Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

Correction to "Carbodiimide Ring-Opening Metathesis Polymerization".

[This corrects the article DOI: 10.1021/acscentsci.3c00032.].

Carbodiimide Ring-Opening Metathesis Polymerization.

Controlled incorporation of nitrogen into macromolecular skeletons is a long-standing challenge whose resolution would enable the preparation of soft materials with the scalability of man-made plastics and functionality of Nature's proteins. Nylons and polyurethanes notwithstanding, nitrogen-rich polymer backbones remain scarce, and their synthesis typically lacks precision. Here we report a strategy that begins to address this limitation founded on a mechanistic discovery: ring-opening metathesis polymerization (ROMP) of carbodiimides followed by carbodiimide derivatization. An iridium guanidinate complex was found to initiate and catalyze ROMP of N-aryl and N-alkyl cyclic carbodiimides. Nucleophilic addition to the resulting polycarbodiimides enabled the preparation of polyureas, polythioureas, and polyguanidinates with varied architectures. This work advances the foundations of metathesis chemistry and opens the door to systematic investigations of structure-folding-property relationships in nitrogen-rich macromolecules.

Percutaneous tracheostomy: Comparison of three different methods with respect to tracheal cartilage injury in cadavers-Randomized controlled study.

Background: Performing tracheostomy improves patient comfort and success rate of weaning from prolonged invasive mechanical ventilation. Data suggest that patients have more benefit of percutaneous technique than the surgical procedure, however, there is no consensus on the percutaneous method of choice regarding severe complications such as late tracheal stenosis. Aim of this study was comparing incidences of cartilage injury caused by different percutaneous dilatation techniques (PDT), including Single Dilator, Griggs' and modified (bidirectional) Griggs' method. Materials and methods: Randomized observational study was conducted on 150 cadavers underwent post-mortem percutaneous tracheostomy. Data of cadavers including age, gender and time elapsed from death until the intervention (more or less than 72 h) were collected and recorded. Primary and secondary outcomes were: rate of cartilage injury and cannula malposition respectively. Results: Statistical analysis revealed that method of intervention was significantly associated with occurrence of cartilage injury, as comparing either standard Griggs' with Single Dilator (p = 0.002; OR: 4.903; 95% CI: 1.834-13.105) or modified Griggs' with Single Dilator (p < 0.001; OR: 6.559; 95% CI: 2.472-17.404), however, no statistical difference was observed between standard and modified Griggs' techniques (p = 0.583; OR: 0.748; 95% CI: 0.347-1.610). We found no statistical difference in the occurrence of cartilage injury between the early- and late post-mortem group (p = 0.630). Neither gender (p = 0.913), nor age (p = 0.529) influenced the rate of cartilage fracture. There was no statistical difference between the applied PDT techniques regarding the cannula misplacement/malposition. Conclusion: In this cadaver study both standard and modified Griggs' forceps dilatational methods were safer than Single dilator in respect of cartilage injury.

T cell immunity ameliorates COVID-19 disease severity and provides post-exposure prophylaxis after peptide-vaccination, in Syrian hamsters.

Background: The emergence of novel SARS-CoV-2 variants that resist neutralizing antibodies drew the attention to cellular immunity and calls for the development of alternative vaccination strategies to combat the pandemic. Here, we have assessed the kinetics of T cell responses and protective efficacy against severe COVID-19 in pre- and post-exposure settings, elicited by PolyPEPI-SCoV-2, a peptide based T cell vaccine. Methods: 75 Syrian hamsters were immunized subcutaneously with PolyPEPI-SCoV-2 on D0 and D14. On D42, hamsters were intranasally challenged with 102 TCID50 of the virus. To analyze immunogenicity by IFN-γ ELISPOT and antibody secretion, lymphoid tissues were collected both before (D0, D14, D28, D42) and after challenge (D44, D46, D49). To measure vaccine efficacy, lung tissue, throat swabs and nasal turbinate samples were assessed for viral load and histopathological changes. Further, body weight was monitored on D0, D28, D42 and every day after challenge. Results: The vaccine induced robust activation of T cells against all SARS-CoV-2 structural proteins that were rapidly boosted after virus challenge compared to control animals (~4-fold, p<0.05). A single dose of PolyPEPI-SCoV-2 administered one day after challenge also resulted in elevated T cell response (p<0.01). The vaccination did not induce virus-specific antibodies and viral load reduction. Still, peptide vaccination significantly reduced body weight loss (p<0.001), relative lung weight (p<0.05) and lung lesions (p<0.05), in both settings. Conclusion: Our study provides first proof of concept data on the contribution of T cell immunity on disease course and provide rationale for the use of T cell-based peptide vaccines against both novel SARS-CoV-2 variants and supports post-exposure prophylaxis as alternative vaccination strategy against COVID-19.

Reliability and clinical correlations of semi-quantitative lung ultrasound on BLUE points in COVID-19 mechanically ventilated patients: The 'BLUE-LUSS'-A feasibility clinical study.

INTRODUCTION: Bedside lung ultrasound has gained a key role in each segment of the treatment chain during the COVID-19 pandemic. During the diagnostic assessment of the critically ill patients in ICUs, it is highly important to maximize the amount and quality of gathered information while minimizing unnecessary interventions (e.g. moving/rotating the patient). Another major factor is to reduce the risk of infection and the workload of the staff. OBJECTIVES: To serve these significant issues we constructed a feasibility study, in which we used a single-operator technique without moving the patient, only assessing the easily achievable lung regions at conventional BLUE points. We hypothesized that calculating this 'BLUE lung ultrasound score' (BLUE-LUSS) is a reasonable clinical tool. Furthermore, we used both longitudinal and transverse scans to measure their reliability and assessed the interobserver variability as well. METHODS: University Intensive Care Unit based, single-center, prospective, observational study was performed on 24 consecutive SARS-CoV2 RT-PCR positive, mechanically ventilated critically ill patients. Altogether 400 loops were recorded, rated and assessed off-line by 4 independent intensive care specialists (each 7+ years of LUS experience). RESULTS: Intraclass correlation values indicated good reliability for transversal and longitudinal qLUSS scores, while we detected excellent interrater agreement of both cLUSS calculation methods. All of our LUS scores correlated inversely and significantly to the P/F values. Best correlation was achieved in the case of longitudinal qLUSS (r = -0.55, p = 0.0119). CONCLUSION: Summarized score of BLUE-LUSS can be an important, easy-to-perform adjunct tool for assessing and quantifying lung pathology in critically ill ventilated patients at bedside, especially for the P/F ratio. The best agreement for the P/F ratio can be achieved with the longitudinal scans. Regarding these findings, assessing BLUE-points can be extended with the BLUE-LUSS for daily routine using both transverse and longitudinal views.

Safety and Activity of PolyPEPI1018 Combined with Maintenance Therapy in Metastatic Colorectal Cancer: an Open-Label, Multicenter, Phase Ib Study.

PURPOSE: Although chemotherapy is standard of care for metastatic colorectal cancer (mCRC), immunotherapy has no role in microsatellite stable (MSS) mCRC, a "cold" tumor. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine derived from 7 tumor-associated antigens (TAA) frequently expressed in mCRC. This study assessed PolyPEPI1018 combined with first-line maintenance therapy in patients with MSS mCRC. PATIENTS AND METHODS: Eleven patients with MSS mCRC received PolyPEPI1018 and Montanide ISA51VG adjuvant subcutaneously, combined with fluoropyrimidine/biologic following first-line induction with chemotherapy and a biologic (NCT03391232). In Part A of the study, 5 patients received a single dose; in Part B, 6 patients received up to three doses of PolyPEPI1018 every 12 weeks. The primary objective was safety; secondary objectives were preliminary efficacy, immunogenicity at peripheral and tumor level, and immune correlates. RESULTS: PolyPEPI1018 vaccination was safe and well tolerated. No vaccine-related serious adverse event occurred. Eighty percent of patients had CD8+ T-cell responses against ≥3 TAAs. Increased density of tumor-infiltrating lymphocytes were detected post-treatment for 3 of 4 patients' liver biopsies, combined with increased expression of immune-related gene signatures. Three patients had objective response according to RECISTv1.1, and 2 patients qualified for curative surgery. Longer median progression-free survival for patients receiving multiple doses compared with a single dose (12.5 vs. 4.6 months; P = 0.017) suggested a dose-efficacy correlation. The host HLA genotype predicted multi-antigen-specific T-cell responses (P = 0.01) indicative of clinical outcome. CONCLUSIONS: PolyPEPI1018 added to maintenance chemotherapy for patients with unresectable, MSS mCRC was safe and associated with specific immune responses and antitumor activity warranting further confirmation in a randomized, controlled setting.

Valorizing the Unexplored Filtration Waste of Brewing Industry for Green Silver Nanocomposite Synthesis.

The brewing industry generates a substantial amount of by-products rich in polyphenols, carbohydrates, sugars, sulfates, nitrogen compounds, organic carbon, and several elements, including chlorine, magnesium, and phosphorus. Although limited quantities of these by-products are used in fertilizers and composts, a large amount is discarded as waste. Therefore, it is crucial to identify different ways of valorizing the by-products. Research regarding the valorization of the brewery by-products is still in its nascent stage; therefore, it still has high potential. Herein, we report the valorization of the brewery by-product from the filtration stage of the brewing process (BW9) to synthesize silver nanocomposites as this waste has remained largely unexplored. The BW9 nanocomposites have been compared to those obtained from the brewery product B. The chemical composition analysis of BW9 and B revealed several organic moieties capable of reducing metal salts and capping the formed nanoparticles. Therefore, the brewery waste from stage 9 was valorized as a precursor and added to silver-based precursor at various temperatures (25, 50, and 80 °C) and for various time periods (10, 30, and 120 min) to synthesize silver nanocomposites. The nanocomposites obtained using BW9 were compared to those obtained using the main product of the brewing industry, beer (B). Synthesized nanocomposites composed of AgCl as a major phase and silver metal (Agmet) was incorporated in minor quantities. In addition, Ag3PO4 was also found in B nanocomposites in minor quantities (up to 34 wt.%). The surface morphology depicted globular nanoparticles with layered structures. Small ball-like aggregates on the layer representative of Ag3PO4 were observed in B nanocomposites. The surface of nanocomposites was capped with organic content and functional groups present in the brewery products. The nanocomposites demonstrated high antibacterial activity against Escherichia coli (E. coli), with BW9 nanocomposites exhibiting a higher activity than B nanocomposites.

In Silico Model Estimates the Clinical Trial Outcome of Cancer Vaccines.

Over 30 years after the first cancer vaccine clinical trial (CT), scientists still search the missing link between immunogenicity and clinical responses. A predictor able to estimate the outcome of cancer vaccine CTs would greatly benefit vaccine development. Published results of 94 CTs with 64 therapeutic vaccines were collected. We found that preselection of CT subjects based on a single matching HLA allele does not increase immune response rates (IRR) compared with non-preselected CTs (median 60% vs. 57%, p = 0.4490). A representative in silico model population (MP) comprising HLA-genotyped subjects was used to retrospectively calculate in silico IRRs of CTs based on the percentage of MP-subjects having epitope(s) predicted to bind ≥ 1-4 autologous HLA allele(s). We found that in vitro measured IRRs correlated with the frequency of predicted multiple autologous allele-binding epitopes (AUC 0.63-0.79). Subgroup analysis of multi-antigen targeting vaccine CTs revealed correlation between clinical response rates (CRRs) and predicted multi-epitope IRRs when HLA threshold was ≥ 3 (r = 0.7463, p = 0.0004) but not for single HLA allele-binding epitopes (r = 0.2865, p = 0.2491). Our results suggest that CRR depends on the induction of broad T-cell responses and both IRR and CRR can be predicted when epitopes binding to multiple autologous HLAs are considered.

Effect of Microwave Treatment in a High Pressure Microwave Reactor on Graphene Oxide Reduction Process-TEM, XRD, Raman, IR and Surface Electron Spectroscopic Studies.

Reduced graphene oxide (rGO) was prepared by chemical reduction of graphene oxide (GO) (with a modified Hummers method) in aqueous solutions of hydrazine (N2H4), formaldehyde (CH2O), formic acid (HCO2H) accompanied by a microwave treatment at 250 °C (MWT) by a high pressure microwave reactor (HPMWR) at 55 bar. The substrates and received products were investigated by TEM, XRD, Raman and IR spectroscopies, XPS, XAES and REELS. MWT assisted reduction using different agents resulted in rGOs of a large number of vacancy defects, smaller than at GO surface C sp3 defects, oxygen groups and interstitial water, interlayer distance and diameter of stacking nanostructures (flakes). The average number of flake layers obtained from XRD and REELS was consistent, being the smallest for CH2O and then increasing for HCO2H and N2H4. The number of layers in rGOs increases with decreasing content of vacancy, C sp3 defects, oxygen groups, water and flake diameter. MWT conditions facilitate formation of vacancies and additional hydroxyl, carbonyl and carboxyl groups at these vacancies, provide no remarkable modification of flake diameter, what results in more competitive penetration of reducing agent between the interstitial sites than via vacancies. MWT reduction of GO using a weak reducing agent (CH2O) provided rGO of 8 layers thickness.

Valorization of Brewery Wastes for the Synthesis of Silver Nanocomposites Containing Orthophosphate.

Brewery wastes from stage 5 (Wort precipitate: BW5) and stage 7 (Brewer's spent yeast: BW7) were valorized for the synthesis of silver phosphate nanocomposites. Nanoparticles were synthesized by converting silver salt in the presence of brewery wastes at different temperatures (25, 50, and 80 °C) and times (10, 30, and 120 min). Unexpectedly, BW7 yielded Ag3PO4 nanoparticles with minor contents of AgCl and Ag metal (Agmet). Contrastingly, BW5 produced AgCl nanoparticles with minor amounts of Ag3PO4 and Agmet. Nanocomposites with different component ratios were obtained by simply varying the synthesis temperature and time. The morphology of the nanocomposites contained ball-like structures representative of Ag3PO4 and stacked layers and fused particles representing AgCl and Agmet. The capping on the nanoparticles contained organic groups from the brewery by-products, and the surface overlayer had a rich chemical composition. The organic overlayers on BW7 nanocomposites were thinner than those on BW5 nanocomposites. Notably, the nanocomposites exhibited high antibacterial activity against Escherichia coli ATCC 25922. The antibacterial activity was higher for BW7 nanocomposites due to a larger silver phosphate content in the composition and a thin organic overlayer. The growth of Agmet in the structure adversely affected the antimicrobial property of the nanocomposites.

A Peptide Vaccine Candidate Tailored to Individuals' Genetics Mimics the Multi-Targeted T Cell Immunity of COVID-19 Convalescent Subjects.

Long-term immunity to coronaviruses likely stems from T cell activity. We present here a novel approach for the selection of immunoprevalent SARS-CoV-2-derived T cell epitopes using an in silico cohort of HLA-genotyped individuals with different ethnicities. Nine 30-mer peptides derived from the four major structural proteins of SARS-CoV-2 were selected and included in a peptide vaccine candidate to recapitulate the broad virus-specific T cell responses observed in natural infection. PolyPEPI-SCoV-2-specific, polyfunctional CD8+ and CD4+ T cells were detected in each of the 17 asymptomatic/mild COVID-19 convalescents' blood against on average seven different vaccine peptides. Furthermore, convalescents' complete HLA-genotype predicted their T cell responses to SARS-CoV-2-derived peptides with 84% accuracy. Computational extrapolation of this relationship to a cohort of 16,000 HLA-genotyped individuals with 16 different ethnicities suggest that PolyPEPI-SCoV-2 vaccination will likely elicit multi-antigenic T cell responses in 98% of individuals, independent of ethnicity. PolyPEPI-SCoV-2 administered with Montanide ISA 51 VG generated robust, Th1-biased CD8+, and CD4+ T cell responses against all represented proteins, as well as binding antibodies upon subcutaneous injection into BALB/c and hCD34+ transgenic mice modeling human immune system. These results have implications for the development of global, highly immunogenic, T cell-focused vaccines against various pathogens and diseases.

Soil fertility management among smallholder farmers in Mount Kenya East region.

Declining soil fertility continues to hinder agricultural production especially among resource-constrained smallholder farmers in sub-Saharan Africa, prompting for evaluation of the strategies used by these farming communities. In this study, we assess soil fertility management among smallholder farmers in Mount Kenya East region. The aim is to examine underlying factors conditioning the uptake of integrated soil fertility management (ISFM) practices in this region; determine the adoption relationship between the practices; and to cluster these techniques. Data for this study was collected between January-March 2019 through a household survey based on a farm household questionnaire and complemented with semi-structured interview with farmers and extension officers. Statistical analyses were generated using SPSS. We use hierarchical clustering analysis to visualize ISFM combination patterns, and correlation matrix in factor analysis to determine the inter-relationship between different ISFM practices. Fisher's exact test and Welch's t-test were used to examine the association between explanatory variables and adoption of ISFM practices. Results show that the decision to invest in fertility practices was correlated with a number of farmers' socio-economic, farm-related factors and institutional characteristics. Fertilizer application correlated significantly with manure use, agroforestry and minimum tillage. ISFM techniques were separated into 3 sets following Ward's hierarchical clustering, namely, manure, fertilizer use and agroforestry (cluster 1 or C1), slash-no-burn, residue burn and fallowing (C2); and residue application and minimum tillage (C3). The study recommends creation of an enabling environment including innovative financing opportunities to facilitate farmers' investment capacities in ISFM and cushion them from potential income loss resulting from implementation of some technologies.

Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells.

There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that reflect phosphoinositide changes in a population of transiently transfected cells. This method is highly sensitive, specific, and capable of semiquantitative characterization of plasma membrane phosphoinositide changes with high temporal resolution.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours.

AIM OF THE STUDY: The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells. MATERIALS AND METHODS: As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized. RESULTS: We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology. CONCLUSIONS: We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

[Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis]. / Kultureller Nachweis von Leptospiren in Glaskörperflüssigkeit und Antikörpernachweis gegen Leptospiren in Glaskörperflüssigkeit und Serum von 225 Pferden mit equiner rezidivierender Uveitis (ERU).

In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU.

[Genetic information from tumor-infiltrating B lymphocytes as a driver tool ("GPS") for anti-tumor T cell CARs]. / A tumorban felhalmozódó B-limfociták genetikai információja, mint az immun T-sejt vezette tumorellenes "autó" (CAR, kiméra antigénreceptor) célba irányító "GPS"-e.

The rapidly growing field of gene therapy techniques to modify T cells with chimeric antigen receptors (CARs) for cancer care solutions, reached considerable achievements. However, there is an urgent need of reliable, well tolerable tumor-associated antigen specific antibodies. Tumor-infiltrating B (TIL-B) cell originated single chain Fv (scFv) gene regions could be selected with tumor specificity. DNA sequences of these antibody variable regions were subjects to get engineered into new CAR constructs. Our novel strategy harnesses tumor-infiltrating B cells' unique capacity to reveal highly tumor-associated disialylated glycosphingolipids (GD3 gangliosides). We used these human antibody fragments for generating GD3 ganglioside specific CAR gene constructs for potential usage in solid tumors.

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors.

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications.

The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.