Placement of a Catheter into the Transverse Sinus in Monitoring Intracranial Lesions: A Technical Note.

High intracranial pressure (ICP) can be induced by stroke, brain trauma, and brain tumor, and lead to cerebral injury. Monitoring the blood flow of a damaged brain is important for detecting intracranial lesions. Blood sampling is a better way to monitor changes in brain oxygen and blood flow than computed tomography perfusion and magnetic resonance imaging. This article describes how to take blood samples from the transverse sinus in a high ICP rat model. Also, it compares the blood samples from the transverse sinus and femoral artery/vein through blood gas analysis and neuronal cell staining. The findings may be of significance to the monitoring of the oxygen and blood flow of intracranial lesions.

Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway.

BACKGROUND: Glycation is an important step in aging and oxidative stress, which can lead to endothelial dysfunction and cause severe damage to the eyes or kidneys of diabetics. Inhibition of the formation of advanced glycation end products (AGEs) and their cell toxicity can be a useful therapeutic strategy in the prevention of diabetic retinopathy (DR). Gardenia jasminoides Ellis (GJE) fruit is a selective inhibitor of AGEs. Genipin is an active compound of GJE fruit, which can be employed to treat diabetes. AIM: To confirm the effect of genipin, a vital component of GJE fruit, in preventing human retinal microvascular endothelial cells (hRMECs) from AGEs damage in DR, to investigate the effect of genipin in the down-regulation of AGEs expression, and to explore the role of the CHGA/UCP2/glucose transporter 1 (GLUT1) signal pathway in this process. METHODS: In vitro, cell viability was tested to determine the effects of different doses of glucose and genipin in hRMECs. Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, immunofluorescence, wound healing assay, transwell assay, and tube-forming assay were used to detect the effect of genipin on hRMECs cultured in high glucose conditions. In vivo, streptozotocin (STZ) induced mice were used, and genipin was administered by intraocular injection (IOI). To explore the effect and mechanism of genipin in diabetic-induced retinal dysfunction, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assays were performed to explore energy metabolism and oxidative stress damage in high glucose-induced hRMECs and STZ mouse retinas. Immunofluorescence and Western blot were used to investigate the expression of inflammatory cytokines [vascular endothelial growth factor (VEGF), SCG3, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-18, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3)]. The protein expression of the receptor of AGEs (RAGE) and the mitochondria-related signal molecules CHGA, GLUT1, and UCP2 in high glucose-induced hRMECs and STZ mouse retinas were measured and compared with the genipin-treated group. RESULTS: The results of CCK-8 and colony formation assay showed that genipin promoted cell viability in high glucose (30 mmol/L D-Glucose)-induced hRMECs, especially at a 0.4 µmol/L dose for 7 d. Flow cytometry results showed that high glucose can increase apoptosis rate by 30%, and genipin alleviated cell apoptosis in AGEs-induced hRMECs. A high glucose environment promoted ATP, ROS, MMP, and 2-NBDG levels, while genipin inhibited these phenotypic abnormalities in AGEs-induced hRMECs. Furthermore, genipin remarkably reduced the levels of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-18, and NLRP3 and impeded the expression of VEGF and SCG3 in AGEs-damaged hRMECs. These results showed that genipin can reverse high glucose induced damage with regard to cell proliferation and apoptosis in vitro, while reducing energy metabolism, oxidative stress, and inflammatory injury caused by high glucose. In addition, ROS levels and glucose uptake levels were higher in the retina from the untreated eye than in the genipin-treated eye of STZ mice. The expression of inflammatory cytokines and pathway protein in the untreated eye compared with the genipin-treated eye was significantly increased, as measured by Western blot. These results showed that IOI of genipin reduced the expression of CHGA, UCP2, and GLUT1, maintained the retinal structure, and decreased ROS, glucose uptake, and inflammation levels in vivo. In addition, we found that SCG3 expression might have a higher sensitivity in DR than VEGF as a diagnostic marker at the protein level. CONCLUSION: Our study suggested that genipin ameliorates AGEs-induced hRMECs proliferation, apoptosis, energy metabolism, oxidative stress, and inflammatory injury, partially via the CHGA/UCP2/GLUT1 pathway. Control of advanced glycation by IOI of genipin may represent a strategy to prevent severe retinopathy and vision loss.

The effect of a-Lipoic acid (ALA) on oxidative stress, inflammation, and apoptosis in high glucose-induced human corneal epithelial cells.

PURPOSE: Oxidative stress and inflammation had been proved to play important role in the progression of diabetic keratopathy (DK). The excessive accumulation of AGEs and their bond to AGE receptor (RAGE) in corneas that cause the formation of oxygen radicals and the release of inflammatory cytokines, induce cell apoptosis. Our current study was aimed to evaluate the effect of ALA on AGEs accumulation as well as to study the molecular mechanism of ALA against AGE-RAGE axis mediated oxidative stress, apoptosis, and inflammation in HG-induced HCECs, so as to provide cytological basis for the treatment of DK. METHODS: HCECs were cultured in a variety concentration of glucose medium (5.5, 10, 25, 30, 40, and 50 mM) for 48 h. The cell proliferation was evaluated by CCK-8 assay. Apoptosis was investigated with the Annexin V- fluorescein isothiocyanate (V-FITC)/PI kit, while, the apoptotic cells were determined by flow cytometer and TUNEL cells apoptosis Kit. According to the results of cell proliferation and cell apoptosis, 25 mM glucose medium was used in the following HG experiment. The effect of ALA on HG-induced HCECs was evaluated. The HCECs were treated with 5.5 mM glucose (normal glucose group, NG group), 5.5 mM glucose + 22.5 mM mannitol (osmotic pressure control group, OP group), 25 mM glucose (high glucose group, HG group) and 25 mM glucose + ALA (HG + ALA group) for 24 and 48 h. The accumulation of intracellular AGEs was detected by ELISA kit. The RAGE, catalase (CAT), superoxide dismutase 2 (SOD2), cleaved cysteine-aspartic acid protease-3 (Cleaved caspase-3), Toll-like receptors 4 (TLR4), Nod-like receptor protein 3 (NLRP3) inflammasome, interleukin 1 beta (IL-1 ß), and interleukin 18 (IL-18) were quantified by RT-PCR, Western blotting, and Immunofluorescence, respectively. Reactive oxygen species (ROS) production was evaluated by fluorescence microscope and fluorescence microplate reader. RESULTS: When the glucose medium was higher than 25 mM, cell proliferation was significantly inhibited and apoptosis ratio was increased (P < 0.001). In HG environment, ALA treatment alleviated the inhibition of HCECs in a dose-dependent manner, 25 µM ALA was the minimum effective dose. ALA could significantly reduce the intracellular accumulation of AGEs (P < 0.001), activate protein and genes expression of CAT and SOD2 (P < 0.001), and therefore inhibited ROS-induced oxidative stress and cells apoptosis. Besides, ALA could effectively down-regulate the protein and gene level of RAGE, TLR4, NLRP3, IL-1B, IL-18 (P < 0.05), and therefore alleviated AGEs-RAGE-TLR4-NLRP3 pathway-induced inflammation in HG-induced HCECs. CONCLUSION: Our study indicated that ALA could be a desired treatment for DK due to its potential capacity of reducing accumulation of advanced glycation end products (AGEs) and down-regulating AGE-RAGE axis-mediated oxidative stress, cell apoptosis, and inflammation in high glucose (HG)-induced human corneal epithelial cells (HCECs), which may provide cytological basis for therapeutic targets that are ultimately of clinical benefit.

Bioresource-derived colloidal nitrogen-doped graphene quantum dots as ultrasensitive and stable nanosensors for detection of cancer and neurotransmitter biomarkers.

Rapid and accurate detection of cancer and neurological diseases is a major issue that has received great attention recently to enable early therapy treatment. In this report, we utilize an atmospheric pressure microplasma system to convert a natural bioresource chitosan into nitrogen-doped graphene quantum dots (NGQDs) for photoluminescence (PL) based selective detection of cancer and neurotransmitter biomarkers. By adjusting the pH conditions during the detection, multiple biomolecules including uric acid (UA), folic acid (FA), epinephrine (EP), and dopamine (DA) can be simultaneously detected with high selectivity and sensitivity using a single material only. Linear relationships between the biomarker concentration and the PL intensity ratio are obtained starting from 0.8 to 100 µM with low limits of detection (LoDs) of 123.1, 157.9, 80.5, and 91.3 nM for UA, EP, FA, and DA, respectively. Our work provides an insight into the multiple biomarker detection using a single material only, which is beneficial for the early detection and diagnosis of cancer and neurological diseases, as well as the development of new drugs.

A case report of possible concurrent vasculitis in vertebral bodies and partial transverse myelitis following COVID-19 vaccination.

INTRODUCTION: Cases with organ-specific and systemic vasculitis associated with corona virus disease 2019 (COVID-19) vaccination have been reported. However, acute partial transverse myelitis (APTM) is rare adverse events following received COVID-19 vaccines. To the best of our knowledge, there is no report on vaccine-associated APTM accompanied by possible concurrent vasculitis. Herein we present a case with possible concurrent spinal vasculitis and APTM following the second dose of inactivated COVID-19 vaccine. CASE SUMMARY: A 33-year-old man presented with weakness of left lower limb and aberrant sensation of his left lower trunk and limb (from T9 level to toes) for 2 days following receipt of an inactivated COVID-19 vaccine. Remarkable demyelinating lesion at T7 spinal cord was showed by 3.0T magnetic resonance imaging (MRI) scan. Moreover, vertebral bodies of T3-T7 also presented high signal in T-2 weighted imaging (T2WI) accompanied by multiple sites of flowing void effect indicating possible vasculitis. Oligoclonal band was positive in cerebrospinal fluid (CSF) while it was negative in sera. Intravenous methylprednisolone (1 g/d) was administrated for 5 days followed by subsequent dose-tapering prednisone. His limb weakness and aberrant sensation both improved and he was able to walk unaided after treatment. The MRI recheck also showed remarkable improvement on the lesions in spinal cord and vertebral bodies. CONCLUSION: this case illustrates the concurrence of possible vasculitis in vertebral bodies and acute transverse myelitis (ATM) following COVID-19 vaccination.

Meroterpenoid Dimers from Ganoderma Mushrooms and Their Biological Activities Against Triple Negative Breast Cancer Cells.

(±)-Dimercochlearlactones A-J (1-10), ten pairs of novel meroterpenoid dimers and one known spirocochlealactone A (11), were isolated from Ganoderma mushrooms. The structural elucidation of new compounds, including their absolute configurations, depends on spectroscopic analysis and electronic circular dichroism (ECD) calculations. Biological studies showed that (+)- and (-)-2, (-)-3, and (+)- and (-)-11 are cytotoxic toward human triple negative breast cancer (TNBC) cells (MDA-MB-231) with IC50 values of 28.18, 25.65, 11.16, 8.18, and 13.02 µM, respectively. Wound healing assay revealed that five pairs of meroterpenoids (±)-5-(±)-8 and (±)-10 could significantly inhibit cell mobility at 20 µM in MDA-MB-231 cells. The results provide a new insight into the biological role of Ganoderma meroterpenoids in TNBC.

ß-Asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

This study aimed to investigate the role and possible mechanism of ß-asarone in regulating neuronal apoptosis and axonal regeneration. A scratch injury was applied to cell cultures of mouse primary cortical neurons to mimic neuronal injury. The neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining and western blot analysis of apoptosis-related proteins. The axonal regeneration was assessed by immunofluorescent staining of ß-tubulin III and western blot analysis of axonal markers. In the results, ß-asarone inhibited neuronal apoptosis and promoted axonal regeneration by suppressing tumor necrosis factor-α (TNF-α) expression in scratch-injured mouse neuronal cells. Research investigating the molecular mechanisms by which ß-asarone inhibited TNF-α expression showed that, on the one hand, ß-asarone suppressed the JNK/c-Jun pathway and thus transcriptionally inhibited TNF-α expression; on the other hand, ß-asarone induced expression of UHRF1 that recruited DNMT1 to induce TNF-α promoter methylation and subsequently decreased the messenger RNA expression of TNF-α. In conclusion, ß-asarone suppresses TNF-α expression through DNA methylation and c-Jun-mediated transcription modulation in scratch-injured neuronal cells.

Protective effect of dabrafenib on renal ischemia-reperfusion injury in vivo and in vitro.

AIMS: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which can lead to poor outcome and increased risk of mortality. Dabrafenib (DAB) is an approved cancer treatment. Little is known about the effect of DAB in prevention or treatment of renal IRI. METHODS: For in vivo experiments, C57BL/6 mice were divided into four groups: sham (no IRI, no DAB), IRI, DAB, and DAB + IRI. IRI was induced by clamping of bilateral renal pedicles for 30 min. For in vitro experiments, HK-2 cells were used to establish the hypoxia/reoxygenation (H/R) injury model, with four groups: control (no H/R, no DAB), H/R, DAB, and DAB + H/R. Renal function and renal histological changes were recorded. Expression of NGAL and KIM-1 proteins and mRNAs were determined by western blotting and qRT-PCR; secretion of inflammatory cytokines (IL-6 and TNF- α) was determined by qRT-PCR; Cell death was determined using the TUNEL assay, measurement of cleaved caspase-3, and flow cytometry. Necroptosis-related proteins were determined by western blotting. RESULTS: In mice, DAB pretreatment improved renal function and also reduced histological injury, inflammation, cell death, and expression of necroptosis-associated proteins. In HK-2 cells, DAB significantly decreased the levels of NGAL and KIM-1, inflammatory cytokines, cell death, and necroptosis-related proteins. CONCLUSION: Our in vitro and in vivo experiments indicated that DAB appears to alleviate renal IRI by suppressing cell death and inhibiting inflammatory responses. DAB has potential use for the clinical prevention and treatment of AKI-induced IRI.

Universal artificial intelligence platform for collaborative management of cataracts.

PURPOSE: To establish and validate a universal artificial intelligence (AI) platform for collaborative management of cataracts involving multilevel clinical scenarios and explored an AI-based medical referral pattern to improve collaborative efficiency and resource coverage. METHODS: The training and validation datasets were derived from the Chinese Medical Alliance for Artificial Intelligence, covering multilevel healthcare facilities and capture modes. The datasets were labelled using a three-step strategy: (1) capture mode recognition; (2) cataract diagnosis as a normal lens, cataract or a postoperative eye and (3) detection of referable cataracts with respect to aetiology and severity. Moreover, we integrated the cataract AI agent with a real-world multilevel referral pattern involving self-monitoring at home, primary healthcare and specialised hospital services. RESULTS: The universal AI platform and multilevel collaborative pattern showed robust diagnostic performance in three-step tasks: (1) capture mode recognition (area under the curve (AUC) 99.28%-99.71%), (2) cataract diagnosis (normal lens, cataract or postoperative eye with AUCs of 99.82%, 99.96% and 99.93% for mydriatic-slit lamp mode and AUCs >99% for other capture modes) and (3) detection of referable cataracts (AUCs >91% in all tests). In the real-world tertiary referral pattern, the agent suggested 30.3% of people be 'referred', substantially increasing the ophthalmologist-to-population service ratio by 10.2-fold compared with the traditional pattern. CONCLUSIONS: The universal AI platform and multilevel collaborative pattern showed robust diagnostic performance and effective service for cataracts. The context of our AI-based medical referral pattern will be extended to other common disease conditions and resource-intensive situations.

Changqin NO. 1 inhibits neuronal apoptosis via suppressing GAS5 expression in a traumatic brain injury mice model.

The present study was designed to investigate the mechanism of the traditional Chinese medicine Changqin NO. 1 on the amelioration of traumatic brain injury (TBI). Adult male C57BL/6J mice and newborn mice were used to generate a mouse TBI model and harvest primary neurons, respectively. The localizations of specific neural markers neuropilin-1 (Nrp-1), growth-associated protein-43 (GAP-43) and microtubule-associated protein Tau (Tau) were examined in brain tissues by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling apoptotic cell detection in tissue sections and the CCK-8 cell viability assay were performed to examine neuronal apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were also carried out in this study. The association between long non-coding RNA (lncRNA) growth-arrest specific 5 (GAS5), miR-335 and RAS p21 GTPase activating protein 1 (Rasa1) was disclosed using the dual-luciferase reporter assay. Changqin NO. 1 inhibited TBI-induced neuronal apoptosis in vivo and in vitro. GAS5 functioned as a competing endogenous RNA (ceRNA) by sponging miR-335 to upregulate Rasa1 expression in mouse neuronal cells. Further investigations demonstrated that GAS5 promoted neuronal apoptosis following TBI via the miR-335/Rasa1 axis. In vivo experiments indicated that Changqin NO. 1 exerted neuroprotection during TBI via the GAS5/miR-335/Rasa1 axis. Changqin NO. 1 promoted neuroprotective effects by inhibiting neuronal apoptosis via the GAS5/miR-335/Rasa1 axis in TBI.

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes.

Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP-Mg(2+). Such activity was enhanced by reconstituted SecYEG-SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca(2+)-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain.

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.

Protein-based MRI contrast agents for molecular imaging of prostate cancer.

PURPOSE: The purpose of this study was to demonstrate a novel protein-based magnetic resonance imaging (MRI) contrast agent that has the capability of targeting prostate cancer and which provides high-sensitivity MR imaging in tumor cells and mouse models. PROCEDURE: A fragment of gastrin-releasing peptide (GRP) was fused into a protein-based MRI contrast agent (ProCA1) at different regions. MR imaging was obtained in both tumor cells (PC3 and H441) and a tumor mouse model administrated with ProCA1.GRP. RESULTS: PC3 and DU145 cells treated with ProCA1.GRPs exhibited enhanced signal in MRI. Intratumoral injection of ProCA1.GRP in a PC3 tumor model displayed enhanced MRI signal. The contrast agent was retained in the PC3 tumor up to 48 h post-injection. CONCLUSIONS: Protein-based MRI contrast agent with tumor targeting modality can specifically target GRPR-positive prostate cancer. Intratumoral injection of the ProCA1 agent in the prostate cancer mouse model verified the targeting capability of ProCA1.GRP and showed a prolonged retention time in tumors.

Calcium-dependent association of calmodulin with the rubella virus nonstructural protease domain.

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca(2+)- and Zn(2+)-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca(2+)-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca(2+)/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152-1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca(2+)/CaM with a dissociation constant of 100-300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a "wrapping around" mode of interaction between RUBpep and Ca(2+)/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.

A single EF-hand isolated from STIM1 forms dimer in the absence and presence of Ca2+.

Stromal interaction molecule 1 (STIM1) is responsible for activating the Ca(2+) release-activated Ca(2+) (CRAC) channel by first sensing the changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) via its luminal canonical EF-hand motif and subsequently oligomerizing to interact with the CRAC channel pore-forming subunit Orai1. In this work, we applied a grafting approach to obtain the intrinsic metal-binding affinity of the isolated EF-hand of STIM1, and further investigated its oligomeric state using pulsed-field gradient NMR and size-exclusion chromatography. The canonical EF-hand bound Ca(2+) with a dissociation constant at a level comparable with [Ca(2+)](ER) (512 +/- 15 microm). The binding of Ca(2+) resulted in a more compact conformation of the engineered protein. Our results also showed that D to A mutations at Ca(2+)-coordinating loop positions 1 and 3 of the EF-hand from STIM1 led to a 15-fold decrease in the metal-binding affinity, which explains why this mutant was insensitive to changes in Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)) and resulted in constitutive punctae formation and Ca(2+) influx. In addition, the grafted single EF-hand motif formed a dimer regardless of the presence of Ca(2+), which conforms to the EF-hand paring paradigm. These data indicate that the STIM1 canonical EF-hand motif tends to dimerize for functionality in solution and is responsible for sensing changes in [Ca(2+)](ER).

A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication.

The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn(2+)-binding ligands. This minidomain bound to Zn(2+) with a stoichiometry of approximately 0.7 and an apparent dissociation constant of <500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn(2+) binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys(1175), Cys(1178), Cys(1225) and Cys(1227) were required for the binding of Zn(2+). Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)(48)CXC Zn(2+)-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication.