Miracle-dz/UniFAR-FTUs huggingface dataset:
The role of the neck linker and neck in kinesin velocity regulation

Kinesins are microtubule-associated motor proteins that convert the chemical energy of ATP into mechanical energy to perform a wide range of intracellular motility functions. Kinesin molecules represent long-stretched structures revealing distinct globular motor domains, composed of a conserved catalytic core and a so-called mechanical element. The catalytic core contains the microtubule- and ATP-binding sites. The mechanical element, comprising the neck linker and the neck, has been known to be crucial for motility generation, in general. Human neuron-specific kinesin-1 (KIF5A) and human mitotic kinesin-5 (Eg5) are kinesins with conserved motor domains, revealing about 40% identity in amino acid sequence. In addition, there is a very high level of similarity in their crystal structures. Regardless, Eg5 moves about 25 times slower than kinesin-1. Yet, the molecular mechanisms involved in velocity regulation of kinesin motors have been unclear. The central goal of this study was to find out which structural elements of the kinesin motor domain are responsible for motility generation, in general and for velocity regulation, in particular. With this intention, C-terminally truncated KIF5A and Eg5 proteins, lacking defined structural elements of the motor domain, were expressed and comparatively analyzed to define the minimal structural organization of the catalytic core required to generate activity. Furthermore, a series of KIF5A and Eg5 chimeras with interchanged neck linker and neck sequences were constructed to determine the structural elements involved in kinesin velocity regulation. The present study provides first evidence that the neck linker and neck elements are involved in the fine-tuning of the velocity at which kinesin motors move. Evolutionary the neck linker and neck might have evolved to adapt the variety of cellular motors to different biological functions.
Large Reductions in Match Play Physical Performance Variables Across a Professional Football Season With Control for Situational and Contextual Variables

This investigation examined match play physical performance across a professional football season using a multicamera computerized tracking system. A linear mixed-effects model, controlling for situational and contextual variables, identified decreases in team average total distance (TD): season quarter 1 (Q1) (11,047 m) > season quarter 2 (Q2) (10,473 m) (P = 0.002; ES = Small), season quarter 3 (Q3) (10,449 m) (P < 0.001; ES = Moderate), and season quarter 4 (Q4) (10,385 m) (P < 0.001; ES = Moderate); work rate (WR): Q1 (115 m/min) > Q3 (108 m/min) (P < 0.001; ES = Moderate), Q4 (107 m/min) (P < 0.001; ES = Moderate); Q2 (109 m/min) > Q4 (107 m/min) (P = 0.003; ES = Small); high-speed running distance (HSR): Q1 (1,051 m) > Q2 (813 m) (P = 0.006; ES = Small); number of high-speed runs (NHSR): Q1 (87) > Q2 (65) (P < 0.001; ES = Small), Q3 (64) (P = 0.002; ES = Small); sprint distance (SD): Q1 (202 m) > Q4 (130 m) (P < 0.001; ES = Moderate), Q2 (179 m) > Q3 (165 m) (P = 0.035; ES = Small), Q4 (130 m) (P < 0.001; ES = Moderate) and number of sprints (NS): Q1 (20.4) > Q3 (10.2) (P < 0.001; ES = Moderate), Q4 (8.3) (P < 0.001; ES = Large); Q2 (14.9) > Q3 (10.2) (P < 0.001; ES = Moderate), Q4 (8.3) (P < 0.001; ES = Large). Within-position changes were observed for WR: Q1 (122 m/min) > Q4 (113 m/min) (P = 0.002; ES = Large) in central midfielders and for NS: Q1 > Q3 in wide defenders (21.7 vs. 10.8) (P = 0.044; ES = Large) and central midfielders (18.1 vs. 8.3) (P = 0.002; ES = Large); Q1 > Q4 in central defenders (13.1 vs. 5.3) (P = 0.014; ES = Large), wide defenders (21.6 vs. 7.1) (P < 0.001; ES = Very Large), central midfielders (18.1 vs. 8.5) (P = 0.005; ES = Large), and wide midfielders (20.8 vs. 12.2) (P = 0.012; ES = Large); Q2 > Q3 in central midfielders (16.9 vs. 8.3) (P = 0.002; ES = Large) and Q2 > Q4 in wide defenders (16.3 vs. 7.1) (P = 0.005; ES = Very Large), central midfielders (16.9 vs. 8.5) (P = 0.004; ES = Large), and wide midfielders (20.8 vs. 12.2) (P = 0.007; ES = Large). The match-play physical performance was reduced across the competitive season. The most notable reductions were observed in wide defenders, central midfielders, and wide midfielders in sprint performance indices.
Microglia Stimulate Zebrafish Brain Repair Via a Tumor Necrosis Factor-α-Initiated Inflammatory Cascade

The adult zebrafish brain, unlike mammals, has a remarkable regenerative capacity. Although inflammation inhibits regeneration in mammals, it is necessary for zebrafish brain repair. Microglia are resident brain immune cells that regulate the inflammatory response. To explore the microglial role in repair, we used liposomal clodronate, colony stimulating factor-1 receptor (csf1r) inhibition to ablate microglia and two genetic mutants that lacks microglia during brain injury. We found that microglial ablation inhibited injury-induced neurogenesis and regeneration. Microglial suppression specifically attenuated cell proliferation at the progenitor cell amplification stage of neurogenesis. Notably, the loss of microglia impaired phospho-stat3 (signal transducer and activator of transcription 3) and ß-catenin signaling by dynamic regulation of tumor necrosis factor-a after injury, and the ectopic activation of stat3 and ß-catenin rescued neurogenesis defects caused by microglial loss. Microglial absence leads to neutrophil accumulation, hindering the resolution of inflammation and macrophages are not sufficient for regeneration. These findings reveal specific roles of microglia and inflammatory signaling during zebrafish telencephalic regeneration that should provide strategies to improve mammalian brain repair.
Humanized Mouse as a Tool to Predict Immunotoxicity of Human Biologics

Advancements in science enable researchers to constantly innovate and create novel biologics. However, the use of non-human animal models during the development of biologics impedes identification of precise in vivo interactions between the human immune system and treatments. Due to lack of this understanding, adverse effects are frequently observed in healthy volunteers and patients exposed to potential biologics during clinical trials. In this study, we evaluated and compared the effects of known immunotoxic biologics, Proleukin®/IL-2 and OKT3 in humanized mice (reconstituted with human fetal cells) to published clinical outcomes. We demonstrated that humanized mice were able to recapitulate in vivo pathological changes and human-specific immune responses, such as elevated cytokine levels and modulated lymphocytes and myeloid subsets. Given the high similarities of immunological side effects observed between humanized mice and clinical studies, this model could be used to assess immunotoxicity of biologics at a pre-clinical stage, without placing research participants and/or patients at risk.
The Polyadenosine RNA Binding Protein ZC3H14 is Required in Mice for Proper Dendritic Spine Density

ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), an evolutionarily conserved member of a class of tandem zinc finger (CCCH) polyadenosine (polyA) RNA binding proteins, is associated with a form of heritable, nonsyndromic autosomal recessive intellectual disability. Previous studies of a loss of function mouse model, Zc3h14Δex13/Δex13, provide evidence that ZC3H14 is essential for proper brain function, specifically for working memory. To expand on these findings, we analyzed the dendrites and dendritic spines of hippocampal neurons from Zc3h14Δex13/Δex13 mice, both in situ and in vitro. These studies reveal that loss of ZC3H14 is associated with a decrease in total spine density in hippocampal neurons in vitro as well as in the dentate gyrus of 5-month old mice analyzed in situ. This reduction in spine density in vitro results from a decrease in the number of mushroom-shaped spines, which is rescued by exogenous expression of ZC3H14. We next performed biochemical analyses of synaptosomes prepared from whole wild-type and Zc3h14Δex13/Δex13 mouse brains to determine if there are changes in steady state levels of postsynaptic proteins upon loss of ZC3H14. We found that ZC3H14 is present within synaptosomes and that a crucial postsynaptic protein, CaMKIIα, is significantly increased in these synaptosomal fractions upon loss of ZC3H14. Together, these results demonstrate that ZC3H14 is necessary for proper dendritic spine density in cultured hippocampal neurons and in some regions of the mouse brain. These findings provide insight into how a ubiquitously expressed RNA binding protein leads to neuronal-specific defects that result in brain dysfunction.
Glabridin Attenuates the Retinal Degeneration Induced by Sodium Iodate In Vitro and In Vivo

Background Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly over the age of 60. AMD is divided into dry AMD and wet AMD. Although there are certain treatment methods for wet age-related macular degeneration (AMD), there are no effective treatments for dry AMD yet, and finding new drugs or treatment methods for dry AMD has become a priority. For this purpose, this study explored Glabridin (Glab), an isoflavane found in the root extract of licorice, which has never been investigated in relation to eye diseases. Purpose To investigate the effect of Glab on the sodium iodate (NaIO3) induced retinal degeneration in vitro and in vivo. Methods In vitro, cell viability and cytotoxicity were tested with methylthiazolyldiphenyl‐tetrazolium bromide (MTT) assay among the groups of ARPE-19 cells. The cell apoptosis was tested with Hoechst 33342 staining and flow cytometry. The level of Reactive oxygen species (ROS) was measured to check the effect on oxidative stress. The protein expressions of phosphorylation of ERK1/2 and p38 were detected by Western blotting. In vivo, C57BL/6J mice were pretreated with Glab intraperitoneally for one week and continued for 4 weeks. NaIO3 was given to mice through tail vein intravenous injection after 1 week of Glab administration. The retinas of mice were monitored by Optical coherence tomography (OCT) and electroretinography (ERG) at 1w, 2w, 3w, and 4w, respectively, followed by H&E staining. Results In vitro, the Glab protected the retinal pigment epithelial (RPE) cells against oxidative stress and apoptosis by inhibiting phosphorylation of ERK1/2 and the p38 MAPK pathway. In vivo, Glab significantly prevented retinal damage by stopping the progression of retinal degeneration and reducing the formation of deposits on the RPE layer induced by NaIO3. According to the findings of electroretinogram (ERG), Glab helped to maintain the normal function of the retina. Conclusion Glabridin has a protective effect against retinal degeneration. It is suggested that Glab be further investigated for the treatment of retinal degeneration diseases.
Field Evaluation of Recombinant Antigen ELISA in Detecting Zoonotic Schistosome Infection Among Water Buffaloes in Endemic Municipalities in the Philippines

In this study, we investigated the use of recombinant antigens thioredoxin peroxidase-1 (rSjTPx-1) and tandem repeat rSj1TR in evaluating the antibody positivity rates of Schistosoma japonicum infection among water buffaloes from four endemic areas in the Philippines, two municipalities with high endemicity (Calatrava, Negros Occidental and Catarman, Northern Samar) and two municipalities nearing elimination with no cases of human schistosomiasis (Talibon and Trinidad, Bohol). These recombinant antigen ELISA assays were compared with other diagnostic tests including SEA-ELISA, FECT, and fecal-based PCR. Results showed that rSj1TR-ELISA has the highest agreement with PCR in all study areas. Furthermore, significant positivity rates among water buffaloes were seen in Talibon and Trinidad, indicating that water buffaloes are maintaining the schistosome parasites in transmission areas even in the absence of human infection. Hence, serological assay using a more sensitive and specific rSj1TR-ELISA can be used for animal surveillance to prevent emergence and re-emergence of human schistosomiasis.
p38 and Casein Kinase 2 Mediate Ribonuclease 1 Repression in Inflamed Human Endothelial Cells via Promoter Remodeling Through Nucleosome Remodeling and Deacetylase Complex

Vascular pathologies, such as thrombosis or atherosclerosis, are leading causes of death worldwide and are strongly associated with the dysfunction of vascular endothelial cells. In this context, the extracellular endonuclease Ribonuclease 1 (RNase1) acts as an essential protective factor in regulation and maintenance of vascular homeostasis. However, long-term inflammation causes strong repression of RNase1 expression, thereby promoting endothelial cell dysfunction. This inflammation-mediated downregulation of RNase1 in human endothelial cells is facilitated via histone deacetylase (HDAC) 2, although the underlying molecular mechanisms are still unknown. Here, we report that inhibition of c-Jun N-terminal kinase by small chemical compounds in primary human endothelial cells decreased physiological RNase1 mRNA abundance, while p38 kinase inhibition restored repressed RNase1 expression upon proinflammatory stimulation with tumor necrosis factor alpha (TNF-α) and poly I:C. Moreover, blocking of the p38 kinase- and HDAC2-associated kinase casein kinase 2 (CK2) by inhibitor as well as small interfering RNA (siRNA)-knockdown restored RNase1 expression upon inflammation of human endothelial cells. Further downstream, siRNA-knockdown of chromodomain helicase DNA binding protein (CHD) 3 and 4 of the nucleosome remodeling and deacetylase (NuRD) complex restored RNase1 repression in TNF-α treated endothelial cells implicating its role in the HDAC2-containing repressor complex involved in RNase1 repression. Finally, chromatin immunoprecipitation in primary human endothelial cells confirmed recruitment of the CHD4-containing NuRD complex and subsequent promoter remodeling via histone deacetylation at the RNASE1 promoter in a p38-dependent manner upon human endothelial cell inflammation. Altogether, our results suggest that endothelial RNase1 repression in chronic vascular inflammation is regulated by a p38 kinase-, CK2-, and NuRD complex-dependent pathway resulting in complex recruitment to the RNASE1 promoter and subsequent promoter remodeling.
Gene network modeling via TopNet reveals robust epistatic interactions between functionally diverse tumor critical mediator genes

Malignant cell transformation and the underlying genomic scale reprogramming of gene expression require cooperation of multiple oncogenic mutations. Notably, this cooperation is reflected in the synergistic regulation of downstream genes, so-called cooperation response genes (CRGs). CRGs impact diverse hallmark features of cancer cells and are not known to be functionally connected. Yet, they act as critical mediators of the cancer phenotype at an unexpectedly high frequency of >50%, as indicated by genetic perturbations. Here we demonstrate that CRGs function within a network of strong genetic interdependencies that are critical to the robustness of the malignant state. Our approach, termed TopNet, utilizes attractor-based ternary network modeling that takes the novel approach of incorporating uncertainty in the underlying gene perturbation data and is capable of identifying non-linear gene interactions. TopNet reveals topological gene network architecture that effectively predicts previously unknown, functionally relevant epistatic gene interactions, and thus, among a broad range of applications, has utility for identification of non-mutant targets for cancer intervention.
Stable Protein Sialylation in Physcomitrella

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrella (Physcomitrium patens, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, P. patens meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed P. patens. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of six mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for synthesis, activation, transport and transfer of sialic acid. Production of free and (CMP)-activated sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.
Systematics of Protosteloid Amoebae

Because of their simple fruiting bodies consisting of one to a few spores atop a finely tapering stalk, protosteloid amoebae, previously called protostelids, were thought of as primitive members of the Eumycetozoa sensu Olive 1975. The studies presented here have precipitated a change in the way protosteloid amoebae are perceived in two ways: (1) by expanding their known habitat range and (2) by forcing us to think of them as amoebae that occasionally form fruiting bodies rather than as primitive fungus-like organisms. Prior to this work protosteloid amoebae were thought of as terrestrial organisms. Collection of substrates from aquatic habitats has shown that protosteloid and myxogastrian amoebae are easy to find in aquatic environments. Also, prior to this work the Eumycetozoa sensu Olive 1975, was a supposedly monophyletic taxon that included protosteloid amoebae as basal to Myxogastria and Dictyostelia. Three studies presented here erode this idea. These studies include a brief review of the diversity in nucleolar ultrastructure present among eumycetozoans in which new transmission electron micrographs of the protosteloid amoeba Echinosteliopsis oligospora are presented. Further, phylogenetic analyses of protosteloid amoebae based on the gene sequences of the small subunit of the ribosomal RNA (SSU rDNA) show that protosteloid amoebae are polyphyletic within the eukaryote supergroup, Amoebozoa, and that some are deeply embedded within well characterized lineages of nonfruiting amoebae, i.e., vannellids and acanthamoebids. As a result, we now call these simple, fruiting amoebae 'protosteloid amoebae' as opposed to 'protostelids' since the latter implies (incorrectly) that these organisms comprise a natural phylogenetic group. These molecular phylogenetic analyses suggest that isolate LHI05 represents a new protosteloid species that branches among the Acanthamoebidae, an amoebozoan taxon in which fruiting amoebae have never previously been described. Thus a new genus and species, Luapeleamoeba hula g. ad interim sp. ad interim, are proposed to accommodate protosteloid isolate LHI05. Suggestions for additional taxonomic revision are presented as are suggestions for future research particularly with respect to molecular phylogenetic, phylogenomic, and evolutionary/developmental biological approaches. Finally, this work has prompted critical thinking about the origins and evolution of simple fruiting bodies and complex life cycles among amoebozoans, and these basic biological questions are discussed. This dissertation is approved for recommendation to the Graduate Council Dissertation Directors: _______________________________________ Dr. Frederick W. Spiegel _______________________________________ Dr. Jeffrey D. Silberman Dissertation Committee: _______________________________________ Dr. Steven L. Stephenson _______________________________________
Freshwater Sediment Microbial Communities Are Not Resilient to Disturbance From Agricultural Land Runoff

Microorganisms are critically important for the function of surface water ecosystems but are frequently subjected to anthropogenic disturbances at either acute (pulse) or long-term (press) scales. Response and recovery of microbial community composition and function following pulse disturbance is well-studied in controlled, laboratory scale experiments but is less well-understood in natural environments undergoing continual press disturbance. The objectives of this study were to determine the drivers of sediment microbial compositional and functional changes in freshwaters receiving continual press disturbance from agricultural land runoff and to evaluate the ability of the native microbial community to resist disturbance related changes as a proxy for freshwater ecosystem health. Freshwater sediments were collected seasonally over 1 year in Kewaunee County, Wisconsin, a region impacted by concentrated dairy cattle farming, manure fertilization, and associated agricultural runoff which together serve as a press disturbance. Using 16S rRNA gene amplicon sequencing, we found that sediments in locations strongly impacted by intensive agriculture contain significantly higher abundances (p < 0.01) of the genera Thiobacillus, Methylotenera, Crenotrhix, Nitrospira, and Rhodoferax compared to reference sediments, and functions including nitrate reduction, nitrite reduction, and nitrogen respiration are significantly higher (p < 0.05) at locations in close proximity to large farms. Nine species-level potential human pathogens were identified in riverine sediments including Acinetobacer lwoffi and Arcobacter skirrowii, two pathogens associated with the cattle microbiome. Microbial community composition at locations in close proximity to intensive agriculture was not resistant nor resilient to agricultural runoff disturbance within 5 months post-disturbance but did reach a new, stable microbial composition. From this data, we conclude that sediment microbial community composition is sensitive and shifts in response to chemical and microbial pollution from intensive agriculture, has a low capacity to resist infiltration by non-native, harmful bacteria and, overall, the natural buffering capacity of freshwater ecosystems is unable to fully resist the impacts from agricultural press disturbance.