Lysosomal agents inhibit store-operated Ca2+ entry.

Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca2+ entry (SOCE) by these agents. Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca2+-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai. Similarly, the K+/H+ ionophore, nigericin, that rapidly elevates lysosomal pH, also inhibited SOCE in a Stim1-dependent manner. In contrast, other strategies for manipulating lysosomes (bafilomycin A1, lysosomal re-positioning) had no effect upon SOCE. Finally, the effects of GPN on SOCE and Stim1 was reversed by a dynamin inhibitor, dynasore. Our data show that lysosomal agents not only release Ca2+ from stores but also uncouple this release from the normal recruitment of Ca2+ influx.

Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation.

RNA polymerase II (RNAPII) is an essential machinery for catalyzing mRNA synthesis and controlling cell fate in eukaryotes. Although the structure and function of RNAPII have been relatively defined, the molecular mechanism of its assembly process is not clear. The identification and functional analysis of assembly factors will provide new understanding to transcription regulation. In this study, we identify that RTR1, a known transcription regulator, is a new multicopy genetic suppressor of mutants of assembly factors Gpn3, Gpn2, and Rba50. We demonstrate that Rtr1 is directly required to assemble the two largest subunits of RNAPII by coordinating with Gpn3 and Npa3. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunit and multiple copies of RTR1 can inhibit the formation of cytoplasmic clump of RNAPII subunit in gpn3-9 mutant, indicating a new layer function of Rtr1 in checking proper assembly of RNAPII. In addition, we find that disrupted activity of Rtr1 phosphatase does not trigger the formation of cytoplasmic clump of RNAPII subunit in a catalytically inactive mutant of RTR1. Based on these results, we conclude that Rtr1 cooperates with Gpn3 and Npa3 to assemble RNAPII core.

Synthetic negative genome screen of the GPN-loop GTPase NPA3 in Saccharomyces cerevisiae.

The GPN-loop GTPase Npa3 is encoded by an essential gene in the yeast Saccharomyces cerevisiae. Npa3 plays a critical role in the assembly and nuclear accumulation of RNA polymerase II (RNAPII), a function that may explain its essentiality. Genetic interactions describe the extent to which a mutation in a particular gene affects a specific phenotype when co-occurring with an alteration in a second gene. Discovering synthetic negative genetic interactions has long been used as a tool to delineate the functional relatedness between pairs of genes participating in common or compensatory biological pathways. Previously, our group showed that nuclear targeting and transcriptional activity of RNAPII were unaffected in cells expressing exclusively a C-terminal truncated mutant version of Npa3 (npa3∆C) lacking the last 106 residues naturally absent from the single GPN protein in Archaea, but universally conserved in all Npa3 orthologs of eukaryotes. To gain insight into novel cellular functions for Npa3, we performed here a genome-wide Synthetic Genetic Array (SGA) study coupled to bulk fluorescence monitoring to identify negative genetic interactions of NPA3 by crossing an npa3∆C strain with a 4,389 nonessential gene-deletion collection. This genetic screen revealed previously unknown synthetic negative interactions between NPA3 and 15 genes. Our results revealed that the Npa3 C-terminal tail extension regulates the participation of this essential GTPase in previously unknown biological processes related to mitochondrial homeostasis and ribosome biogenesis.

An integrative review of primary health care nurses' mental health knowledge gaps and learning needs.

Background: The global COVID-19 pandemic has escalated the prevalence of mental illness in the community. While specialist mental health nurses have advanced training and skills in mental health care, supporting mental health is a key role for all nurses. As front-line health care professionals, primary health care (PHC) nurses need to be prepared and confident in managing mental health issues. Aim: To critically analyse and synthesise international literature about the knowledge gaps and learning needs of PHC nurses in providing mental health care. Design and methods: An integrative review. The quality of papers was assessed using the Mixed Methods Appraisal Tool. Data were extracted into a summary table and analysed using narrative analysis. Data sources: CINAHL, Ovid MEDLINE, Web of Science and EBSCO electronic databases were searched between 1999 and 2019. Papers were included if they reported original research which explored mental health education/training of nurses working in PHC. Findings: Of the 652 papers identified, 13 met the inclusion criteria. Four themes were identified: preparedness; addressing knowledge gaps, education programs, and facilitators and barriers. Discussion: Despite increasing integration of physical and mental health management in PHC, there is limited evidence relating to knowledge gaps and skills development of PHC nurses or their preparedness to provide mental health care. Conclusion: Findings from this review, together with the global increase in mental illness in communities arising from COVID-19, highlight the need for PHC nurses to identify their mental health learning needs and engage in education to prepare them to meet rising service demands.

GPN does not release lysosomal Ca2+ but evokes Ca2+ release from the ER by increasing the cytosolic pH independently of cathepsin C.

The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.

Npa3 interacts with Gpn3 and assembly factor Rba50 for RNA polymerase II biogenesis.

RNA polymerase II is one of the most vital macromolecular complexes in eukaryotes and the assembly of such complete enzyme requires many factors. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNA polymerase II with nonredundant roles. We show here that rapid degradation of each GPN protein in yeast leads to cytoplasmic accumulation of Rpb1 and defects in the assembly of RNA polymerase II, suggesting conserved functions of GPN paralogs for RNA polymerase II biogenesis as in humans. Taking advantage of a multicopy genetic screening, we identified GPN3 and assembly factor RBA50 among others as strong suppressors of npa3ts mutants. We further demonstrated that Npa3 interacts with Gpn3 and Rba50, similarly human Gpn1 physically interacts with Gpn3 and RPAP1 (human analog of Rba50). Moreover, a mutual dependency of protein levels of Npa3 and Gpn3 was also clearly presented in yeast using an auxin-inducible degron (AID) system. Interestingly, Rpb2, the second largest subunit of RNA polymerase II was determined to be the subunit that interacts with both Gpn1 and Rba50, indicating a close association of Npa3 and Rba50 in Rpb2 subcomplex assembly. Based on these results, we conclude that Npa3 interacts with Gpn3 and Rba50, for RNA polymerase II biogenesis. We therefore propose that multiple factors may coordinate through conserved regulatory mechanisms in the assembly of RNA polymerase complex.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail.

The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.

Nucleocytoplasmic shuttling of the GPN-loop GTPase Gpn3 is regulated by serum and cell density in MCF-12A mammary cells.

The best-known function of the essential GPN-loop GTPase Gpn3 is to contribute to RNA polymerase II assembly, a prerequisite for its nuclear targeting. Although this process occurs in the cytoplasm, we have previously shown that Gpn3 enters the cell nucleus before being polyubiquitinated. Here, we show that inhibiting Crm1-mediated nuclear export with leptomycin B, or the proteasome with MG132, caused the nuclear accumulation of recombinant and endogenous Gpn3 in MCF-12A cells. When added simultaneously, leptomycin B and MG132 had an additive effect. Analysis of Gpn3 primary sequence revealed the presence of at least five nuclear export sequence (NES) motifs, with some having a higher exposure to the solvent in the GTP-bound than GDP-bound state in a Gpn3 structural model. Inactivation of any of these NESes led to some degree of Gpn3 nuclear accumulation, although mutating NES1 or NES3 had the more robust effect. MCF-12A cells expressing exclusively a NES-deficient version of Gpn3R-Flag proliferated slower than cells expressing Gpn3R-Flag wt, indicating that nuclear export is important for Gpn3 function. Next, we searched for physiological conditions regulating Gpn3 nucleocytoplasmic shuttling. Interestingly, whereas Gpn3R-Flag was both nuclear and cytoplasmic in low-density growing MCF-12A cells, it was exclusively cytoplasmic in high-density areas. Furthermore, Gpn3R-Flag was cytoplasmic, mostly perinuclear, in sparse but starved MCF-12A cells, and serum-stimulation caused a rapid, although transient, Gpn3R-Flag nuclear accumulation. We conclude that Gpn3 nucleocytoplasmic shuttling is regulated by cell density and growth factors, and propose that Gpn3 has an unknown nuclear function positively linked to cell growth and/or proliferation.

Inflicting, Monitoring, Visualizing, and Quantitating Various Sterile Membrane Damages and the Repair Response in Dictyostelium discoideum.

Eukaryotic cells have been constantly challenged throughout their evolution by pathogens, mechanical stresses, or toxic compounds that induce plasma membrane (PM) or endolysosomal membrane damage. The survival of the wounded cells depends on damage detection and repair machineries that are evolutionary conserved between protozoan, plants, and animals. We use the social amoeba Dictyostelium discoideum as a model system to study bacteria, mechanical or sterile membrane damage that allows us to identify and monitor factors involved in PM, endolysosomal damage response (ELDR), and endolysosomal homeostasis. Importantly, the sterile damage techniques presented here homogenously affect cell populations, which allows to phenotype mutant strains and quantify various aspects of cell fitness using live cell microscopy. This is instrumental to functionally assess genes involved in the repair of damaged plasma membrane or intracellular compartments and the degradation of extensively damaged compartments. Here, we describe how to inflict sterile PM or endolysosomal membrane damage, how to monitor the cell-intrinsic response to damage, and how to proxy proton leakage from damaged acidic compartments and quantify cell viability.

Archaeal GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis.

IMPORTANCE: GPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea, the mechanism by which these GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

Holstein Cattle Face Re-Identification Unifying Global and Part Feature Deep Network with Attention Mechanism.

In precision dairy farming, computer vision-based approaches have been widely employed to monitor the cattle conditions (e.g., the physical, physiology, health and welfare). To this end, the accurate and effective identification of individual cow is a prerequisite. In this paper, a deep learning re-identification network model, Global and Part Network (GPN), is proposed to identify individual cow face. The GPN model, with ResNet50 as backbone network to generate a pooling of feature maps, builds three branch modules (Middle branch, Global branch and Part branch) to learn more discriminative and robust feature representation from the maps. Specifically, the Middle branch and the Global branch separately extract the global features of middle dimension and high dimension from the maps, and the Part branch extracts the local features in the unified block, all of which are integrated to act as the feature representation for cow face re-identification. By performing such strategies, the GPN model not only extracts the discriminative global and local features, but also learns the subtle differences among different cow faces. To further improve the performance of the proposed framework, a Global and Part Network with Spatial Transform (GPN-ST) model is also developed to incorporate an attention mechanism module in the Part branch. Additionally, to test the efficiency of the proposed approach, a large-scale cow face dataset is constructed, which contains 130,000 images with 3000 cows under different conditions (e.g., occlusion, change of viewpoints and illumination, blur, and background clutters). The results of various contrast experiments show that the GPN outperforms the representative re-identification methods, and the improved GPN-ST model has a higher accuracy rate (up by 2.8% and 2.2% respectively) in Rank-1 and mAP, compared with the GPN model. In conclusion, using the Global and Part feature deep network with attention mechanism can effectively ameliorate the efficiency of cow face re-identification.

Npa3-Gpn3 cooperate to assemble RNA polymerase II and prevent clump of its subunits in the cytoplasm.

RNA polymerase II (RNAPII) is an essential machinery in eukaryotes that catalyzes mRNA synthesis and controls cell fate. Although the structure and function of RNAPII are relatively well defined, the molecular mechanism of its assembly process is poorly understood. Three members of GPN-loop GTPase family Npa3/Gpn1, Gpn2, and Gpn3 participate in the biogenesis of RNAPII with non-redundant roles. In this study, we demonstrate that Gpn3 and Npa3 directly participate in the assembly of the two largest subunits during biogenesis of RNAPII. When Gpn3 is defective, assembly of RNAPII is disrupted, leading to cytoplasmic foci of RNAPII subunits. Long-term assembly factor defects will lead to the accumulation of different kind of newly synthesized RNAPII subunits in the cytoplasm to form foci, and this can be prevented by recovery of the defective assembly factor. Cytoplasmic foci of RNAPII subunits in mutants of these assembly factors reveals a new cellular rescue response named the 'RNAPII assembly stress response'.

A nuclear proteome localization screen reveals the exquisite specificity of Gpn2 in RNA polymerase biogenesis.

The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.

Does lysosomal rupture evoke Ca2+ release? A question of pores and stores.

Lysosomotropic agents have been used to permeabilize lysosomes and thereby implicate these organelles in diverse cellular processes. Since lysosomes are Ca2+ stores, this rupturing action, particularly that induced by GPN, has also been used to rapidly release Ca2+ from lysosomes. However, a recent study has questioned the mechanism of action of GPN and concluded that, acutely, it does not permeabilize lysosomes but releases Ca2+ directly from the ER instead. We therefore appraise these provocative findings in the context of the existing literature. We suggest that further work is required to unequivocally rule out lysosomes as contributors to GPN-evoked Ca2+ signals.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant-negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function.

Awake microvascular decompression with fat-teflon sandwich technique: Clinical implications of a novel approach for cranial nerve neuralgias.

Re-appearance of trigeminal neuralgia (TN) pain following microvascular decompression (MVD) is a challenging issue. A selective ablation with MVD provides the best response in such recurrences. The absence of intra-operative indicator for immediate correction of sub-optimal decompression is the primary factor for failure. We analysed the effectiveness and safety of awake MVD in minimizing failure, by tailoring the procedure according to intra-operative response with re-exploration or additional procedure like internal neurolysis in the same setting, especially in patients without vascular compression and those unfit for General Anesthesia (GA). The prospective study from June 2016 to June 2017 includes one glossopharyngeal neuralgia (GPN) and 6 trigeminal neuralgia (TN). Five cases responded with immediate complete pain relief but in 2 cases, incomplete pain relief resulted in alteration of intraoperative decision. In one case, a partial pain relief, mandated an additional internal neurolysis in the same setting, resulting in complete pain relief while in the other, re-exploration revealed a hidden venous conflict, not identified on MRI following which an additional IN was performed. All cases were followed up with BNI PIS for a minimum of one year without recurrence. Awake MVD is safe and reliable intraoperative neurophysiological prognostic marker of immediate pain relief and provides a window for an immediate correction of sub-optimal decompression with Internal Neurolysis when needed, in the same setting, especially in neuroimaging negative and elderly cases unfit for GA. It has the potential to reduce the rate of re-intervention and increase the overall effectiveness of MVD by specifically ameliorating the pain burden and quality of life.

Gpn3 Is Essential for Cell Proliferation of Breast Cancer Cells Independent of Their Malignancy Degree.

Successful therapies for patients with breast cancer often lose their initial effectiveness. Thus, identifying new molecular targets is a constant goal in the fight against breast cancer. Gpn3 is a protein required for RNA polymerase II nuclear targeting in both yeast and human cells. We investigated here the effect of suppressing Gpn3 expression on cell proliferation in a progression series of isogenic cell lines derived from the nontumorigenic MCF-10A breast cells that recapitulate different stages of breast carcinogenesis. Gpn3 protein levels were comparable in all malignant derivatives of the nontumorigenic MCF-10A cells. shRNA-mediated inhibition of Gpn3 expression markedly decreased cell proliferation in all MCF-10A sublines. A fraction of the largest RNA polymerase II subunit Rpb1 was retained in the cytoplasm, but most Rpb1 remained nuclear after suppressing Gpn3 in all cell lines studied. Long-term proliferation experiments in cells with suppressed Gpn3 expression resulted in the eventual loss of all isogenic cell lines but MCF-10CA1d.cl1. In MCF-10CA1d.cl1 cells, Gpn3 knockdown reduced the proliferation of breast cancer stem cells as evaluated by mammosphere assays. After the identification that Gpn3 plays a key role in cell proliferation in mammary epithelial cells independent of the degree of transformation, we also analyzed the importance of Gpn3 in other human breast cancer cell lines from different subtypes. Gpn3 was also required for cell proliferation and nuclear translocation of RNA polymerase II in such cellular models. Altogether, our results show that Gpn3 is essential for breast cancer cell proliferation regardless of the transformation level, indicating that Gpn3 could be considered a molecular target for the development of new antiproliferative therapies. Importantly, our analysis of public data revealed that Gpn3 overexpression was associated with a significant decrease in overall survival in patients with estrogen receptor-positive and Human epidermal growth factor receptor 2 (HER2+) breast cancer, supporting our proposal that targeting Gpn3 could potentially benefit patients with breast cancer.

Evaluation of iron deposition in brain basal ganglia of patients with Parkinson's disease using quantitative susceptibility mapping.

AIM OF THE STUDY: Parkinson's disease is associated with iron deposition in the brain. The QSM (quantitative susceptibility mapping) is more sensitive than T2-weighted imaging, T2* and R2. Few studies have been used QSM to evaluate the iron in the basal ganglia of patients with Parkinson's disease. Our aim was to evaluate the iron deposition in the basal ganglia using QSM and determination of diagnostic value of this method and evaluation of the association between disease stage with QSM and age with QSM in all nuclei, separately. MATERIALS AND METHODS: Thirty patients were tested using Hoehn and Yahr test in three different stages. Fifteen healthy subjects were considered as control group. MRI sequences were performed using SIEMENS 3 T scanner.The Signal Processing in NMR software was used to process and analyze the images. The QSM in every of the basal ganglia was measured separately. RESULTS: There was a significant difference for QSM in the Subtania Nigera, Red Nucleus, Thalamic Nucleus and Globus Pallidus nucleus between two groups. The relationship between disease stage with QSM was significant in Subtania Nigera, Red Nucleus, and Globus Pallidus nucleus. The QSM values had a significant association with disease stage in all nuclei. The results showed that QSM has a higher accuracy in Subtania Nigera, Globus Pallidus, Red Nucleus and Thalamic Nucleus, respectively. CONCLUSIONS: Using QSM in Red Nucleus, Subtania Nigera, and Globus Pallidus nuclei can help diagnosis and staging the patients with Parkinson's disease. In future, studies with emphasis on the disease stage can be helpful in evaluation the different parts of these three nuclei.

Gpn2 and Rba50 Directly Participate in the Assembly of the Rpb3 Subcomplex in the Biogenesis of RNA Polymerase II.

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII.