A potential code of luteinizing hormone-releasing hormone-induced calcium ion responses in the regulation of luteinizing hormone secretion among individual gonadotropes

Luteinizing hormone-releasing hormone (LHRH) induces two Ca2+ responses in single gonadotropes: a Ca2+ spike/plateau or oscillation. Similar receptor-mediated Ca2+ signals have been reported in many cell types but their functional significance is obscure. Accordingly, we have determined the concentration-response properties of LHRH-induced luteinizing hormone (LH) release at the single cell level. We demonstrate a critical single cell LHRH threshold for LH release. Each gonadotrope had a particular LHRH threshold value and a range of different single cell thresholds was distributed in the gonadotrope population. The physiological significance of the threshold was demonstrated by a striking reduction (delta ED50 = 153 nM) of the LHRH threshold immediately before the preovulatory surge of LH release. The metestrous phenotype of secretion resembled a quantal process in contrast with the graded process of the proestrous phenotype. That is, the quantity of hormone secreted per metestrous gonadotrope was independent of LHRH concentration and more all-or-none than graded. The LHRH threshold and the quantal secretion process of metestrous gonadotropes was further studied by measuring cytosolic Ca2+ using fura-2 and digital imaging microscopy. We provide evidence suggesting that the Ca2+ spike/plateau and oscillation are the respective responses to subthreshold and suprathreshold concentrations of LHRH. It is proposed therefore that the Ca2+ oscillation and spike/plateau response form a binary intracellular signaling code that functions as an on-off switch. It is further proposed that this potential code unraveled here for the regulation of hormone secretion may also regulate other gonadotrope functions. Thus, while the Ca2+ spike/plateau response is strongly associated with LH release, it may be associated with reduced levels of LH-beta mRNA, and reduced numbers of LHRH receptors. Conversely, while the Ca2+ oscillation appears to be unrelated to LH release, it may be associated with increased levels of LH-beta mRNA, and increased numbers of LHRH receptors. This model may explain in molecular terms the long-standing observation that an invariant, albeit pulsatile, pattern of LHRH release is sufficient to support the preovulatory surge of LH release.     

Inhibition of Janus Kinase 1 synergizes docetaxel sensitivity in prostate cancer cells

Prostate cancer (PCa) is the second most common malignancy and is the fifth leading cause of cancer mortality among men globally. Docetaxel-based therapy remains the first-line treatment for metastatic castration-resistant prostate cancer. However, dose-limiting toxicity including neutropenia, myelosuppression and neurotoxicity is the major reason for docetaxel dose reductions and fewer cycles administered, despite a recent study showing a clear survival benefit with increased total number of docetaxel cycles in PCa patients. Although previous studies have attempted to improve the efficacy and reduce docetaxel toxicity through drug combination, no drug has yet demonstrated improved overall survival in clinical trial, highlighting the challenges of improving the activity of docetaxel monotherapy in PCa. Herein, we identified 15 lethality hits for which inhibition could enhance docetaxel sensitivity in PCa cells via a high-throughput kinome-wide loss-of-function screen. Further drug-gene interactions analyses identified Janus kinase 1 (JAK1) as a viable druggable target with existing experimental inhibitors and FDA-approved drugs. We demonstrated that depletion of endogenous JAK1 enhanced docetaxel-induced apoptosis in PCa cells. Furthermore, inhibition of JAK1/2 by baricitinib and ruxolitinib synergizes docetaxel sensitivity in both androgen receptor (AR)–negative DU145 and PC3 cells, but not in the AR-positive LNCaP cells. In contrast, no synergistic effects were observed in cells treated with JAK2-specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.     

Flux boundary measurements for the study of tree stability

The shear strength of soil is an important parameter that affects tree stability and can vary depending on the magnitude of the soil’s negative pore-water pressure (matric suction). The surface flux boundary condition affects the matric suction of soil, and therefore is important for tree stability. Field measurements were performed around a roadside tree for 2 years. The instrumentation results show that the matric suction in the soil fluctuated between 0 and 35 kPa. Matric suction changes in the soil could lead to a decrease in the tree resistive moment of up to 80 %.     

Using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling in order to predict clinical outcomes of patients with heart failure

Background

Current risk prediction models in heart failure (HF) including clinical characteristics and biomarkers only have moderate predictive value. The aim of this study was to use matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling to determine if a combination of peptides identified with MALDI-MS will better predict clinical outcomes of patients with HF.

Methods

A cohort of 100 patients with HF were recruited in the biomarker discovery phase (50 patients who died or had a HF hospital admission vs. 50 patients who did not have an event). The peptide extraction from plasma samples was performed using reversed phase C18. Then samples were analysed using MALDI-MS. A multiple peptide biomarker model was discovered that was able to predict clinical outcomes for patients with HF. Finally, this model was validated in an independent cohort with 100 patients with HF.

Results

After normalisation and alignment of all the processed spectra, a total of 11,389 peptides (m/z) were detected using MALDI-MS. A multiple biomarker model was developed from 14 plasma peptides that was able to predict clinical outcomes in HF patients with an area under the receiver operating characteristic curve (AUC) of 1.000 (p?=?0.0005). This model was validated in an independent cohort with 100 HF patients that yielded an AUC of 0.817 (p?=?0.0005) in the biomarker validation phase. Addition of this model to the BIOSTAT risk prediction model increased the predictive probability for clinical outcomes of HF from an AUC value of 0.643 to an AUC of 0.823 (p?=?0.0021). Moreover, using the prediction model of fourteen peptides and the composite model of the multiple biomarker of fourteen peptides with the BIOSTAT risk prediction model achieved a better predictive probability of time-to-event in prediction of clinical events in patients with HF (p?=?0.0005).

Conclusions

The results obtained in this study suggest that a cluster of plasma peptides using MALDI-MS can reliably predict clinical outcomes in HF that may help enable precision medicine in HF.

     

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein

With the development of de novo binders for protein targets from non‐related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single‐chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking “disembodied” amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein‐antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen‐based detection agents for typhoid diagnostics.