Published: June 2021

Many non‐long terminal repeat (non‐LTR) retrotransposons lack internal promoters and are co‐transcribed with their host genes. These transcripts need to be liberated before inserting into new loci. Using structure‐based bioinformatics, we have recently shown that several classes of retrotransposons in phyla spanning arthropods, nematodes, and chordates utilize self‐cleaving ribozymes of the hepatitis delta virus (HDV) family for processing their 5′ termini. Ribozyme‐terminated retrotransposons include rDNA‐specific R2, R4 and R6; telomere‐specific SART; and Baggins and RTE elements. The R2 and R6 ribozymes were recently shown to promote translation initiation of downstream open reading frames in translation reactions in vitro with rabbit reticulocyte lysate and they also demonstrate in vivo activity in Drosophila S2 cell transfections. Translation initiation now extends to other retrotransposon‐associated ribozymes including those that do not insert site‐specifically. Additionally, mutagenesis and deletion experiments have been used to establish the functions of various parts of the ribozyme in ribosome binding and translation. Novel roles, such as those presented here, highlight the biological importance of self‐cleaving ribozymes as well as functional RNAs in general.
The roles of peripheral domains in HDV‐like ribozymes
Authors: Riccitelli Nathan James , Webb Judy , Luptak Andrej
Published: June 2021

Hepatitis delta virus (HDV)‐like ribozymes represent a class of self‐cleaving molecules found in a plethora of species that perform a diverse set of biological roles, ranging from processing of concatameric transcripts to essential steps in non‐LTR retrotransposition. The diversity of biological functions of these ribozymes is reflected in the variety of structures found in the peripheral domains of these molecules. These regions, are not directly responsible for catalysis, yet strongly influence the rate of cleavage. We have determined that as the number of base‐pairs increases in the J1/2 and L2 regions, cleavage rate also increases. Enhanced activity is also observed when the P2 helix is extended, so long as the J1/2 domain is enlarged to accommodate the additional base‐pairs from the ribozyme core. Conversely, cleavage rate is significantly reduced if the J1/2 and L2 are too short, with no activity observable with less than one nucleotide in J1/2 and four nucleotides in L2. Using structure‐based searches, we show that ribozymes consisting of the catalytic core alone, flanked by short peripheral domains, do occur in nature. Interestingly, these ribozymes retain biochemical characteristics of the HDV and the mammalian CPEB3 ribozymes, but develop selectivity for divalent ions, suggesting that while peripheral domains are not required for catalysis, they provide enhanced stability that permits the ribozymes to function in a wider range of environments.
A normal‐tumor switch alters the activity of KLF4‐miR‐206 signaling in breast cancer
Authors: Lin Chen-Chung , Ruppert John Michael
Published: June 2021

The zinc finger protein Kruppel‐like factor 4 (KLF4) is a contextdependent regulator of tumorigenesis and stem cell properties in cancer. KLF4 suppresses the growth of colorectal tumors, but acts as an oncogene in cancers of the mammary gland or skin. Endogenous KLF4 promotes breast cancer cell migration and invasion in vitro and tumorigenesis in animals. We recently reported that microRNA‐206 (miR‐206) expression in breast cancer cells is largely dependent on KLF4, and have now examined its role downstream of KLF4. Like other miRs, miR‐206 regulates the translation of bound transcripts. Surprisingly, miR‐206 promotes protein translation in normal mammary epithelial cells, but inhibits translation in breast cancer cells. Associated with this switched activity in translational control, miR‐206 suppressed the migration of normal breast cells, but promoted cell migration and invasion in cancer cells. As observed for KLF4, knockdown of endogenous miR‐206 in mammary cancer cells inhibited the growth of tumor xenografts. Consistent with these results, miR‐206 can posttranscriptionally suppress Connexin43, a known breast tumor suppressor. Taken together, our study suggests that KLF4‐‐ miR‐206 signaling has an oncogenic role in mammary tumorigenesis, and identifies the normal‐tumor switch in the activity of miR‐206 as a potential determinant of KLF4 activity as either an oncogene or a tumor suppressor.
ZBP1 KH34 consensus RNA‐binding site identifies posttranscriptional regulatory networks
Authors: Chao Jeffrey , Singer Robert
Published: June 2021

ZBP1 is a multifunctional regulator of RNA metabolism that has been shown to control aspects of localization, stability and translation for many mRNAs. To reveal the basis for ZBP1 recognition of its RNA targets, we have biochemically characterized the interaction between ZBP1 and the β‐actin zipcode. We have determined that ZBP1 KH34 recognizes a bipartite RNA element located within the first 28 nucleotides of the zipcode. The 5′ sequence of the bipartite element was determined to be CGGAC while the 3′ sequence was C/A‐CA‐C/U. The spacing between these RNA sequences is consistent with the structure of KH34 that we solved by X‐ray crystallography where the tandem KH domains are arranged in an anti‐parallel pseudodimer conformation. NMR chemical shift mapping of KH34 in complex with zipcode RNA fragments identified that KH4 recognizes the 5′ region of the bipartite element and KH3 binds to the 3′ region. In order to identify other mRNA targets of ZBP1, the KH34 consensus sequence was used to search the 3′ UTRs of human and mouse and the bipartite element was conserved in 114 mRNAs. Many of the predicted mRNA targets were found to coimmunoprecipitate with ZBP1 from mouse embryonic brain lysate. Gene ontology analysis of the ZBP1 target mRNAs indicated that these mRNAs were significantly enriched in categories involved in cellular development, assembly and motility, which correspond to the known cellular functions of ZBP1.
The RNA interference pathway from a trypanosome point of view
Authors: Ullu Elisabetta
Published: June 2021

In most eukaryotic cells expression or delivery of long double‐stranded RNA (dsRNA) signals the presence of foreign and/or potentially dangerous nucleic acids, such as viruses or transcripts derived from retroposons and transposons. As a consequence cells go on red alert and activate specific defense mechanisms to eliminate the invaders. Among such mechanisms is the RNA interference (RNAi) pathway, which is triggered by long dsRNAs, destroys target transcripts in a sequence‐specific manner and is widespread throughout eukaryotic evolution. In the late 90s our laboratory reported the discovery of RNAi in Trypanosoma brucei, a protozoan parasite that causes devastating diseases in sub‐Saharan Africa. Since then the way experiments are carried out in T. brucei has undergone a major revolution, and gene silencing by RNAi has found wide applications, including forward genetic screens and global analysis of gene function. I will present a historical recount of the discovery and mechanistic understanding of the trypanosome RNAi pathway with emphasis on its biological significance.
Construction and characterization of a null allele of the hfq gene of the dissimilatory metal reducing bacterium Shewanella oneidensis
Authors: Brennan Christopher , Keane Meaghan , D'Agostino Christine , Goulet Matthew , Sexton Zachary , Hoegler Karl , Tjaden Brian , Pellock Brett
Published: June 2021

Shewanella oneidensis is a dissimilatory metal reducing bacterium that can utilize a range of terminal electron acceptors when grown under anaerobic conditions. Our primary goal is to identify and characterize genes that encode small regulatory non‐coding RNAs (sRNAs) in S. oneidensis. sRNA genes are regulated by changes in environmental conditions and can themselves regulate other genes by inexact base pairing to their mRNA targets. We are particularly interested in sRNAs that may regulate the process of metal reduction.

The highly conserved RNA chaperone protein Hfq has been widely implicated in bacterial sRNA function. We have constructed a null allele of the S. oneidensis hfq gene. The S. oneidensis hfq mutant has a modest delayed growth phenotype. In addition, our mutant displays reduced survival in stationary phase. Our hfq mutant also exhibits a decreased rate of Cr(VI) reduction when grown under anaerobic conditions compared to the rate of Cr(VI) reduction by wild type S. oneidensis. We are currently evaluating the growth and survival of the Shewanella hfq null mutant. We are also investigating the mutant's sensitivity to a variety of physical stresses, evaluating the capacity of the hfq mutant to reduce metals other than Cr(VI), and determining the role of Shewanella hfq in sRNA function.

This research is funded by a grant from the RI‐INBRE program, award # P20RR016457‐10.
Rapidly Evolving MicroRNAs Retain Their Targets by a Co‐Evolution Mechanism
Authors: Ramaiah Madhuvanthi , Shum Eleen Y. , Wilkinson Miles F.
Published: June 2021

MicroRNAs (miRNAs) are small regulatory molecules with diverse roles in many biological systems. Most studies to date have focused on the function of conserved miRNAs. In contrast, little is known about rapidly evolving miRNAs despite their preponderance in some biological systems, including the reproductive tract. Here, we asked whether rapidly evolving miRNAs have changed their target repertoire in different species or whether they have co‐evolved with their targets to maintain regulation. As a model system to address this question, we used a set of rapidly evolving X‐linked miRNA genes selectively expressed in the testis: the X‐mir1 gene cluster. Using reporter analysis, we identified several targets regulated by both human and mouse X‐miR1 family members despite their having little or no sequences in common. Intriguingly, 3 of the 5 targets we identified with this characteristic encode translation regulatory factors—FMRP, eIF4e and CYFIP1—suggesting that the X‐mir1 cluster has a role in translational regulation in male germ cells. We found that multiple members of the X‐miR1 cluster regulated these targets, suggesting that this miRNA cluster acts as a functional unit. We propose that rapidly evolving miRNAs create a flexible regulatory environment, but at the same time, they retain crucial targets through co‐evolutionary mechanisms to maintain their physiological roles.

Study supported by NIH.
The small RNA complement of salamander limb regeneration
Authors: Lovci Michael Thomas , Zhu Wei , Pao Gerald , Kuo Dwight , Smith Jeramiah , Verma Inder , Voss S Randal , Bryant Susan , Gardiner David , Harkins Timothy , Yeo Gene , Hunter Tony
Published: June 2021

The exceptional regenerative capacities of the Mexican axolotl (Ambystoma mexicanum) have made it one of the most prominent organisms in the study of vertebrate regeneration. Here we present results of a genomic survey of small RNAs during limb regeneration using 454 high‐throughput sequencing technology. We mapped small RNAs to an axolotl transcriptome assembly and to a representative library of repetitive DNA sequences sampled from the axolotl genome. Intriguingly, the majority of the axolotl small RNAs (~72%) mapped to repetitive DNA elements while only ~8% corresponded to microRNAs. We identified several microRNAs which appear to be regulated over the regeneration time‐course, several of which are conserved in a zebrafish fin regeneration model. We identified unusual small RNAs that emerge during the regenerative process which could be endogenous siRNAs or piRNAs; several of these were successfully mapped to assembled, protein‐coding transcripts. These observations suggest a small‐RNA mediated mechanism for regeneration that is conserved in vertebrates which employs small RNA classes which have been previously ascribed to pluripotent cell types.
Specific microRNA (miRNA) species regulate the expression of the beta‐site APP‐cleaving enzyme (BACE1) in relevant human cell types with implications for Alzheimer disease
Authors: Lahiri Debomoy K , Long Justin M
Published: June 2021

MiRNAs are small RNAs that mediate potent inhibitory effects on gene expression and which recent studies implicate in the regulationof gene products involved in Alzheimer's disease (AD) (Long & Lahiri, Exp. Neurol., 2011). Aberrations in AD may result from the over‐production of amyloid‐β peptide (Aβ), a product of Aβ precursor protein (APP). Dysregulation of proteins involved in Aβ production, such as APP and BACE1, may contribute to excess Aβ deposition. Using multiple bioinformatic tools and functional studies, we reported a specific microRNA (miR‐101) that regulates APP levels (Long & Lahiri, BBRC, 2011). Here we report the discovery of a novel BACE1‐specific miRNA regulatory interaction. First, we prepared a BACE1 3′‐UTR reporter construct by inserting the 3.9 kb BACE1 3′‐UTR downstream of a Renilla reporter luciferase gene. The reporter construct was delivered along with several miRNAs predicted to target the BACE1 3′‐UTR into human cultures. Several “hits” (e.g. miR‐339‐5p) resulted in reduced reporter expression. We validated the reporter expression for miR‐339‐5p by Western analysis of native BACE1 levels, which were significantly reduced following miR‐339‐5p delivery (with anticipated reductions in toxic Aβ levels). Our results reveal a novel regulatory interaction between BACE1 and specific endogenously expressed miRNA species that may serve as putative therapeutic targets for AD.

This work is supported by grants from Alzheimer's Association and NIH to Dr. D.K. Lahiri.
Manipulating the ERAD pathway to regulate GABAA receptor protein homeostasis
Authors: Han Dongyun , Mu Tingwei
Published: June 2021

GABAA (γ‐aminobutyric acid type A) receptors are major inhibitory ligand‐gated ion channels in the mammalian central nervous system. To function, GABAA receptors need to fold into their native structures, assemble correctly in the endoplasmic reticulum (ER), and traffic properly to plasma membrane. Terminally misfolded GABAA receptors are degraded through the ER associated protein degradation (ERAD) pathway. Extensive ERAD leads to loss of function of GABAA receptors, causing idiopathic epilepsy. We intend to manipulate the ERAD pathway to regulate GABAA receptor protein homeostasis in the cell, as a therapeutic strategy to ameliorate idiopathic epilepsy. A number of critical ERAD factors were enriched after the immunoprecipitation of GABAA receptors followed by affinity purification‐mass spectrometry analysis using HEK293 overexpressing GABAA receptors. Inhibition of critical ERAD factors increased the total protein expression level for both wild type α1β2γ2 and α1(A322D)β2γ2 GABAA receptors. However, trafficking and function of GABAA receptors were only enhanced with selective ERAD factor inhibition for α1(A322D)β2γ2 GABAA receptors. It appears that selective ERAD factor inhibition upregulates both cellular folding and cellular trafficking capacity. Manipulation of the ERAD pathway might be a general strategy to ameliorate a variety of ion channel misfolding diseases.
Regulation of chaperone activity in the endoplasmic reticulum
Authors: Gehring Kalle , Kozlov Guennadi
Published: June 2021

The synthesis and folding of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a large family of ER proteins. The PDI family includes general enzymes such as PDI‐1 that recognize unfolded proteins, and others, including ERp57 and ERp72, that are selective for specific proteins. PDI‐1 consists of four thioredoxin‐like domains termed a‐b‐ b’‐a’. The a and a’ domains contain catalytic CxxC motifs that confer the disulfide oxidoreductase activity, while the b and b’ domains contain the principal binding site for unfolded proteins. Here, we determined the crystal structure of a dimeric form of a bb’ fragment of human PDI at 2.3 Å resolution. The structure shows that the b’ domain is mediates substrate interactions through a hydrophobic binding site for unfolded proteins. In ERp57, this site is conserved but binds lectin co‐chaperones that are specific for the folding of glycoproteins. Recent results with PDI‐1 suggest the hydrophobic binding site of PDI‐1 may also be involved in regulation of the redox state of ER chaperones.
Regulation of levels and localization of peptidases in prostate cancer cells by steroid hormones
Authors: DeFries Christa C. , Liu Yu , Bruce Lisa A. , Gambe Rutendo G. , Belayneh Najat S. , Tetel Marc J. , Wolfson Adele J.
Published: June 2021

The enzyme thimet oligopeptidase (TOP) is present in a wide variety of cell types and hydrolyzes many peptides of physiological importance in neuroendocrine pathways. TOP has been identified in prostate cancer cells, and its activity is increased by androgens in androgen‐responsive cells. Using the androgen‐sensitive cell line DU145‐AR, we have found that TOP levels and activity were significantly increased after 24 hours incubation with either dihydrotestosterone or estradiol. TOP was found both intracellularly and in the medium. These increases were not observed with an androgen‐insensitive line (DU145‐T). TOP works in concert with the peptidase prolyl endopeptidase (PEP) in breakdown of gonadotropin‐releasing hormone (GnRH), a peptide known to influence growth of prostate cells. We have identified PEP in DU145‐AR cells and seen an increase in PEP levels with androgen treatment. Furthermore, immunocytochemistry revealed that TOP was more clearly localized to nuclei at high levels of estradiol, compared to its distribution between cytoplasm and nucleus in the absence of steroid or at low levels of estradiol. These results suggest that peptidases, especially those involved in GnRH metabolism, may be involved in regulation of growth of prostate cancer cells and, hence, be targets for cancer therapy.
Deletion of the Cathepsin B gene improves memory deficits in a Alzheimer's disease mouse model expressing APP containing the wild‐type β‐secretase site sequence
Authors: Hook Vivian , Yu Jin , Zhu Hong , El-Amour Salim , Hook Gregory , Kindy Mark
Published: June 2021

Severe memory loss is detrimental in Alzheimer's disease (AD). We hypothesize that cathepsin B (CatB) participates in memory deficits of AD. Therefore, goal of this study was to assess the effects of cathepsin B gene knockout (CatB) on the memory deficits of a transgenic AD mouse model expressing human APP containing the wild‐type (WT) β‐secretase site and the London mutant γ‐secretase site sequences (AβPPWT/Lon). Results show that CatB gene knockout in AβPPWT/Lon mice results in significant improvement in memory, assessed by the Morris water maze test. Moreover, the CatB gene knockout reduces brain leevels of amyloid‐β peptides (Aβ) and carboxyl‐terminal β‐fragment (CTFβ); these data are consistent with CatB possibly having a role as β‐secretase for the WT β‐site of AβPP expressed in the majority of AD patients. In contrast, deleting the BACE1 gene in the AβPPWT/Lon mice had no effect on memory deficits or on the biomarkers. In conclusion, these data validate CatB as a drug target for improving memory deficits in AβPPWT/Lon mice and suggest that CatB inhibitors may be effective for improving memory in the majority of AD patients.

This work was supported by NIH grants R21AG0311963, R44AG030865, and R44AG032784 to GH, and a grant from the Alzheimer's Association to VH.