Acquisition of the mcr-1 gene by a high-risk clone of KPC-2-producing Klebsiella pneumoniae ST437/CC258, Brazil

We identified one clinical isolate of K. pneumoniae harboring the mcr 1 (plasmid of IncX4 family) and bla_KPC-2 (plasmid of IncFIB family) genes in southern Brazil. These findings highlight that K. pneumoniae isolates carrying both mcr-1 and bla_KPC-2 may emergence as a serious threat to antimicrobial therapy.     

Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.     

Chlorambucil therapy in hairy cell leukemia: effects on lipid composition and lymphocyte subpopulations

Two patients with progressive hairy cell leukemia following splenectomy were treated with low-dose daily chlorambucil. Both had an objective hematologic response as determined by a return to normal hematocrit and platelet count. This was also reflected in the mononuclear cell fraction by the normalization of cholesterol content, cholesterol/phospholipid ratio, and the lymphocyte subpopulations. This article confirms previous reports on the efficacy of chlorambucil in this setting and describes some morphological, and biochemical concomitant events.     

Trastuzumab-induced cardiotoxicity in elderly women with HER-2-positive breast cancer: a meta-analysis of real-world data

Background: We conducted a meta-analysis to assess the overall risk of cardiac toxicity associated with trastuzumab treatment in elderly breast cancer patients.

Methods: We searched databases from PubMed, EMBASE and Cochrane Central Registry of Controlled Trials to identify relevant studies. Statistical analyses were conducted to calculate the incidence rate, overall hazard ratio (HR) and 95% CIs using a fixed effects model.

Results: A total of 116,342 and 360 elderly patients from five cohort studies and two randomized clinical trials (RCTs) were included for analysis. The pooled incidences of symptomatic congestive heart failure (CHF) and CHF/HF/CM were 6.4% (95% CI 4.1% – 9.4) and 16.4% (95% CI 16.19% – 16.61) in patients with median age of 67.5 years from two RCTs and in patients with median age of 67.5 (60 – 75), 71 (66 – 80+), 74.5 (65 – 89), 75 (66 – 81+) and 79.5 (60 – 99) from five cohort studies, respectively. Trastuzumab was significantly correlated with an increased risk of defined cardiac toxicities in five cohort studies (HR = 1.89, 95% CI 1.72 – 2.07, p < 0.00001) and two RCTs (HR = 3.00, 95% CI 1.71, 5.26, p < 0.00001). Sub-group analysis showed that the anthracycline-based chemotherapy increased the risk of CHF/HF and CM in patients among five cohort studies (HR = 2.16, 95% CI: 1.8 – 1.87, p < 0.00001).

Conclusion: Trastuzumab is likely associated with an increased risk of cardiac toxicity in elderly patients with HER-2-positive breast cancer. Carefully monitoring cardiac function in elderly patients receiving trastuzumab, particularly with concurrent use of anthracycline, is warranted.     

Understanding the frustration arising from the competition between function,misfolding, and aggregation in a globular protein

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron–sulfur clusters but whose mutations are associated with Friedreich’s ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.Frustration is a general condition that arises in the presence of conflicting requirements. A system is frustrated when it is impossible to fully minimize its energy by optimizing simultaneously all of the possible interactions among its components (1). Although complex systems tend in general to exhibit frustration because of the large number and heterogeneity of their components, protein molecules are remarkable in that their folding process involves interactions that express a minimal level of frustration. According to the so-called principle of minimal frustration, the energy of proteins decreases as they explore conformations increasingly similar in structure to the native state (2). Consequently, the free energy landscape of proteins is characterized by the presence of a well-defined global minimum and very few other local minima, which are typically intermediate states along the folding pathway. This organization of conformational space normally ensures rapid and reliable folding (2–6).Proteins, however, have evolved not only to fold, but also to function. Because the evolutionary constraints that select for a given function may be in conflict with the folding process, it is possible that local frustration patterns may localize in specific regions of proteins, in particular in their active sites. Indeed, a statistical survey of different proteins has shown that frustrated interactions tend to cluster at binding sites and that such frustration decreases upon complex formation (7). Because frustration is associated with the presence of local minima in the free energy landscape, it is important to understand how proteins have evolved to minimize the possible effects associated with these local minima, which are likely to contain misfolded elements and thus to potentially give rise to aggregation.To address this question we studied frataxin, a mitochondrial protein that binds both Fe²⁺ and Fe³⁺ ions and forms a ternary complex with the two main components of the iron–sulfur cluster biogenesis machinery (8–11). This protein offers good opportunities for investigating the relationships between folding, misfolding, and disease. Indeed, its dysfunction is related to a neurodegenerative disease called Friedreich’s ataxia (12). Frataxin is also capable of binding different divalent and trivalent cations, whose recognition sites have been mapped (13). Furthermore, frataxin is involved in donating iron to ferrochelatease via direct interaction through an extended binding site involving some of the residues implicated in metal binding (14).We have previously shown that frataxin folds via a complex mechanism, which we described through a broad free energy barrier (15). This feature, which has been associated with frustration (16), allows the experimental characterization of both the early and late events of folding (16–19). In this work we explored the mechanistic details of the folding reaction of frataxin at residue-level resolution. This result was achieved by characterizing the structures of both the early and late events of folding using Φ-value analysis (20) and restrained molecular dynamics simulations (21). By analyzing the structures of the different states along the folding process we found an unexpected number of nonnative interactions that slow down folding and superpose with the highly frustrated regions, as detected by the frustratometer server (22). The nonnative regions, which display peculiar Φ values, either negative or greater than unity, were predicted on the basis of the transition state structures determined from the Φ values, and subsequently confirmed by a second round of amino acid substitutions rationally designed to probe misfolded regions along the folding pathway.The characterization of the folding pathway of frataxin and of its misfolded elements enables us to discuss the competition between folding and function and its consequences for misfolding and aggregation.