Achiral‐to‐Chiral Transition in Benzil Solidification: Analogies with Racemic Conglomerates Systems Showing Deracemization

Experimental results show that benzil (1,2‐diphenyl‐1,2‐ethanedione), an achiral compound that crystallizes as a racemic conglomerate, yields by solidification polycrystalline scalemic mixtures of high enantiomeric excesses. These results are related to those previously reported in this type of compounds on deracemizations of racemic mixtures of crystal enantiomorphs obtained by wet grinding. However, the present results strongly suggest that these experiments cannot be explained without taking into account chiral recognition interactions at the level of precritical clusters. The conditions that would define a general thermodynamic scenario for such deracemizations are discussed. Chirality 25:393–399, 2013. © 2013 Wiley Periodicals, Inc.     

DNA alkylating therapy induces tumor regression through an HMGB1-mediated activation of innate immunity

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.     

POTATO LEAF EXTRACT AND ITS COMPONENT, α‐SOLANINE,EXERT SIMILAR IMPACTS ON DEVELOPMENT AND OXIDATIVE STRESS IN Galleria mellonella L.

Plants synthesize a broad range of secondary metabolites that act as natural defenses against plant pathogens and herbivores. Among these, potato plants produce glycoalkaloids (GAs). In this study, we analyzed the effects of the dried extract of fresh potato leaves (EPL) on the biological parameters of the lepidopteran, Galleria mellonella (L.) and compared its activity to one of the main EPL components, the GA α‐solanine. Wax moth larvae were reared from first instar on a diet supplemented with three concentrations of EPL or α‐solanine. Both EPL and α‐solanine affected survivorship, fecundity, and fertility of G. mellonella to approximately the same extent. We evaluated the effect of EPL and α‐solanine on oxidative stress in midgut and fat body by measuring malondialdehyde (MDA) and protein carbonyl (PCO) contents, both biomarkers of oxidative damage. We evaluated glutathione S‐transferase (GST) activity, a detoxifying enzyme acting in prevention of oxidative damage. EPL and α‐solanine altered MDA and PCO concentrations and GST activity in fat body and midgut. We infer that the influence of EPL on G. mellonella is not enhanced by synergistic effects of the totality of potato leaf components compared to α‐solanine alone.     

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.     

In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin''s barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.Proteases play pivotal roles in complicated tissue processes by influencing immune responses, epithelial and mesenchymal cell integrity, proliferation and migration, as well as extracellular matrix maturation and remodeling. As a prime example, they control all phases of cutaneous wound healing by participating in coagulation, complement activation, recruitment of immune cells, migration of keratinocytes and fibroblasts, angiogenesis, and formation of the scar tissue (1, 2). Immediately after injury a blood clot is formed through a series of interconnected proteolytic processing events of coagulation factors to initially seal the site of damage and to provide a provisional fibrin matrix (3, 4). Soon after and interfacing with coagulation, the complement system is activated to fight invading bacteria. During the inflammatory phase (day 1 to 3) the kallikrein–kinin axis controls vasodilation and vascular permeability, and leukocytes enter the wounded tissue in response to pro-inflammatory chemo-attractants whose activity is regulated by limited proteolysis (5). Upon activation of the plasmin system, the fibrin clot is proteolytically degraded to facilitate migration of keratinocytes from the epidermis and the hair follicles as well as of macrophages and fibroblasts in the granulation tissue. These migratory events that occur during the phase of new tissue formation (days 3–10) are further promoted by matrix metalloproteinases (MMPs)¹ that are activated by plasmin and concomitantly modulate tissue influx of immune cells and resolution of inflammation (6, 7). MMPs are also heavily involved in matrix remodeling and scar formation as the final step of skin repair that starts 1 to 2 weeks after injury, but may continue for up to 1 year or more (8).As a consequence of their crucial roles in the healing skin, wound proteases have been implicated in the pathogenesis of healing disorders (9). Impaired wound healing has detrimental consequences and often leads to the development of chronic, nonhealing ulcers. In particular, patients suffering from vascular disease, diabetes, or autoimmune disorders frequently develop chronic skin ulcers. Chronic wounds have become a major problem in industrialized western countries with their rising rates of obesity and the increasing life expectancy, putting also an enormous burden on health systems (10). Because MMPs received much attention in chronic wound repair (11, 12), current diagnostic tests rely on assessment of general MMP activity in wound swabs (13), but suffer from lack of specificity and fail in many cases in predicting the actual wound status. Hence, novel multiparameter point-of-care tests are needed that integrate multiple proteolytic events to deliver robust results on aberrant proteolysis as an indicator for chronic wound progression (14).Proteolytic cleavages in major cascades, such as blood coagulation and complement activation, have been mapped in great detail through seminal biochemical studies (15, 16). In vitro studies used purified or recombinant proteins or monitored processing of radioactively labeled components spiked into activated blood plasma (17, 18). Later, the invention of monoclonal antibodies and/or active site labels also enabled the analysis of endogenous proteolytically activated coagulation factors and complement components in in vivo samples (19). However, none of these techniques allowed directly recording the actual interconnected cleavage events of these complex proteolytic activation cascades in vivo and in response to a natural incidence like tissue injury, a prerequisite to better understand their disturbances in pathology. Addressing this limitation, mass spectrometry-based degradomics technologies have been developed that identify and relatively quantify protein N termini in complex biological samples (20–22). One of these methods, Terminal Amine Isotopic Labeling of Substrates (TAILS), was successfully applied in vitro to identify novel substrates of individual proteases (23–28) and more recently also in vivo to systematically assess protease activity in complex tissue samples (29, 30). TAILS has unique multiplexing capabilities and thus is particularly suited for analyzing the N-terminome at multiple time points after the stimulus (31) as required for the time-resolved analysis of proteolytic events at critical turning points after skin injury (32).An optimal sample for the system-wide analysis of protease activity in cutaneous wound healing should be easily and preferentially noninvasively accessible, cover most cleavage events, and be ideally obtained from the same wound at multiple time points after wounding. This is the case for wound exudates, which can be either directly collected from the wound site (33, 34) or extracted from wound dressings (35). Several proteomic analyses of wound fluids have been performed that mostly focused on the quantitative comparison of proteins in fluids from normal and impaired healing (33, 35). The most recent studies covered a significant proportion of the wound proteome and recorded differential protein abundances at single states of chronic manifestation or normal healing (35). However, these analyses did not integrate data on healing progression and/or functional modifications to the wound proteome along the healing process. Importantly, several studies suggest a higher predictive power of post-translational modifications than relative protein abundances for disease progression (36, 37). Hence, recording proteolytic signatures at critical time points after wounding is a promising approach to define pivotal events in acute healing that might be disturbed in healing impairments.Here, we exploited the power of multiplexed iTRAQ-TAILS to globally analyze the wound fluid proteome and N-terminome at multiple time points after injury. We identified more than 650 proteins and almost 1300 protein N termini from exudates collected in a clinically relevant pig wound healing model. By combining quantitative proteome and N-terminome analyses, we temporally discerned major phases of acute wound healing and mapped key cleavages in blood coagulation and complement activation. Further, we revealed protease dynamics through identification, quantification, and relative weighting of multiple cleavages in complement C3. Finally, by integrating data on known cleavage site specificities we related groups of proteases to identified cleavage sites and established direct cleavage of the integrin adapter protein kindlin-3 by caspase-3, which might play an important role in immune cell apoptosis during cutaneous wound healing.     

Apolipoprotein A5-1131T>C polymorphism, but not APOE genotypes, increases susceptibility for dyslipidemia in children and adolescents

Apolipoprotein A5 (APOA5) and apolipoprotein E (APOE) play important roles in the metabolism of cholesterol and triglycerides. The aim of this study was to determine the allelic and genotypic distributions of the APOA5-1131T>C (rs 662799) and the APOE HhaI polymorphisms and to identify the association of both individual and combined APOA5-APOE genetic variants and the risk for dyslipidemia in children and adolescents. We genotyped 53 dyslipidemic and 77 normolipidemic individuals. The total cholesterol, triglycerides and HDL cholesterol were determined enzymatically. For APOA5 polymorphism, the presence of the allele C confers an individual risk for dyslipidemia (OR = 2.38, 95% CI = 1.15-4.89; P = 0.018). No significant differences were observed for lipid parameters among the APOA5 groups, except for a higher value of HDLc (P = 0.024) in C-carriers. The allelic and genotypic frequencies of APOE polymorphism were similar between groups and did not increase the susceptibility for dyslipidemia. None of the combined APOA5-APOE polymorphisms increased risk for dyslipidemia. We demonstrated an association between APOA5-1131T>C polymorphism and dyslipidemia in children and adolescents. This finding may be useful to guide new studies with genetic markers down a path toward a better characterization of the genetic risk factors for dyslipidemia and atherosclerotic diseases.     

Faster HIV-1 disease progression among Brazilian individuals recently infected with CXCR4-utilizing strains

Introduction

Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus''s co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression.

Methods

A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/µL.

Results

Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4<350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4>350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4>350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype.

Conclusions

Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.