Abscisic acid induces PpeKIL1 to terminate fruit growth and promote fruit abortion in peach (Prunus persica).

Abnormal pollination from chance events or hybridization between species leads to unusual embryo development, resulting in fruit abortion. To elucidate the mechanism underlying fruit abortion, we conducted a comprehensive analysis of the transcriptome and hormone profiles in aborting fruits (AF) derived from an interspecific cross between the peach cultivar 'Huangjinmi 3' and the Prunus mume cultivar 'Jiangmei', as well as in normal-seeded fruits (NF) resulting from an intraspecific cross of 'Huangjinmi 3' with the 'Manyuanhong' peach cultivars. Growth of AF was inhibited during the exponential growth phase, with up-regulation of oxidative stress related genes and down-regulation of DNA replication and cell cycle genes. Accumulation of the tissue growth-related hormones auxin and cytokinin was reduced in AF, while levels of the growth inhibiting hormone abscisic acid (ABA) were higher compared to NF. The increased ABA concentration aligned with down-regulation of the ABA catabolism gene CYP707A2, which encodes abscisic acid 8'-hydroxylase. Correlation analysis showed ABA could explain the maximum proportion of differently expressed genes between NF and AF. We also showed that expression of KIRA1-LIKE1 (PpeKIL1), a peach ortholog of the Arabidopsis KIRA1 gene, was up-regulated in AF. PpeKIL1 promotes senescence or delays normal growth in tobacco and Arabidopsis, and its promoter activity increases with exogenous ABA treatment. Our study demonstrates a candidate mechanism where ABA induces expression of PpeKIL1, which further blocks normal fruit growth and triggers fruit abscission.

Profiling of Unfolded Protein Response Markers and Effect of IRE1α-specific Inhibitor in Pituitary Neuroendocrine Tumor.

CONTEXT: Inositol-requiring enzyme 1α (IRE1α) and PKR-like ER kinase (PERK), which are endoplasmic reticulum (ER) membrane proteins, regulate the unfolded protein response (UPR). These molecules have recently gained attention as a novel therapeutic target in secretory tumors. The roles of the UPR in pituitary neuroendocrine tumors (PitNETs) are unclear. OBJECTIVE: To clarify UPR profiling of PitNETs and to investigate the effect of pharmacological modulation of UPR by KIRA8, a newly developed IRE1α-specific inhibitor. METHODS: In 131 patients with PitNETs, we evaluated RNA expression of UPR markers in PitNETs and their clinical phenotypes. Using GH3 cells, we examined the effects of KIRA8 and its combination with octreotide on UPR profiling, cell growth, and apoptosis. RESULTS: Cytoprotective adaptive-UPR (A-UPR) markers were more increased in functioning PitNETs (FPitNETs, n = 112) than in nonfunctioning PitNETs (NFPitNETs, n = 19), while there was no difference in proapoptotic terminal-UPR (T-UPR) markers. Similarly, overt somatotroph tumors (STs, acromegaly, n = 11) increased A-UPR compared with silent STs (n = 10). In STs, serum IGF-1 levels were inversely correlated with Txnip mRNA expression, a representative T-UPR marker. KIRA8 inhibited cell growth and facilitated apoptosis in GH3 cells with increased expressions of T-UPR markers, which was enhanced by the combination with octreotide. Octreotide increased mRNA expression of Txnip and Chop, but decreased spliced Xbp1 under ER stress. Octreotide is suggested to inhibit activation of IRE1α but to reciprocally induce T-UPR under PERK. CONCLUSION: UPR markers in FPitNETs are implicated as dominant A-UPR but blunted T-UPR. KIRA8, enhanced with octreotide, unbalances the UPR, leading to antitumor effects. Targeting IRE1α may provide a novel strategy to treat PitNETs.

Isolated lateral extra-articular tenodesis in ACL-deficient knees: in vivo knee kinematics and clinical outcomes.

PURPOSE: To carry out an in vivo kinematic analysis of isolated modified Lemaire lateral extra-articular tenodesis (LET) to explore its ability to modify the stability of anterior cruciate ligament (ACL) deficient knees. The secondary aim was to look at the clinical outcomes of the isolated LET to analyze whether biomechanical changes have an influence on clinical improvement or not. METHODS: A total of 52 patients who underwent an isolated modified Lemaire LET were prospectively studied. Twenty-two were over 55-year-old patients with ACL rupture and subjective instability (group 1). They were followed up for 2 years postoperatively. Thirty were patients underwent a two-stage ACL revision (group 2). They were followed up for 4 months postoperatively (up to the second stage of the ACL revision). Preoperative, intraoperative, and postoperative kinematic analyses were carried out using the KiRA accelerometer and KT1000 arthrometer to look for residual anterolateral rotational instability and residual anteroposterior instability. Functional outcomes were measured with the single-leg vertical jump test (SLVJT) and the single-leg hop test (SLHT). Clinical outcomes were evaluated using the IKDC 2000, Lysholm, and Tegner scores. RESULTS: A significant reduction of both rotational and anteroposterior instability was detected. It was present both with the patient under anesthesia (p < 0.001 and p = 0.007 respectively) as well as with the patient awake (p = 0.008 and p = 0.018 respectively). Postoperative analysis of knee laxity did not show any significant variation from the first to the last follow-up. Both the SLVJT and SLHT improved significantly at the last follow-up (p < 0.001 and p = 0.011 respectively). The mean values of both the IKDC and Lysholm and Tegner scores showed an improvement (p = 0.008; p = 0.012; p < 0.001). CONCLUSION: The modified Lemaire LET improves the kinematics of ACL-deficient knees. The improvement in the kinematics leads to an improvement in subjective stability as well as in the function of the knee and in the clinical outcomes. At the 2-year follow-up, these improvements were maintained in a cohort of patients over 55 years. Following our findings, to reduce knee instability, an isolated LET in ACL-deficient knees may be used when ACL reconstruction in patients over 55 years is not indicated. LEVEL OF EVIDENCE: Level IV.

KIRA8 attenuates non-alcoholic steatohepatitis through inhibition of the IRE1α/XBP1 signalling pathway.

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH.

The IRE1α Inhibitor KIRA6 Blocks Leukotriene Biosynthesis in Human Phagocytes.

The ER stress and Unfolded Protein Response (UPR) component inositol-requiring enzyme 1α (IRE1α) has been linked to inflammation and lipid mediator production. Here we report that the potent IRE1α inhibitor, KIRA6, blocks leukotriene biosynthesis in human phagocytes activated with lipopolysaccharide (LPS) plus N-formyl-methionyl-leucyl-phenylalanine (fMLP) or thapsigargin (Tg). The inhibition affects both leukotriene B4 (LTB4) and cysteinyl leukotriene (cys-LTs) production at submicromolar concentration. Macrophages made deficient of IRE1α were still sensitive to KIRA6 thus demonstrating that the compound's effect on leukotriene production is IRE1α-independent. KIRA6 did not exhibit any direct inhibitory effect on key enzymes in the leukotriene pathway, as assessed by phospholipase A2 (PLA2), 5-lipoxygenase (5-LOX), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S) enzyme activity measurements in cell lysates. However, we find that KIRA6 dose-dependently blocks phosphorylation of p38 and ERK, mitogen-activated protein kinases (MAPKs) that have established roles in activating cytosolic PLA2α (cPLA2α) and 5-LOX. The reduction of p38 and ERK phosphorylation is associated with a decrease in cPLA2α phosphorylation and attenuated leukotriene production. Furthermore, KIRA6 inhibits p38 activity, and molecular modelling indicates that it can directly interact with the ATP-binding pocket of p38. This potent and unexpected, non-canonical effect of KIRA6 on p38 and ERK MAPKs and leukotriene biosynthesis may account for some of the immune-modulating properties of this widely used IRE1α inhibitor.

Lateral extraarticular tenodesis improves stability in non-anatomic ACL reconstructed knees: in vivo kinematic analysis.

PURPOSE: To carry out an in vivo kinematic analysis to determine whether adding a lateral extraarticular tenodesis (LET) for those patients with subjective instability and objective residual laxity after a transtibial (TT) anterior cruciate ligament reconstruction (ACLR) reduces anteroposterior and rotational laxity and to evaluate the 2-year follow-up clinical outcomes to analyze whether biomechanical changes determine clinical improvement or not. METHODS: A total of 19 patients with residual knee instability after TT ACLR who underwent a modified Lemaire LET were prospectively evaluated for at least 2-year follow-up. Preoperative, intraoperative, and 6 and 24-month postoperative kinematic analyses were carried out using the KiRA accelerometer and KT1000 arthrometer to look for residual anterolateral rotational instability and residual anteroposterior instability. Functional outcomes were measured with the single-leg vertical jump test and the single-leg hop test. Clinical outcomes were evaluated using the IKDC 2000, Lysholm, and Tegner scores. RESULTS: A significant reduction in anterolateral rotational instability was detected with the patient under anesthesia (from 3 ± 1.2 to 1.1 ± 1.1 m/s2; p < 0.05) as well as with the patient awake (from 2.1 ± 0.8 to 0.7 ± 1.4 m/s2; p < 0.05). A significant reduction in anteroposterior instability was only present under anesthesia (from 3.4 ± 1.9 to 2.1 ± 1.1 mm; p < 0.05), while no difference was present without anesthesia (from 2.3 ± 1.1 to 1.6 ± 1 mm; n.s.). Postoperative analysis of knee laxity did not show any significant variation from the first to the last follow-up. Both the single-leg vertical jump test and single-leg hop test improved significantly at the last follow-up (both p < 0.05). The mean values of both the IKDC and Tegner scores showed an improvement (p < 0.05 and p < 0.05, respectively), whereas that was not the case with the Lysholm score (n.s.). CONCLUSIONS: The modified Lemaire LET can improve the kinematics of a non-anatomic ACL reconstructed knee with residual subjective and objective instability. These kinematic changes were able to lead to an improvement in subjective stability as well as the function of the knee in a small cohort of recreationally active patients. At 2-year follow-up, the kinematic changes as well as the level of activity of the patients and the IKDC score show their improvement sustained. LEVEL OF EVIDENCE: Level IV.

IRE1α-Driven Inflammation Promotes Clearance of Citrobacter rodentium Infection.

Endoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.

The evaluation of Rolimeter, KLT, KiRA and KT-1000 arthrometer in healthy individuals shows acceptable intra-rater but poor inter-rater reliability in the measurement of anterior tibial knee translation.

PURPOSE: To assess measurement equivalence, inter- and intra-rater reliability, standard error of measurements (SEM) and false positive measurements (FPM) of four different knee arthrometers (KLT,Karl Storz; KiRA, I + ; KT-1000 MEDmetric Corp; Rolimeter, Aircast) in healthy patients. METHODS: Four different investigators (two advanced (AR) and two beginners (BR)) examined 12 participants with healthy knees at two time points with regards to anterior tibial translation (ATT) and side-to-side difference (SSD). Test equivalence was assessed using the TOST (two-one-sided t test) procedure with ± 1 mm equivalence boundaries. Intraclass correlation coefficients (ICCs) were calculated using two-way mixed effects models. Furthermore, false positive-(SSD > 3 mm) and SEMs were assessed. RESULTS: A total of 2304 Lachman Tests were performed. Between-rater SSDs were equivalent between AR and BR raters for the Rolimeter only. Inter-rater ICC values (SSD, ATT) were graded as "poor" to "moderate" for all devices. Equivalent test-retest results were observed for all raters using the Rolimeter, KLT and KT-1000, whereas measurement consistency with KiRA was given in the advanced examiners group only. Intra-rater ICC values (Range: SSD, ATT) were graded as "poor" to "moderate" for SSD values and "moderate" to "good" for ATT. SEMs were lowest for the Rolimeter and highest for KiRA. FPM were never obtained with the Rolimeter (0%), twice (2.1%) with the KT-1000, three times (3.1%) with the KLT and 33 times (34.4%) using KiRA. CONCLUSION: There is acceptable intra-rater but poor inter-rater reliability with all tested arthrometers. Measures of knee laxity are comparable between Rolimeter, KLT and KT-1000 but higher for KiRA. Clinically, the present study shows that repeated arthrometry measurements should always be performed by the same investigators.

IRE1α Is a Therapeutic Target for Cystic Fibrosis Airway Inflammation.

New anti-inflammatory treatments are needed for CF airway disease. Studies have implicated the endoplasmic reticulum stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway inflammation. The activation of IRE1α promotes activation of its cytoplasmic kinase and RNase, resulting in mRNA splicing of X-box binding protein-1 (XBP-1s), a transcription factor required for cytokine production. We tested whether IRE1α kinase and RNase inhibition decreases cytokine production induced by the exposure of primary cultures of homozygous F508del CF human bronchial epithelia (HBE) to supernatant of mucopurulent material (SMM) from CF airways. We evaluated whether IRE1α expression is increased in freshly isolated and native CF HBE, and couples with increased XBP-1s levels. A FRET assay confirmed binding of the IRE1α kinase and RNase inhibitor, KIRA6, to the IRE1α kinase. F508del HBE cultures were exposed to SMM with or without KIRA6, and we evaluated the mRNA levels of XBP-1s, IL-6, and IL-8, and the secretion of IL-6 and IL-8. IRE1α mRNA levels were up-regulated in freshly isolated CF vs. normal HBE and coupled to increased XBP-1s mRNA levels. SMM increased XBP-1s, IL-6, and IL-8 mRNA levels and up-regulated IL-6 and IL-8 secretion, and KIRA6 blunted these responses in a dose-dependent manner. Moreover, a triple combination of CFTR modulators currently used in the clinic had no effect on SMM-increased XBP-1s levels coupled with increased cytokine production in presence or absence of KIRA6. These findings indicate that IRE1α mediates cytokine production in CF airways. Small molecule IRE1α kinase inhibitors that allosterically reduce RNase-dependent XBP-1s may represent a new therapeutic strategy for CF airway inflammation.

Is the KiRA Device Useful in Quantifying the Pivot Shift in Anterior Cruciate Ligament-Deficient Knees?

BACKGROUND: Various technologies have been developed to quantify the pivot shift, as it is regarded as a key indicator of anterolateral rotatory laxity of the knee. PURPOSE: To determine the usefulness of a commercially available triaxial accelerometer (Kinematic Rapid Assessment [KiRA]) in numerically quantifying the pivot shift in patients under anesthesia with an anterior cruciate ligament (ACL)-deficient knee. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 3. METHODS: Both knees of 50 patients (26 male [mean age, 30.4 years], 24 female [mean age, 26.6 years]) under anesthesia were assessed immediately before unilateral ACL reconstruction by an orthopaedic fellow and 1 of 3 experienced knee surgeons. The pivot-shift grade and 2 KiRA outputs (range of acceleration and slope of acceleration change) were compared. RESULTS: The surgeon and fellow recorded the same pivot-shift grade for 45 of 50 patients (90%). Data from the 5 patients with no agreement and 1 patient with extreme outlying data were excluded from subsequent analysis. Using the KiRA range and slope data, the surgeon identified the injured knee in 74% and 76% of patients, respectively, while the fellow's rate of injured knee identification was 74% and 80%, respectively. A correlation could be found only between pivot-shift grade and surgeon-derived range data (ρ = 0.40; P < .01) but not slope data or any fellow-derived outputs. Using the surgeon-derived range data, there was a significant difference between a grade 3 pivot (>5 m/s2) and a grade 1 or 2 pivot (<5 m/s2) (P = .01). CONCLUSION: Although a correlation between KiRA output data and pivot-shift grade was found when the device was used by an experienced surgeon, there was no correlation when used by a well-trained but less experienced orthopaedic fellow. Furthermore, the KiRA output data identified the ACL-deficient knee correctly in only 74% of patients. Although a threshold acceleration range value could be identified, above which the value was associated with a grade 3 pivot shift, this was dependent on the examiner, and distinction between other grades could not be made.

Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib.

BACKGROUND: Inositol-requiring enzyme 1α (IRE1α), along with protein kinase R-like endoplasmic reticulum kinase (PERK), is a principal regulator of the unfolded protein response (UPR). Recently, the 'mono'-specific IRE1α inhibitor, kinase-inhibiting RNase attenuator 6 (KIRA6), demonstrated a promising effect against multiple myeloma (MM). Side-stepping the clinical translation, a detailed UPR phenotype in patients with MM and the mechanisms of how KIRA8 works in MM remains unclear. METHODS: We characterized UPR phenotypes in the bone marrow of patients with newly diagnosed MM. Then, in human MM cells we analyzed the possible anti-tumor mechanisms of KIRA8 and a Food and Drug Administration (FDA)-approved drug, nilotinib, which we recently identified as having a strong inhibitory effect against IRE1α activity. Finally, we performed an RNA-sequence analysis to detect key IRE1α-related molecules against MM. RESULTS: We illustrated the dominant induction of adaptive UPR markers under IRE1α over the PERK pathway in patients with MM. In human MM cells, KIRA8 decreased cell viability and induced apoptosis, along with the induction of C/EBP homologous protein (CHOP); its combination with bortezomib exhibited more anti-myeloma effects than KIRA8 alone. Nilotinib exerted a similar effect compared with KIRA8. RNA-sequencing identified Polo-like kinase 2 (PLK2) as a KIRA8-suppressed gene. Specifically, the IRE1α overexpression induced PLK2 expression, which was decreased by KIRA8. KIRA8 and PLK2 inhibition exerted anti-myeloma effects with apoptosis induction and the regulation of cell proliferation. Finally, PLK2 was pathologically confirmed to be highly expressed in patients with MM. CONCLUSION: Dominant activation of adaptive IRE1α was established in patients with MM. Both KIRA8 and nilotinib exhibited anti-myeloma effects, which were enhanced by bortezomib. Adaptive IRE1α signaling and PLK2 could be potential therapeutic targets and biomarkers in MM.

Glycosylation deletion of hemagglutinin head in the H5 subtype avian influenza virus enhances its virulence in mammals by inducing endoplasmic reticulum stress.

Hemagglutinin (HA) glycosylation of avian influenza virus (AIV) effects differently depending on the variation of glycosylation position and numbers. The natural mutation on the glycosylation sites of the AIV HA head occurs frequently. Our previous study shows that deletion of 158 or 169 glycosylation site on the HA head of the H5 subtype AIV strain rS-144-/158+/169+ increases the viral virulence in mammals; however, the mechanism remains unknown. In this study, several AIVs with different deletions at HA head glycosylation sites 144, 158 or 169 were tested for their biological characteristics to clarify the possible mechanism. We found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of inflammatory cytokines than rS-144-/158+/169+ did in the infected cells, but the TCID50 , EID50 and MDT of the viruses showed no difference. Moreover, we found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of endoplasmic reticulum (ER) stress in the cells. Inhibition of inositol-requiring enzyme 1α (IRE1α) phosphorylation reduced the inflammation induced by AIV infection. Furthermore, we found that rS-144-/158-/169+ virus activated the c-Jun N-terminal kinase (JNK), X-box binding protein 1 (XBP1), and nuclear factor-κB pathways by activating IRE1α phosphorylation under ER stress, whereas the rS-144-/158+/169- virus activated only the JNK pathway by altering IRE1α phosphorylation. In vivo analysis of Kira6 intervention further confirmed that ER stress played a key role in higher virulence for HA head 158 or 169 site de-glycosylation AIV. Our findings reveal that deletion of additional HA head glycosylation sites 158 or 169 enhanced the AIV virulence via activating of strong ER stress and inflammation.

Geospatial Distribution of Family Planning Services in Kira Municipality, Wakiso District, Uganda.

Introduction: Access to family planning (FP) services remains a challenge, particularly in informal urban settlements. The unmet need for FP in these settings is high, with a correspondingly high prevalence of unintended pregnancies that may lead to unsafe abortions. However, there is a paucity of quality data on the distribution of FP services in such settings in Uganda. This paper described the geospatial distribution of FP services in Kira Municipality, Wakiso District, Uganda. Methods: This was a cross-sectional study in which we determined the availability and distribution of FP services in Kira Municipality. Community mapping and analysis were conducted using ArcGIS (version 10.1) and ArcGIS Online. Stata version 13.1 was used for data analysis. Chi-square test was used to compare the contraceptive provision and availability among facilities from informal and formal settlements. Results: Of the 176 healthcare facilities surveyed, only 42% (n = 74) offered contraceptives in informal settlements. The majority of the facilities were privately owned small clinics (95%). At least 80% of the facilities provided three or more modern contraceptive methods, with no difference (p = 0.107) between facilities in informal and formal settlements. Only 30.7% (p = 0.001) of the facilities provided at least one long-acting contraceptive. Similarly, 20 and 12% (p = 0.001) of the facilities had implants and intrauterine devices (IUDs) on the day of the survey. Almost 25% of the facilities did not offer contraceptive services (counseling and commodities) to unmarried adolescents. Conclusions: Most facilities were small privately-owned clinics, offering at least three modern contraceptive methods. The unavailability of long-acting reversible methods in the informal settings may affect the quality of FP services due to limited choice. The inequity in service provision that disfavors the unmarried adolescent may increase unwanted/unintended pregnancies. We recommend that local governments and partners work toward filling the existing commodities gap and addressing the discrimination against unmarried adolescents in such settings.

Effect of Kinase Inhibiting RNase Attenuator (KIRA) Compounds on the Formation of Face-to-Face Dimers of Inositol-Requiring Enzyme 1: Insights from Computational Modeling.

Inositol-requiring enzyme 1α (IRE1α) is a transmembrane dual kinase/ribonuclease protein involved in propagation of the unfolded protein response (UPR). Inositol-requiring enzyme 1α is currently being explored as a potential drug target due to the growing evidence of its role in variety of disease conditions. Upon activation, IRE1 cleaves X-box binding protein 1 (XBP1) mRNA through its RNase domain. Small molecules targeting the kinase site are known to either increase or decrease RNase activity, but the allosteric relationship between the kinase and RNase domains of IRE1α is poorly understood. Subsets of IRE1 kinase inhibitors (known as "KIRA" compounds) bind to the ATP-binding site and allosterically impede the RNase activity. The KIRA compounds are able to regulate the RNase activity by stabilizing the monomeric form of IRE1α. In the present work, computational analysis, protein-protein and protein-ligand docking studies, and molecular dynamics simulations were applied to different IRE1 dimer systems to provide structural insights into the perturbation of IRE1 dimers by small molecules kinase inhibitors that regulate the RNase activity. By analyzing structural deviations, energetic components, and the number of hydrogen bonds in the interface region, we propose that the KIRA inhibitors act at an early stage of IRE1 activation by interfering with IRE1 face-to-face dimer formation thus disabling the activation of the RNase domain. This work sheds light on the mechanism of action of KIRA compounds and may assist in development of further compounds in, for example, cancer therapeutics. The work also provides information on the sequence of events and protein-protein interactions initiating the unfolded protein response.

The insulin-like growth factor-I receptor stimulating activity (IRSA) in health and disease.

Determination of true IGF-I bioactivity in serum and other biological fluids is still a substantial challenge. The IGF-IR Kinase Receptor Activation assay (IGF-IR KIRA assay) is a novel tool to asses IGF-IR stimulating activity (IRSA) and has opened a new era in studying the IGF system. In this paper we discuss many studies showing that measuring IRSA by the IGF-IR KIRA assay often provides fundamentally different information about the IGF system than the commonly used total IGF-I immunoassays. With the IGF-IR KIRA assay phosphorylation of tyrosine residues of the IGF-IR is used as read out to quantify IRSA in unknown (serum) samples. The IGF-IR KIRA assay gives information about net overall effects of circulating IGF-I, IGF-II, IGFBPs and IGFBP-proteases on IGF-IR activation and seems especially superior to immunoreactive total IGF-I in monitoring therapeutic interventions. Although the IRSA as measured by the IGF-IR KIRA assay probably more closely reflects true bioactive IGF-I than measurements of total IGF-I in serum, the IGF-IR KIRA assay in its current form does not give information about all the post-receptor intracellular events mediated by the IGF-IR. Interestingly, in several conditions in health and disease IRSA measured by the IGF-IR KIRA assay is considerably higher in interstitial fluid and ascites than in serum. This suggests that both the paracrine (local) and endocrine (circulating) IRSA should be measured to get a complete picture about the role of the IGF system in health and disease.

Revisiting the Role of Insulin-Like Growth Factor-I Receptor Stimulating Activity and the Apolipoprotein E in Alzheimer's Disease.

Background: Alterations in insulin-like growth factor I (IGF-I) signaling have been associated with dementia and Alzheimer's disease (AD). Studies on the association between IGF-I levels and dementia risk have been inconclusive. We reported earlier that higher levels of IGF-I receptor stimulating activity are associated with a higher prevalence and incidence of dementia. Objective: In the present study, we test the robustness of the association between IGF-I receptor stimulating activity and dementia by extending the follow-up period to 16 years and investigate possible effect modification by apolipoprotein E (ApoE). Methods: At baseline, circulating IGF-I receptor stimulating activity was determined by the IGF-I kinase receptor activation (KIRA) assay in 1,014 elderly from the Rotterdam Study. Dementia was assessed from baseline (1997-1999) to follow-up in January 2015. Associations of IGF-I receptor stimulating activity and incident dementia were assessed with Cox proportional hazards models. Results: During 10,752 person-years of follow-up, 174 people developed dementia. In the extended follow-up we no longer observed a dose-response relationship between IGF-I receptor stimulating activity and risk of dementia [adjusted odds ratio 1.11; 95% confidence interval (CI) 0.97-1.28]. Interestingly, we found evidence of an interaction between ApoE-ε4 and tertiles of IGF-I receptor stimulating activity. IGF-I receptor stimulating activity in the median and top tertiles was related to increased dementia incidence in hetero- and homozygotes of the ApoE-ε4 allele, but did not show any association with dementia risk in people without the ApoE-ε4 allele (adjusted odds ratio medium vs. low IGF-I receptor stimulating activity in ApoE-ε4 carriers: 1.45; 95% CI 1.00-2.12). These findings suggest a threshold effect in ApoE-ε4 carriers. In line with the hypothesis that downregulation of IGF-I signaling is associated with increased dementia risk, ApoE-ε4 homozygotes without prevalent dementia displayed lower levels of IGF-I receptor stimulating activity than heterozygotes and non-carriers. Conclusion: The findings shed new light on the association between IGF-I signaling and the neuropathology of dementia and ask for replication in other cohorts, using measures of IGF-I receptor stimulating activity rather than total serum levels as putative markers of dementia risk.

Triaxial accelerometer can quantify the Lachman test similarly to standard arthrometers.

PURPOSE: To assess the relationship between the KiRA triaxial accelerometer and the KT-1000 measurements in the intact, anterior cruciate ligament (ACL) deficient, and ACL reconstructed knee joint for the quantification of the Lachman test. Moreover, the intra- and inter-examiner repeatability of the KiRA device will be determined. It was hypothesized that the side-to-side difference of the anterior tibial translation as measured by the KiRA device would be equivalent to the one measured by the KT-1000 during the Lachman test. METHODS: Sixty patients were divided into three groups and have been prospectively included in the present study. Group_A composed of 20 patients with a diagnosis of an isolated ACL tear. Group_B composed of 20 patients who underwent ACL reconstruction with a Single-Bundle Lateral Plasty (SBLP) technique with at least 20 years of follow-up. Group_C was the control group and included 20 patients with no history of ACL lesion. Lachman test has been performed at manual-maximum load on both sides, the involved and the contralateral and analyzed with the two different devices. RESULTS: The KiRA device in terms of side-to-side difference resulted not statistically different from the measurement of the KT-1000 arthrometer for the three study groups (n.s): Group_A: (4 ± 2 mm KiRA, 4 ± 2 mm KT1000), Group_B: (4 ± 2 mm KiRA, 4 ± 2 mm KT-1000), Group_C: (4 ± 2 mm KiRA, 4 ± 2 mm KT-1000), an excellent intra- (ICC = 0.88-0.89) and inter-examiner (ICC = 0.79) agreement was found for KiRA measurements. CONCLUSION: The KiRA (I+, Italy) device offers a valid method to quantify the Lacham test. LEVEL OF EVIDENCE: II.

Combined reconstruction of the anterior cruciate ligament associated with anterolateral tenodesis effectively controls the acceleration of the tibia during the pivot shift.

PURPOSE: The pivot shift test is quantified subjectively during assessment of patients presenting with suspected Anterior Cruciate Ligament (ACL) tears and has a low interobserver reproducibility. The Kinematic Rapid Assessment (KiRA) is a triaxial accelerometer that makes it possible to non-invasively quantify tibial acceleration during the pivot shift test. Abolishing pivot shift is considered to be a key element in surgical reconstruction but is incomplete in 25-38% of patients. METHODS: Patients were included prospectively. Inclusion criteria were patients requiring ACL reconstruction associated with at least one of the following factors corresponding to the patient who have a high risk of rupture either by their sports activity, a failure case, or the notion of important rotational laxity: the patient practiced a competitive pivot-contact sport, revision ACL reconstruction (besides STG (semitendinosus-gracilis graft) repair), subjective explosive rotational laxity, Segond fracture, and TELOS value of >10 mm. Standardized pre- and postoperative pivot shift tests were immediately performed under anesthesia in both knees. RESULTS: Forty-three patients were included. Mean preoperative variations in tibial acceleration in the healthy and injured knees were 1.2 ± 0.1 and 2.7 ± 0.3 m/s2, respectively, p < 0.01. A statistically significant decrease in immediate postoperative mean variations in acceleration in the injured knee occurred: 1.5 ± 0.3 m/s2, p < 0.01. There was no longer any statistical difference between postoperative contralateral healthy knees and operated knees (n.s). CONCLUSIONS: Combined ACL reconstruction associated with anterolateral tenodesis suppress acute pathologic tibial acceleration in the pivot shift. LEVEL OF EVIDENCE: III.

PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma.

Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared serum and ascites from 22 women with ovarian carcinoma. We found that ascites contained 46-fold higher PAPP-A levels as compared to serum (P < 0.001). The majority (80%) of PAPP-A was enzymatically active. This is supported by the finding that ascites contained more cleaved than intact IGFBP-4 (P < 0.03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P < 0.05); in 8 of 22 patients by more than two-fold. In contrast, ascites contained similar levels of immunoreactive IGF-I, and lower levels of IGF-II (P < 0.001). Immunohistochemistry demonstrated the presence of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P < 0.05). In conclusion, human ovarian tumors express PAPP-A, IGFBP-4 and IGFs and these proteins are also present in ascites. We suggest that both soluble PAPP-A in ascites and tissue-associated PAPP-A serve to increase IGF bioactivity and, thereby, to stimulate IGF-IR-mediated tumor growth.

Instability of IGF-IGFBP complex as a cause of the different performance of serum and EDTA-plasma after storage: EDTA-plasma is preferable for evaluating bioactive IGF especially in the mouse.

OBJECTIVE: The insulin-like growth factor (IGF) signaling pathway is recognized as a potential target for treating several cancers, and strategies targeting the IGF type 1 receptor (IGF-1R) have been evaluated in many clinical trials. These suggested that the pretreatment level of circulating free IGF gives an estimate of IGF bioactivity and might be a predictive biomarker of the response to anti-IGF-1R antibodies. However, there is no defined protocol for measuring free and bioactive IGF concentrations, partly because the measurement procedures, including sample collection and handling, have not been standardized. We investigated the effects of sample collection methods and storage conditions on bioactive IGF measurement using a modified kinase receptor activation (KIRA) assay in human and mouse samples. DESIGN: Blood samples were obtained from healthy men and women, and from healthy male and female wild-type BALB/c mice. Serum and ethylenediaminetetraacetic acid (EDTA)-plasma samples were collected and used immediately or stored in small quantities at 4 °C or -80 °C for 3, 7, or 14 days. A bioassay directed against the phosphorylated IGF-1R using western blot analysis was developed as a modification of the KIRA assay, in which the level of phosphorylation of IGF-1R represented the IGF bioactivity in blood samples. RESULTS: The levels of bioactive IGFs in mouse serum stored at 4 °C increased markedly in a time-dependent manner; the increase was slightly reduced in samples stored at -80 °C. Analysis of mouse EDTA-plasma stored at 4 °C showed a similar pattern, but the time-dependent increase was less than in the serum samples. By contrast, the levels of bioactive IGFs in EDTA-plasma stored at -80 °C were stable over 14 days. The levels of human bioactive IGFs in both serum and EDTA-plasma stored at 4 °C increased slightly with time, but the increases were much smaller than in mouse samples. The levels of human bioactive IGF in both serum and EDTA-plasma stored at -80 °C were stable over 14 days. CONCLUSIONS: The use of EDTA-plasma avoids the problems with long-term storage. Therefore, EDTA-plasma should be used when measuring circulating IGF bioactivity, especially in mouse samples. All samples should be stored at -80 °C when long-term storage is unavoidable. Because of the large difference in the stability of the IGF-IGF-binding protein complex between the human and mouse in vitro, all samples should be handled carefully to ensure the accurate evaluation of IGF bioactivity, especially in mouse samples.