Sex-Specific Absolute Delta Thresholds for High-Sensitivity Cardiac Troponin T.

BACKGROUND: Sex differences in high-sensitivity cardiac troponin (hs-cTn) concentrations from healthy populations have led to the establishment of sex-specific upper reference limits for hs-cTn assays. This study assessed the performance of sex-specific delta (i.e., changes in concentrations) thresholds for the hs-cTnT assay for ruling in acute myocardial infarction (AMI) in different emergency department (ED) populations. METHODS: This retrospective study consisted of 2 cohorts (Cohort 1 derivation and Cohort 2 validation). Cohort 1 consisted of 18 056 ED patients who had serial hs-cTnT measured using a 0-h/3-h algorithm at a US medical center, with Cohort 2 consisting of 1137 ED patients with 0-h/3-h sampling at a Canadian medical center. The primary outcome was AMI diagnosis with sex-specific deltas derived based on the Youden index and specificity estimates (i.e., ≥90%) in Cohort 1 and validated in Cohort 2. RESULTS: In Cohort 1, 42% of all patients had 0-h hs-cTnT above the sex-specific 99th percentile. Males had higher 0-h hs-cTnT (median 17 ng/L) and absolute deltas (median 2 ng/L) than females (0-h median 11 ng/L, P < 0.0001; deltas median 1 ng/L, P < 0.0001) in non-AMI patients but not in patients with AMI. For ruling in AMI, the sex-specific delta thresholds based on 90% specificity (14 ng/L for males, 11 ng/L for females) performed best and resulted in 91% diagnostic accuracy in both males and females. The sex-specific delta thresholds yielding high specificity estimates were confirmed in the validation data set. CONCLUSIONS: Sex-specific absolute delta thresholds can be used to rule in AMI and are robust across different study populations.

Caspase-11 Mediates Pyroptosis of Tubular Epithelial Cells and Septic Acute Kidney Injury.

BACKGROUND/AIMS: Acute kidney injury (AKI) is a serious complication of sepsis and has a high morbidity and mortality rate. Caspase-11 induces pyroptosis, a form of programmed cell death that plays a critical role in endotoxic shock, but its role in tubular epithelial cell death and whether it contributes to sepsis-associated AKI remains unknown. METHODS: The caspase-11-/- mouse received an intraperitoneal injection of lipopolysaccharide (LPS, 40 mg/kg body weight). Caspase-11-/- renal tubular epithelial cells (RTECs) form C57BL caspase-11-/- mice were treated with LPS in vitro. The IL-1ß ELISA kit and Scr assay kit were used to measure the level of interleukin-1ß and serum creatinine. Annexin V-FITC assay and TUNEL staining assay were used to detect the cell death in different groups in vitro and in vivo. Western blot was performed to analyze the protein expression of caspase-11 and Gsdmdc1. RESULTS: LPS-induced sepsis results in lytic death of RTECs, accompanied by increased expression of the pyroptosis-related proteins caspase-11 and Gsdmd. However, the increase in pyroptosis-related protein expression induced by LPS was attenuated with caspase-11 knockout, both in vitro and in vivo. Furthermore, when challenged with lethal doses of systemic LPS, pathologic abnormalities in renal structure, increased serum and kidney interleukin-1ß, increased serum creatinine, and animal death were observed in wild-type mice but prevented in caspase-11-/- mice. CONCLUSIONS: Caspase-11-induced pyroptosis of RTECs is a key event during septic AKI, and targeting of caspase-11 in RTECs may serve as a novel therapeutic target in septic AKI.

Urine and oral fluid drug testing in support of pain management.

In recent years, the abuse of opioid drugs has resulted in greater prevalence of addiction, overdose, and deaths attributable to opioid abuse. The epidemic of opioid abuse has prompted professional and government agencies to issue practice guidelines for prescribing opioids to manage chronic pain. An important tool available to providers is the drug test for use in the initial assessment of patients for possible opioid therapy, subsequent monitoring of compliance, and documentation of suspected aberrant drug behaviors. This review discusses the issues that most affect the clinical utility of drug testing in chronic pain management with opioid therapy. It focuses on the two most commonly used specimen matrices in drug testing: urine and oral fluid. The advantages and disadvantages of urine and oral fluid in the entire testing process, from specimen collection and analytical methodologies to result interpretation are reviewed. The analytical sensitivity and specificity limitations of immunoassays used for testing are examined in detail to draw attention to how these shortcomings can affect result interpretation and influence clinical decision-making in pain management. The need for specific identification and quantitative measurement of the drugs and metabolites present to investigate suspected aberrant drug behavior or unexpected positive results is analyzed. Also presented are recent developments in optimization of test menus and testing strategies, such as the modification of the standard screen and reflexed-confirmation testing model by eliminating some of the initial immunoassay-based tests and proceeding directly to definitive testing by mass spectrometry assays.

Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1.

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

Interpretation of urine drug testing results in patients using transdermal buprenorphine preparations for the treatment of chronic noncancer pain.

OBJECTIVE: To determine whether the prevailing liquid chromatography and tandem mass spectroscopy assay (LC-MS/MS) assay designed to monitor buprenorphine compliance of the sublingual formulation used in the substance abuse treatment setting can be extrapolated to the transdermal formulation used in the chronic pain treatment setting, which is 1000-fold less concentrated. DESIGN: Retrospective chart review. SUBJECTS: Self-reported compliant patients using the transdermal or sublingual formulations of buprenorhphine. Transdermal patch application was also visually confirmed during clinic visits. METHODS: Urine drug test results from a LC-MS/MS were compared between samples from transdermal and sublingual patients. RESULTS: While all sublingual patients tested positive for at least one metabolite of buprenorphine, only 69% of the transdermal patients did so. In addition, the most abundant metabolite in the transdermal patients was buprenorphine-glucuronide, as compared with norbuprenorphine-glucuronide in sublingual patients. CONCLUSIONS: These data suggest that currently available urine drug tests for buprenorphine, including the more expensive LC-MS/MS based assays, may not be sufficiently sensitive to detect the metabolites from transdermal buprenorphine patients. This study highlights the need to evaluate the value and sensitivity of urine drug tests given the wide range of buprenorphine dosing in clinical practice. These results underscore the need for additional cost benefit analyses comparing different confirmatory drug testing techniques including many commercially available drug testing options. © 2014 Wiley Periodicals, Inc.

STIM1 and STIM2 protein deficiency in T lymphocytes underlies development of the exocrine gland autoimmune disease, Sjogren's syndrome.

Primary Sjögren's Syndrome (pSS) is an autoimmune disease involving salivary and other exocrine glands that leads to progressive lymphocytic infiltration into the gland, tissue damage, and secretory defects. The mechanism underlying this disease remains poorly understood. Here we report that mice with T-cell-targeted deletion of Stromal Interaction Molecule (STIM) 1 and STIM2 [double-knockout (DKO)] mice develop spontaneous and severe pSS-like autoimmune disease, displaying major hallmarks of the disease. In DKO mice, diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by age 6 wk, progressing to severe inflammation by age 12 wk. Sjögren's syndrome-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, and progressive salivary gland destruction and loss of fluid secretion were also seen. Importantly, we report that peripheral blood mononuclear cells as well as lymphocytic infiltrates in submandibular glands from patients with pSS demonstrated significant reductions in STIM1 and STIM2 proteins. Store-operated calcium entry was also reduced in peripheral blood mononuclear cells from pSS patients compared with those from healthy controls. Thus, deficiency of STIM1 and STIM2 proteins in T cells, and consequent defects in Ca(2+) signaling, are associated with salivary gland autoimmunopathy in DKO mice and pSS patients. These data reveal a previously unreported link between STIM1 and STIM2 proteins and pSS.

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

Store-operated Ca²+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I(SOC), activated in response to Ca²+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I(CRAC); the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³+, removal of extracellular Ca²+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²+-containing, but not Ca²+-free, medium. Consistent with this, I(CRAC) is activated in cells pretreated with thapsigargin in Ca²+-free medium while I(SOC) is activated in cells pretreated in Ca²+-containing medium. Significantly, TRPC1 function is required for sustained K(Ca) activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

Physiological functions and regulation of TRPC channels.

The TRP-canonical (TRPC) subfamily, which consists of seven members (TRPC1-TRPC7), are Ca(2+)-permeable cation channels that are activated in response to receptor-mediated PIP2 hydrolysis via store-dependent and store-independent mechanisms. These channels are involved in a variety of physiological functions in different cell types and tissues. Of these, TRPC6 has been linked to a channelopathy resulting in human disease. Two key players of the store-dependent regulatory pathway, STIM1 and Orai1, interact with some TRPC channels to gate and regulate channel activity. The Ca(2+) influx mediated by TRPC channels generates distinct intracellular Ca(2+) signals that regulate downstream signaling events and consequent cell functions. This requires localization of TRPC channels in specific plasma membrane microdomains and precise regulation of channel function which is coordinated by various scaffolding, trafficking, and regulatory proteins.

Polarized but differential localization and recruitment of STIM1, Orai1 and TRPC channels in secretory cells.

Polarized Ca(2+) signals in secretory epithelial cells are determined by compartmentalized localization of Ca(2+) signaling proteins at the apical pole. Recently the ER Ca(2+) sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store-dependent Ca(2+) influx. STIM1 also gates the transient receptor potential-canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca(2+) influx and frequency of Ca(2+) oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP(3) receptors (IP(3) Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2-aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca(2+) entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca(2+) permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca(2+) ] oscillations and thus impact critical cellular functions.

Contribution and regulation of TRPC channels in store-operated Ca2+ entry.

Store-operated calcium entry (SOCE) is activated in response to depletion of the endoplasmic reticulum-Ca(2+) stores following stimulation of plasma membrane receptors that couple to PIP2 hydrolysis and IP3 generation. Search for the molecular components of SOCE channels led to the identification of mammalian transient receptor potential canonical (TRPC) family of calcium-permeable channels (TRPC1-TRPC7), which are all activated in response to stimuli that result in PIP2 hydrolysis. While several TRPCs, including TRPC1, TRPC3, and TRPC4, have been implicated in SOCE, the data are most consistent for TRPC1. Extensive studies in cell lines and knockout mouse models have established the contribution of TRPC1 to SOCE. Furthermore, there is a critical functional interaction between TRPC1 and the key components of SOCE, STIM1, and Orai1, which determines the activation of TRPC1. Orai1-mediated Ca(2+) entry is required for recruitment of TRPC1 and its insertion into surface membranes while STIM1 gates the channel. Notably, TRPC1 and Orai1 generate distinct patterns of Ca(2+) signals in cells that are decoded for the regulation of specific cellular functions. Thus, SOCE appears to be a complex process that depends on temporal and spatial coordination of several distinct steps mediated by proteins in different cellular compartments. Emerging data suggest that, in many cell types, the net Ca(2+) entry measured in response to store depletion is the result of the coordinated regulation of different calcium-permeable ion channels. Orai1 and STIM1 are central players in this process, and by mediating recruitment or activation of other Ca(2+) channels, Orai1-CRAC function can elicit rapid changes in global and local [Ca(2+)]i signals in cells. It is most likely that the type of channels and the [Ca(2+)]i signature that are generated by this process reflect the physiological function of the cell that is regulated by Ca(2+).

Contribution of TRPC1 and Orai1 to Ca(2+) entry activated by store depletion.

Store-operated Ca(2+) entry (SOCE) is activated in response to depletion of the ER-Ca(2+) stores by the ER Ca(2+) sensor protein, STIM1 which oligomerizes and moves to ER/PM junctional domains where it interacts with and activates channels involved in SOCE. Two types of channel activities have been described. I(CRAC), via Ca(2+) release-activated Ca(2+) (CRAC) channel, which displays high Ca(2+) selectivity and accounts for the SOCE and cell function in T lymphocytes, mast cells, platelets, and some types of smooth muscle and endothelial cells. Orai1 has been established as the pore-forming component of CRAC channels and interaction of Orai1 with STIM1 is sufficient for generation of the CRAC channel. Store depletion also leads to activation of relatively non-selective cation currents (referred to as I(SOC)) that contribute to SOCE in several other cell types. TRPC channels, including TRPC1, TRPC3, and TRPC4, have been proposed as possible candidate channels for this Ca(2+) influx. TRPC1 is the best characterized channel in this regard and reported to contribute to endogenous SOCE in many cells types. TRPC1-mediated Ca(2+) entry and cation current in cells stimulated with agonist or thapsigargin are inhibited by low [Gd(3+)] and 10-20 µM 2APB (conditions that block SOCE). Importantly, STIM1 also associates with and gates TRPC1 via electrostatic interaction between STIM1 ((684)KK(685)) and TRPC1 ((639)DD(640)). Further, store depletion induces dynamic recruitment of a TRPC1/STIM1/Orai1 complex and knockdown of Orai1 completely abrogates TRPC1 function. Despite these findings, there has been much debate regarding the activation of TRPC1 by store depletion as well as the role of Orai1 and STIM1 in SOC channel function. This chapter summarizes recent studies and concepts regarding the contributions of Orai1 and TRPC1 to SOCE. Major unresolved questions regarding functional interaction between Orai1 and TRPC1 as well as possible mechanisms involved in the regulation of TRPC channels by store depletion will be discussed.

Label-free porous silicon immunosensor for broad detection of opiates in a blind clinical study and results comparison to commercial analytical chemistry techniques.

In this work, we evaluate for the first time the performance of a label-free porous silicon (PSi) immunosensor assay in a blind clinical study designed to screen authentic patient urine specimens for a broad range of opiates. The PSi opiate immunosensor achieved 96% concordance with liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS) results on samples that underwent standard opiate testing (n = 50). In addition, successful detection of a commonly abused opiate, oxycodone, resulted in 100% qualitative agreement between the PSi opiate sensor and LC-MS/MS. In contrast, a commercial broad opiate immunoassay technique (CEDIA) achieved 65% qualitative concordance with LC-MS/MS. Evaluation of important performance attributes including precision, accuracy, and recovery was completed on blank urine specimens spiked with test analytes. Variability of morphine detection as a model opiate target was <9% both within-run and between-day at and above the cutoff limit of 300 ng mL(-1). This study validates the analytical screening capability of label-free PSi opiate immunosensors in authentic patient samples and is the first semiquantitative demonstration of the technology's successful clinical use. These results motivate future development of label-free PSi technology to reduce complexity and cost of diagnostic testing particularly in a point-of-care setting.

CNGA2 contributes to ATP-induced noncapacitative Ca2+ influx in vascular endothelial cells.

BACKGROUND/AIMS: ATP can activate several Ca(2+) influx channels in vascular endothelial cells. For example, it stimulates TRPC channels via capacitative and noncapacitative Ca(2+) entry (CCE and non-CCE, respectively) mechanisms; it also directly acts on P2X purinoceptors, resulting in Ca(2+) influx. In the present study, we tested the hypothesis that cyclic nucleotide-gated (CNG) channels also contribute to ATP-induced non-CCE. METHODS: Two selective inhibitors of CNG channels, L-cis-diltiazem and LY-83583, and CNGA2-specific siRNA were used to study the involvement of CNGA2 in ATP-induced non-CCE in endothelial cells. Ca(2+) influx was studied using Ca(2+)-sensitive fluorescence dyes Fluo-3 and Fluo-4. RESULTS/CONCLUSION: L-cis-diltiazem and LY-83583 markedly reduced ATP-induced non-CCE in 3 types of endothelial cells including the H5V endothelial cell line, the primary cultured bovine aortic endothelial cells and the endothelial cells within isolated mouse aortic strips. The CNGA2-specific siRNA also reduced the ATP-induced non-CCE in H5V endothelial cells. The Ca(2+) influx was inhibited by Rp-8-CPT-cAMPS, MDL-12330A, SQ-22536 and MRS-2179, but not by ODQ or NF-157. Taken together, the present study demonstrated that CNGA2 channels contribute to ATP-induced non-CCE in vascular endothelial cells. It is likely that ATP acts through P2Y(1)receptors and adenylyl cyclases to stimulate CNGA2.

Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity.

hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding but defective for proteasome interaction. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole-cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

CNGA2 channels mediate adenosine-induced Ca2+ influx in vascular endothelial cells.

OBJECTIVE: Adenosine is a cAMP-elevating vasodilator that induces both endothelium-dependent and -independent vasorelaxation. An increase in cytosolic Ca(2+) ([Ca(2+)](i)) is a crucial early signal in the endothelium-dependent relaxation elicited by adenosine. This study explored the molecular identity of channels that mediate adenosine-induced Ca(2+) influx in vascular endothelial cells. METHODS AND RESULTS: Adenosine-induced Ca(2+) influx was markedly reduced by L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated (CNG) channels, in H5V endothelial cells and primary cultured bovine aortic endothelial cells (BAECs). The Ca(2+) influx was also inhibited by 2 adenylyl cyclase inhibitors MDL-12330A and SQ-22536, and by 2 A(2B) receptor inhibitors MRS-1754 and 8-SPT, but not by an A(2A) receptor inhibitor SCH-58261 or a guanylyl cyclase inhibitor ODQ. Patch clamp experiments recorded an adenosine-induced current that could be inhibited by L-cis-diltiazem and LY-83583. A CNGA2-specific siRNA markedly decreased the Ca(2+) influx and the cation current in H5V cells. Furthermore, L-cis-diltiazem inhibited the endothelial Ca(2+) influx in mouse aortic strips, and it also reduced 5-N-ethylcarboxamidoadenosine (NECA, an A(2) adenosine receptor agonist)-induced vasorelaxation. CONCLUSIONS: CNGA2 channels play a key role in adenosine-induced endothelial Ca(2+) influx and vasorelaxation. It is likely that adenosine acts through A(2B) receptors and adenylyl cyclases to stimulate CNGA2.

Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies.

BACKGROUND: Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL. METHODS: By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL. RESULTS: Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly. CONCLUSIONS: This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels.

Epinephrine, through its action on beta-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca(2+) ([Ca(2+)](i)) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca(2+) influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca(2+) dye, it was found that epinephrine and isoprenaline, a beta-adrenoceptor agonist, induced endothelial Ca(2+) influx in the presence of bradykinin. This Ca(2+) influx was inhibited by L-cis-diltiazem and LY-83583, and by a beta(2)-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca(2+) influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca(2+) influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate beta-adrenoceptor agonist-induced endothelial Ca(2+) influx and subsequent vascular dilation.

Pharmacokinetics of tacrolimus in living donor liver transplant and deceased donor liver transplant recipients.

INTRODUCTION: Hepatic dysfunction is an important determinant of the clearance of tacrolimus; however, the impact of reduced hepatic mass in living donor liver transplant (LDLT) patients on the drug exposure and clearance of tacrolimus is not known. AIM.: The aim of the present study is to compare the dosage, concentration and pharmacokinetics parameters of tacrolimus between LDLT and deceased donor liver transplant (DDLT) recipients. PATIENTS AND METHODS: Daily doses used and trough concentrations measured were compared in 12 LDLT and 12 DDLT patients. Multiple blood samples were taken over one dosing interval after oral tacrolimus administration, and pharmacokinetics differences were compared. RESULTS: The mean tacrolimus dosage in first 14 postoperative days was (0.06 mg/kg/day) for LDLT and (0.09 mg/kg/day) for DDLT (P=0.0001). Despite the lower doses used, mean trough concentration was significantly greater in LDLT as compared with DDLT (8.8+/-2.5 ng/mL vs. 6.79+/-1.5 ng/mL, respectively, P=0.013). On the day of the pharmacokinetic study, minimum Concentration (Cmin), 12-hr postdose concentration (Clast), and average concentration (Cavg) were significantly greater in LDLT as compared with DDLT (LDLT: 6.6+/-2.4 ng/mL, 7.2+/-1.8 ng/mL, 8.9+/-3.0 ng/mL; DDLT: 4.3+/-1.0 ng/mL, 4.9+/-1.6 ng/mL, 5.9+/-1.4 ng/mL, P=0.02, 0.04, and 0.02, respectively). Dose normalized AUC was 37.7% greater and clearance, 47.5% lower in LDLT as compared with DDLT. CONCLUSION: Although not statistically significant, the dose normalized AUC was 37.7% greater and clearance 47.5% lower in LDLT as compared with DDLT. An initial tacrolimus dose reduction of about 30-40% may be prudent in LDLT compared with DDLT recipients.

Pharmacokinetics of mycophenolic acid in live donor liver transplant patients vs deceased donor liver transplant patients.

The exposure of mycophenolic acid in live donor liver transplant patients (those receiving a partial hepatic volume) in comparison to deceased donor liver transplant patients (those receiving the whole hepatic volume) after administration of mycophenolate mofetil has not been reported earlier. The aim of the present study is to compare the pharmacokinetics parameters of mycophenolic acid and mycophenolic acid glucuronide in live donor liver transplant patients versus deceased donor liver transplant patients. Twelve live donor liver transplant and 12 deceased donor liver transplant recipients were studied over a dosing interval after intravenous administration of mycophenolate mofetil. The maximum concentration (Cmax) and the area under the plasma concentration versus time curve (AUC) for mycophenolic acid in live donor liver transplant patients were significantly higher than in deceased donor liver transplant patients (Cmax/AUC: live donor liver transplant patients: 16.1 +/- 6.6 microg/mL/43.9 +/- 12.6 microg/mL.h vs deceased donor liver transplant patients: 10.7 +/- 2.0 microg/mL/28.9 +/- 7.1 microg/mL.h; P = .046/.002). The volume of distribution was higher in the deceased donor liver transplant patients compared with live donor liver transplant patients. However, the mean plasma concentration at 12 hours (Clast), drug disposition rate constant, half-life (t 1/2), and mean residence time were similar in both groups. The mean plasma concentration of mycophenolic acid glucuronide was 1.4 to 2.0 times higher in deceased donor liver transplant patients compared with live donor liver transplant patients. These observations point to the need to use a lower dosage (approximately 30%) of mycophenolate mofetil in live donor liver transplant patients compared with deceased donor liver transplant patients.