A genetically modified Plasmodium berghei parasite as a surrogate for whole-sporozoite vaccination against P. vivax malaria.

Two malaria parasite species, Plasmodium falciparum (Pf) and P. vivax (Pv) are responsible for most of the disease burden caused by malaria. Vaccine development against this disease has focused mainly on Pf. Whole-sporozoite (WSp) vaccination, targeting pre-erythrocytic (PE) parasite stages, is a promising strategy for immunization against malaria and several PfWSp-based vaccine candidates are currently undergoing clinical evaluation. In contrast, no WSp candidates have been developed for Pv, mainly due to constraints in the production of Pv sporozoites in the laboratory. Recently, we developed a novel approach for WSp vaccination against Pf based on the use of transgenic rodent P. berghei (Pb) sporozoites expressing immunogens of this human-infective parasite. We showed that this platform can be used to deliver PE Pf antigens, eliciting both targeted humoral responses and cross-species cellular immune responses against Pf. Here we explored this WSp platform for the delivery of Pv antigens. As the Pv circumsporozoite protein (CSP) is a leading vaccine candidate antigen, we generated a transgenic Pb parasite, PbviVac, that, in addition to its endogenous PbCSP, expresses PvCSP under the control of a strictly PE promoter. Immunofluorescence microscopy analyses confirmed that both the PbCSP and the PvCSP antigens are expressed in PbviVac sporozoites and liver stages and that PbviVac sporozoite infectivity of hepatic cells is similar to that of its wild-type Pb counterpart. Immunization of mice with PbviVac sporozoites elicits the production of anti-PvCSP antibodies that efficiently recognize and bind to Pv sporozoites. Our results warrant further development and evaluation of PbviVac as a surrogate for WSp vaccination against Pv malaria.

Virchow node of an unusual aetiology - the experience of a clinical case.

BACKGROUND: The cause of adult adenomegalies may be defiant. On the other hand, ectopic thyroid is a rare condition that happens in every 1:100000 to 300,000 of healthy individuals. Here, we present a case report that joins these two clinical rare and defiant challenges. CLINICAL CASE: Forty-seven-year-old woman, with known thyroid nodules for several years. She had no other relevant personal or familiar history. At our appointment she had no complaints. At the physical examination she had a palpable right thyroid nodule (previously known). The routine blood analysis showed normal thyroid function. The routine cervical ultrasonography showed no dimensional progression of the known thyroid nodules and identified a 31x18mm nodule at the left supraclavicular fossa. The patient underwent a cervical, thoracic, and abdominal computed tomography that exhibited no relevant findings, such as abdominal malignancies. The cytology of the nodule showed characteristics that were "compatible with a benign follicular nodule in ectopic thyroid tissue". CONCLUSION: This is a rare case in which we incidentally found a follicular nodule in ectopic thyroid tissue in the left supraclavicular fossa. Given the rarity of the situation, clinical sense is the mainstay of treatment and follow-up.

Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.

Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitors.

The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.

Systolic function recovery in Heart Failure: Frequency, prognostic impact and predictors.

BACKGROUND: Systolic function recovery in patients with Heart failure (HF) with reduced ejection fraction (EF) is well recognized but not completely understood. We aimed to characterize HF patients with systolic function recovery, its prognostic impact and predictors. METHODS: We analysed patients followed in a HF clinic (2006-2015) with 2 echocardiograms performed. Partial recovery: EF recovery without attaining EF ≥ 50%; total recovery: patients reached EF ≥ 50%. Median follow-up from first echocardiogram: 69 months. Multivariate logistic regression models to determine recovery predictors. RESULTS: We analysed 304 patients with at least mild left ventricular dysfunction. During a median 34 months between echocardiogram re-evaluation 150 (49.3%) patients showed no EF recovery; 55 (18.1%) had partial recovery and 99 (32.6%) totally recovered. Mean patients age: 66; 71.1% men, high comorbidity burden; ischemic HF: 35.5%. Non-recovered patients were mostly men (80.7% vs 61.8% in partially; 61.6% in fully-recovered) with ischemic HF (46.0% vs 32.5% in partially; 21.2% in fully-recovered). Comorbidity burden, NYHA class and therapy were similar. During follow-up, 156 patients (46.7%) died. Patients with total recovery had a multivariate-adjusted 54% lower risk of dying when compared to non-recovered. Partially-recovered patients showed a non-significant adjusted 8% mortality reduction. Independent predictors of systolic function recovery were female gender(OR: 2.17, 95% CI 1.11-4.35), non-ischemic aetiology (OR: 2.78, 95% CI 1.35-5.56), and end diastolic left ventricular diameter < 60 mm (OR: 3.12, 95% CI 1.56-6.25). CONCLUSIONS: HF-recovered patients were mainly women with non-ischemic HF and smaller left ventricles. These patients had significantly better prognosis than those with persistently reduced EF.

Encephalopathy in a kidney transplant recipient.

A 60-year-old man presented several times to the emergency department due to confusion and behavioral changes. He was a kidney transplant recipient dependent on hemodialysis due, presumably, to chronic nephropathy of the transplanted kidney, and was not under any immunosuppressive therapy. He was admitted to the hospital ward due to elevation of C reactive protein and severe proteinuria, leukocyturia and erythrocyturia. The alterations found in the spot urine examination were suggestive of nephritic syndrome, consistent with chronic nephropathy of the transplanted kidney. The neurologic deterioration, however, remained unexplained. CT of the brain and cerebrospinal fluid examination were unremarkable. Infection, auto-immune disease and malignancy were excluded. Corticoid therapy was started for rejection nephropathy. The patient improved dramatically and ultimately the transplanted kidney was removed. Chronic nephropathy of the transplanted kidney was confirmed histologically and the patient remained clinically asymptomatic, without corticoid therapy.

Proteus mirabilis endocarditis.

A 62-year-old man was admitted to the emergency department due to fever and acute heart failure. A transthoracic echocardiogram revealed severe aortic valve obstruction. He was an hepatic transplant recipient and was medicated with everolimus. He underwent mitral and aortic valve replacement with prosthetic valves 4 years ago. Due to his medical background, therapy and clinical presentation, empirical therapy for infective endocarditis was started. Transoesophageal echocardiogram showed severe aortic valve regurgitation but no other findings suggestive of endocarditis. Computed tomography (CT) revealed pulmonary infiltrates compatible with infection and no evidence of septic embolisation. Multiple sets of blood cultures were negative. Proteus mirabilis was isolated in bronchial lavage and antibiotic therapy was adjusted. The patient underwent aortic valve replacement, with no macroscopic findings suggestive of endocarditis. P. mirabilis was isolated in the surgically removed valve. Dual antibiotic therapy was successfully administered for 6 weeks.

Overexpression of Osmosensitive Ca2+-Permeable Channel TMEM63B Promotes Migration in HEK293T Cells.

The recent discovery of the osmosensitive calcium (Ca2+) channel OSCA has revealed the potential mechanism by which plant cells sense diverse stimuli. Osmosensory transporters and mechanosensitive channels can detect and respond to osmotic shifts that play an important role in active cell homeostasis. Members of the TMEM63 family of proteins are described as the closest homologues of OSCAs. Here, we characterize TMEM63B, a mammalian homologue of OSCAs, recently classified as mechanosensitive. In HEK293T cells, TMEM63B localizes to the plasma membrane and is associated with F-actin. This Ca2+-permeable channel specifically induces Ca2+ influx across the membrane in response to extracellular Ca2+ concentration and hyperosmolarity. In addition, overexpression of TMEM63B in HEK293T cells significantly enhanced cell migration and wound healing. The link between Ca2+ osmosensitivity and cell migration might help to establish TMEM63B's pathogenesis, for example, in cancer in which it is frequently overexpressed.

Original Intracerebral Hemorrhage Score for the Prediction of Short-Term Mortality in Cerebral Hemorrhage: Systematic Review and Meta-Analysis.

OBJECTIVES: To systematically assess the discrimination and calibration of the Intracerebral Hemorrhage score for prediction of short-term mortality in intracerebral hemorrhage patients and to study its determinants using heterogeneity analysis. DATA SOURCES: PubMed, ISI Web of Knowledge, Scopus, and CENTRAL from inception to September 15, 2018. STUDY SELECTION: Adult studies validating the Intracerebral Hemorrhage score for mortality prediction in nontraumatic intracerebral hemorrhage at 1 month/discharge or sooner. DATA EXTRACTION: Data were collected on the following aspects of study design: population studied, level of care, timing of outcome measurement, mean study year, and mean cohort Intracerebral Hemorrhage score. The summary measures of interest were discrimination as assessed by the C-statistic and calibration as assessed by the standardized mortality ratio (observed:expected mortality ratio). Random effect models were used to pool both measures. Heterogeneity was measured using the I statistic and explored using subgroup analysis and meta-regression. DATA SYNTHESIS: Fifty-five studies provided data on discrimination, and 35 studies provided data on calibration. Overall, the Intracerebral Hemorrhage score discriminated well (pooled C-statistic 0.84; 95% CI, 0.82-0.85) but overestimated mortality (pooled observed:expected mortality ratio = 0.87; 95% CI, 0.78-0.97), with high heterogeneity for both estimates (I 80% and 84%, respectively). Discrimination was affected by study mean Intracerebral Hemorrhage score (ß = -0.05), and calibration was affected by disease severity, with the score overestimating mortality for patients with an Intracerebral Hemorrhage score greater than 3 (observed:expected mortality ratio = 0.84; 95% CI, 0.78-0.91). Mortality rates were reproducible across cohorts for patients with an Intracerebral Hemorrhage score 0-1 (I = 15%). CONCLUSIONS: The Intracerebral Hemorrhage score is a valid clinical prediction rule for short-term mortality in intracerebral hemorrhage patients but discriminated mortality worse in more severe cohorts. It also overestimated mortality in the highest Intracerebral Hemorrhage score patients, with significant inconsistency between cohorts. These results suggest that mortality for these patients is dependent on factors not included in the score. Further studies are needed to determine these factors.

NT-pro-BNP correlates with disease severity and predicts outcome in cerebral haemorrhage patients: Cohort study.

BACKGROUND: Intracerebral haemorrhage (ICH) is a devastating condition, with more than half of patients dying or becoming dependent after such an event. Natriuretic peptides, frequently used in the management of heart failure, have been shown to correlate with disease severity and prognosis in brain disorders. The aim of this study was to test the hypothesis that NT-pro-BNP correlates with disease severity and is an independent prognostic marker for non-traumatic ICH patients. METHODS: A consecutive sample of 201 non-traumatic ICH patients, who were non-comatose on admission and medically treated in a stroke unit, were evaluated for in-hospital mortality and three-month functional dependency (modified Rankin Scale >2). NT-pro-BNP measurement was performed after admission. Independent predictors of the outcomes in study were assessed using logistic regression and the incremental value of NT-pro-BNP on three previously validated severity scores was evaluated using the variation in C-statistic (Δc). Values of p < .05 were considered significant. RESULTS: In-hospital mortality rate was 8.0%, and 40.3% of patients achieved good functional outcome. NT-pro-BNP correlated with hematoma volume (r = 0.186) and amount of intraventricular blood (r = 0.240). Higher levels of NT-pro-BNP were independently associated with death (Expß = 1.650) and functional dependency (Expß = 1.449). NT-pro-BNP increased the discrimination of the ICH-GS for mortality prediction (Δc = 0.043) and of FUNC and ICH scores for functional outcome prediction (Δc = 0.060 and 0.055 respectively). Admission NT-pro-BNP levels were independently associated with hematoma size. CONCLUSIONS: NT-pro-BNP is an independent prognostic factor for low-risk non-traumatic ICH patients and a valid marker of disease severity in this patient population.

Assessment and Comparison of the Four Most Extensively Validated Prognostic Scales for Intracerebral Hemorrhage: Systematic Review with Meta-analysis.

BACKGROUND/OBJECTIVE: Intracerebral hemorrhage (ICH) is a devastating disorder, responsible for 10% of all strokes. Several prognostic scores have been developed for this population to predict mortality and functional outcome. The aim of this study was to determine the four most frequently validated and most widely used scores, assess their discrimination for both outcomes by means of a systematic review with meta-analysis, and compare them using meta-regression. METHODS: PubMed, ISI Web of Knowledge, Scopus, and CENTRAL were searched for studies validating the ICH score, ICH-GS, modified ICH, and the FUNC score in ICH patients. C-statistic was chosen as the measure of discrimination. For each score and outcome, C-statistics were aggregated at four different time points using random effect models, and heterogeneity was evaluated using the I2 statistic. Score comparison was undertaken by pooling all C-statistics at different time points using robust variance estimation (RVE) and performing meta-regression, with the score used as the independent variable. RESULTS: Fifty-three studies were found validating the original ICH score, 14 studies were found validating the ICH-GS, eight studies were found validating the FUNC score, and five studies were found validating the modified ICH score. Most studies attempted outcome prediction at 3 months or earlier. Pooled C-statistics ranged from 0.76 for FUNC functional outcome prediction at discharge to 0.85 for ICH-GS mortality prediction at 3 months, but heterogeneity was high across studies. RVE showed the ICH score retained the highest discrimination for mortality (c = 0.84), whereas the modified ICH score retained the highest discrimination for functional outcome (c = 0.80), but these differences were not statistically significant. CONCLUSIONS: The ICH score is the most extensively validated score in ICH patients and, in the absence of superior prediction by other scores, should preferably be used. Further studies are needed to validate prognostic scores at longer follow-ups and assess the reasons for heterogeneity in discrimination.

Prognostic models for intracerebral hemorrhage: systematic review and meta-analysis.

BACKGROUND: Prognostic tools for intracerebral hemorrhage (ICH) patients are potentially useful for ascertaining prognosis and recommended in guidelines to facilitate streamline assessment and communication between providers. In this systematic review with meta-analysis we identified and characterized all existing prognostic tools for this population, performed a methodological evaluation of the conducting and reporting of such studies and compared different methods of prognostic tool derivation in terms of discrimination for mortality and functional outcome prediction. METHODS: PubMed, ISI, Scopus and CENTRAL were searched up to 15th September 2016, with additional studies identified using reference check. Two reviewers independently extracted data regarding the population studied, process of tool derivation, included predictors and discrimination (c statistic) using a predesignated spreadsheet based in the CHARMS checklist. Disagreements were solved by consensus. C statistics were pooled using robust variance estimation and meta-regression was applied for group comparisons using random effect models. RESULTS: Fifty nine studies were retrieved, including 48,133 patients and reporting on the derivation of 72 prognostic tools. Data on discrimination (c statistic) was available for 53 tools, 38 focusing on mortality and 15 focusing on functional outcome. Discrimination was high for both outcomes, with a pooled c statistic of 0.88 for mortality and 0.87 for functional outcome. Forty three tools were regression based and nine tools were derived using machine learning algorithms, with no differences found between the two methods in terms of discrimination (p = 0.490). Several methodological issues however were identified, relating to handling of missing data, low number of events per variable, insufficient length of follow-up, absence of blinding, infrequent use of internal validation, and underreporting of important model performance measures. CONCLUSIONS: Prognostic tools for ICH discriminated well for mortality and functional outcome in derivation studies but methodological issues require confirmation of these findings in validation studies. Logistic regression based risk scores are particularly promising given their good performance and ease of application.

Modular Pore-Forming Immunotoxins with Caged Cytotoxicity Tailored by Directed Evolution.

Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.

A Plasmodium berghei sporozoite-based vaccination platform against human malaria.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with enhanced metabolic stability.

Hybrid compounds may play a critical role in the context of the malaria eradication agenda, which will benefit from therapeutic tools active against the symptomatic erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage parasites. To address the need for efficient multistage antiplasmodial compounds, a small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant Plasmodium falciparum W2 strain, with EC50 values in the nM range, and with low cytotoxicity against mammalian cells. The compounds also inhibited the development of P. berghei liver stage parasites, with the most potent compounds displaying EC50 values in the low µM range. SAR analysis revealed that unbranched linkers between the endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1 mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-aminoquinoline moiety improve the compounds' metabolic stability when compared with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be used in malaria eradication campaigns.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

Laying Waste to Mercury: Inexpensive Sorbents Made from Sulfur and Recycled Cooking Oils.

Mercury pollution threatens the environment and human health across the globe. This neurotoxic substance is encountered in artisanal gold mining, coal combustion, oil and gas refining, waste incineration, chloralkali plant operation, metallurgy, and areas of agriculture in which mercury-rich fungicides are used. Thousands of tonnes of mercury are emitted annually through these activities. With the Minamata Convention on Mercury entering force this year, increasing regulation of mercury pollution is imminent. It is therefore critical to provide inexpensive and scalable mercury sorbents. The research herein addresses this need by introducing low-cost mercury sorbents made solely from sulfur and unsaturated cooking oils. A porous version of the polymer was prepared by simply synthesising the polymer in the presence of a sodium chloride porogen. The resulting material is a rubber that captures liquid mercury metal, mercury vapour, inorganic mercury bound to organic matter, and highly toxic alkylmercury compounds. Mercury removal from air, water and soil was demonstrated. Because sulfur is a by-product of petroleum refining and spent cooking oils from the food industry are suitable starting materials, these mercury-capturing polymers can be synthesised entirely from waste and supplied on multi-kilogram scales. This study is therefore an advance in waste valorisation and environmental chemistry.

Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells.

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Inhibition of Plasmodium Liver Infection by Ivermectin.

Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.

Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents.

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.