Chemical Analysis of Gunpowder and Gunshot Residues.

The identification of firearms is of paramount importance for investigating crimes involving firearms, as it establishes the link between a particular firearm and firearm-related elements found at a crime scene, such as projectiles and cartridge cases. This identification relies on the visual comparison of such elements against reference samples from suspect firearms or those existing in databases. Whenever this approach is not possible, the chemical analysis of the gunpowder and gunshot residue can provide additional information that may assist in establishing a link between samples retrieved at a crime scene and those from a suspect or in the identification of the corresponding model and manufacturer of the ammunition used. The most commonly used method for the chemical analysis of gunshot residue is scanning electron microscopy with energy dispersive X-ray, which focuses on the inorganic elements present in ammunition formulation, particularly heavy metals. However, a change in the legal paradigm is pushing changes in these formulations to remove heavy metals due to their potential for environmental contamination and the health hazards they represent. For this reason, the importance of the analysis of organic compounds is leading to the adoption of a different set of analytical methodologies, mostly based on spectroscopy and chromatography. This manuscript reviews the constitution of primer and gunpowder formulations and the analytical methods currently used for detecting, characterising, and identifying their compounds. In addition, this contribution also explores how the information provided by these methodologies can be used in ammunition identification and how it is driving the development of novel applications within forensic ballistics.

GC-MS Silylation Derivative Method to Characterise Black BIC® Ballpoint 2-Phenoxyethanol Ratio Evaporation Profile-A Contribution to Ink Ageing Estimation.

One of the major challenges in forensic document analysis is estimating the age of ink deposition on a manually written document. The present work aims to develop and optimise a methodology, based on the evaporation of 2-phenoxyethanol (PE) over time, that can contribute to ink age estimation. A black BIC® Crystal Ballpoint Pen was purchased in a commercial area, and ink deposition began in September 2016 over 1095 days. For each ink sample, 20 microdiscs were subjected to n-hexane extraction in the presence of an internal standard (ethyl benzoate) followed by derivatisation with a silylation reagent. A gas chromatography-mass spectrometry (GC/MS) method was optimised for PE-trimethylsilyl (PE-TMS) to characterise the ageing curve. The developed method presented good linearity between 0.5 and 50.0 µg mL-1, as well as limits of detection and quantification of 0.026 and 0.104 µg mL-1, respectively. It was possible to characterise PE-TMS concentration over time, which reveals a two-phase decay behaviour. First, there was a substantial decline between the 1st and the 33rd day of deposition, followed a by a stabilisation of the signal, which allowed to detect the presence of PE-TMS up to 3 years. Two unknown compounds were also present and allowed to identify three dating time frames for the same ink stroke: (i) between time 0 and 33 days, (ii) between time 34 and 109 days, and (iii) more than 109 days. The developed methodology allowed to characterise the behaviour of PE over time and to establish a relative dating of three-time frames.

Inflammatory factors, genetic variants, and predisposition for preterm birth.

Preterm birth is a major clinical and public health challenge, with a prevalence of 11% worldwide. It is the leading cause of death in children younger than 5 years old and represents 70% of neonatal deaths and 75% of neonatal morbidity. Despite the clinical and public health significance, this condition's etiology is still unclear, and most of the cases are spontaneous. There are several known preterm birth risk factors, including inflammatory diseases and the genetic background, although the underlying molecular mechanisms are far from understood. The present review highlights the research advances on the association between inflammatory-related genes and the increased risk for preterm delivery. The most associated genetic variants are the TNFα rs1800629, the IL1α rs17561, and the IL1RN rs2234663. Moreover, many of the genes discussed in this review are also implicated in pathologies involving inflammatory or autoimmune systems, such as periodontal disease, bowel inflammatory disease, and autoimmune rheumatic diseases. This review presents evidence suggesting a common genetic background to preterm birth, autoimmune and inflammatory diseases susceptibility.

Extraction Procedures for Hair Forensic Toxicological Analysis: A Mini-Review.

In forensic toxicology, drugs of abuse are routinely analyzed due to legal statute in medical or legal investigation, such as death cases, poisoning cases, and drug misuse, determining potential exposure to controlled substances. Despite the widespread use of blood and urine as matrices for forensic toxicological analysis, the use of hair samples has grown as the limitations associated with this matrix are overcome and new areas of application emerge. Hair provides several advantages over urine and blood, such as the easiness of sample collection and the ability to provide a retrospective analysis of drug consumption. Drug analysis in hair is following a path where less amount of sample has been needed to detect drugs of abuse. Improvement in sample preparation methods and lowering detection limits in instrumental analysis is providing forensic scientists with the tools to use drug analysis with hair as a routine approach in the forensic laboratory. Actually, besides the chronological exposition to a drug, hair may provide information about the day a chemical was used. This should become part of the standard information sent by forensic scientists to criminal investigators and courts. The aim of this review is to summon the extraction procedures used to perform forensic toxicological analysis in hair as well as the major advances in this field once a sample preparation step is critical and takes up most of the total analysis time.

The synthetic cannabinoid JWH-018 modulates Saccharomyces cerevisiae energetic metabolism.

Synthetic cannabinoids are a group of novel psychoactive substances with similar properties to Δ9-THC. Among the vast number of synthetic cannabinoids, designed to be tested in clinical trials, JWH-018 was the first novel psychoactive substance found in the recreational drug marketplace. The consumption of JWH-018 shows typical effects of CB1 agonists including sedation, cognitive dysfunction, tachycardia, postural hypotension, dry mouth, ataxia and psychotropic effects, but appeared to be more potent than Δ9-THC. However, studies on human cells have shown that JWH-018 toxicity depends on the cellular line used. Despite these studies, the underlying molecular mechanisms to JWH-018 action has not been clarified yet. To understand the impact of JWH-018 at molecular and cellular level, we used Saccharomyces cerevisiae as a model. The results showed an increase in yeast growth rate in the presence of this synthetic cannabinoid due to an enhancement in the glycolytic flux at expense of a decrease in pentose phosphate pathway, judging by 2D-Gel proteomic analysis, qRT-PCR experiments and ATP measurements. Overall, our results provide insights into molecular mechanisms of JWH-018 action, also indicating that Saccharomyces cerevisiae is a good model to study synthetic cannabinoids.

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

Transthyretin proteins regulate angiogenesis by conferring different molecular identities to endothelial cells.

Familial amyloidotic polyneuropathy (FAP) has a high prevalence in Portugal, and the most common form of hereditary amyloidosis is caused by an amyloidogenic variant of transthyretin (TTR) with a substitution of methionine for valine at position 30 (V30M). Until now, the available efficient therapy is liver transplantation, when performed in an early phase of the onset of the disease symptoms. However, transplanted FAP patients have a significantly higher incidence of early hepatic artery thrombosis compared with non-FAP transplanted patients. Because FAP was described as an independent risk factor for early hepatic artery thrombosis, more studies to understand the underlying mechanisms involved in this outcome are of the utmost importance. Knowing that the liver is the major site for TTR production, we investigated the biological effects of TTR proteins in the vasculature and on angiogenesis. In this study, we identified genes differentially expressed in endothelial cells exposed to the WT or V30M tetramer. We found that endothelial cells may acquire different molecular identities when exposed to these proteins, and consequently TTR could regulate angiogenesis. Moreover, we show that V30M decreases endothelial survival by inducing apoptosis, and it inhibits migration. These findings provide new knowledge that may have critical implications in the prevention of early hepatic artery thrombosis in FAP patients after liver transplantation.

Insights into the molecular mechanism of protein native-like aggregation upon glycation.

Several human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Familial Amyloidotic Polyneuropathy, have long been associated with, structural and functional changes in disease related proteins leading to aggregation into amyloid fibrils. Such changes can be triggered by post-translational modifications. Methylglyoxal modifications have been shown to induce the formation of small and stable native-like aggregates in the case of the amyloidogenic proteins insulin and α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this work cytochrome c (Cyt c) was used as a model protein for the characterization of specific glycation targets and to study their impact on protein structure, stability, and ability to form native-like aggregates. Our results show that methylglyoxal covalently modifies Cyt c at a single residue and induces early conformational changes that lead to the formation of native-like aggregates. Furthermore, partially unfolded species are formed, but do not seem to be implicated in the aggregation process. This shows a clear difference from the amyloid fibril mechanisms which involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments show that glycation strongly decreases Cyt c conformational stability, which is balanced with an increase of conformational stability upon aggregation. Data collected from analytical and spectroscopic techniques, along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination are used to help to understand the driving force underlying glycation-induced native-like aggregation, and enable the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated Cyt c.

Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation.

Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response.

BACKGROUND: Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4⁺ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. RESULTS: CD4⁺ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a ß-hairpin structure were related with rate of escape from the neutralizing antibodies. CONCLUSION: Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Determination of phytocannabinoids in cannabis samples by ultrasound-assisted solid-liquid extraction and high-performance liquid chromatography with diode array detector analysis.

The characterisation of cannabis plants, especially the determination of specific phytocannabinoids, has gained enormous importance in the last decade, mainly due to the recent changes in cannabis control in several countries or states. This is particularly relevant for the forensic, medical or recreative industry to have a rapid, inexpensive, and reliable methodology to identify and quantify phytocannabinoids. Furthermore, spiking cannabis products with Δ8-tetrahydrocannabinol (THC) is a contemporary trend that demands improving or replacing current methods to include this cannabinoid. The current study presents an ultrasound-assisted solid-liquid extraction followed by high-performance liquid chromatography with diode array detection (HPLC-DAD) methodology to identify and quantify Δ9-THC, Δ8-THC, cannabidiol, cannabinol, Δ9-tetrahydrocannabinolic acid and cannabidiolic acid in cannabis products. The herbal samples were extracted with ethanol:acetonitrile (50:50, v:v) by ultrasonication using only 50 mg of sample. The plant oils were diluted in ethanol. The optimised procedure allowed ≈100% extraction efficiency of the target cannabinoids. The validation assays showed that the method is linear (R2 > 0.997), selective, sensitive, precise and accurate, with suitable limits of detection (0.125-0.250 µg mL-1) and quantification (0.500 µg mL-1). The method was successfully applied to cannabis samples, demonstrating its suitability for routine analyses. This contribution follows the current demand for fast and straightforward analysis services of this plant and its derivatives, using small amounts of sample. The present study compares very favourably against other works, particularly in regards to the extraction efficiency, speed of the overall procedure, method sensitivity, and ability to monitor Δ8-THC spiked samples using a novel solvent mixture.

Novel Psychoactive Substances: The Razor's Edge between Therapeutical Potential and Psychoactive Recreational Misuse.

BACKGROUND: Novel psychoactive substances (NPS) are compounds of natural and synthetic origin, similar to traditional drugs of abuse. NPS are involved in a contemporary trend whose origin lies in a thinner balance between legitimate therapeutic drug research and legislative control. The contemporary NPS trend resulted from the replacement of MDMA by synthetic cathinones in 'ecstasy' during the 2000s. The most common NPS are synthetic cannabinoids and synthetic cathinones. Interestingly, during the last 50 years, these two classes of NPS have been the object of scientific research for a set of health conditions. METHODS: Searches were conducted in the online database PubMed using boolean equations. RESULTS: Synthetic cannabinoids displayed protective and therapeutic effects for inflammatory, neurodegenerative and oncologic pathologies, activating the immune system and reducing inflammation. Synthetic cathinones act similarly to amphetamine-type stimulants and can be used for depression and chronic fatigue. CONCLUSIONS: Despite the scientific advances in this field of research, pharmacological application of NPS is being jeopardized by fatalities associated with their recreational use. This review addresses the scientific achievements of these two classes of NPS and the toxicological data, ending with a reflection on Illicit and NPS control frames.

An Umbrella Review of the Evidence of Sex Determination Procedures in Forensic Dentistry.

Sex determination in forensic dentistry is a major step towards postmortem profiling. The most widely recognized method is DNA, yet its application in the dental field of forensic sciences is still impractical. Depending on the conditions of the remains, teeth are often the only surviving organ. Some systematic reviews (SRs) have been recently produced; hence this umbrella review critically assesses their level of evidence and provides an overall comprehensive view. An electronic database search was conducted in four databases (PubMed, Cochrane, Web of Science, and LILACS) and three grey search engines up to December 2021, focusing on SRs of sex determination through forensic dentistry procedures. The methodological quality of the SRs was analyzed using the measurement tool to assess SRs criteria (AMSTAR2). Five SRs were included, two of critically low quality and three of low quality. The SRs posited that canines are the most dimorphic teeth; oral tissue remnants are a rich source for sex determination by DNA tracing; and artificial intelligence tools demonstrate high potential in forensic dentistry. The quality of evidence on sex determination using dental approaches was rated as low. Well-designed clinical trials and high standard systematic reviews are essential to corroborate the accuracy of the different procedures of sex determination in forensic dentistry.

Potent and broadly reactive HIV-2 neutralizing antibodies elicited by a vaccinia virus vector prime-C2V3C3 polypeptide boost immunization strategy.

Human immunodeficiency virus type 2 (HIV-2) infection affects about 1 to 2 million individuals, the majority living in West Africa, Europe, and India. As for HIV-1, new strategies for the prevention of HIV-2 infection are needed. Our aim was to produce new vaccine immunogens that elicit the production of broadly reactive HIV-2 neutralizing antibodies (NAbs). Native and truncated envelope proteins from the reference HIV-2ALI isolate were expressed in vaccinia virus or in bacteria. This source isolate was used due to its unique phenotype combining CD4 independence and CCR5 usage. NAbs were not elicited in BALB/c mice by single immunization with a truncated and fully glycosylated envelope gp125 (gp125t) or a recombinant polypeptide comprising the C2, V3, and C3 envelope regions (rpC2-C3). A strong and broad NAb response was, however, elicited in mice primed with gp125t expressed in vaccinia virus and boosted with rpC2-C3. Serum from these animals potently neutralized (median 50% neutralizing titer, 3,200) six of six highly divergent primary HIV-2 isolates. Coreceptor usage and the V3 sequence of NAb-sensitive isolates were similar to that of the vaccinating immunogen (HIV-2ALI). In contrast, NAbs were not reactive on three X4 isolates that displayed major changes in V3 loop sequence and structure. Collectively, our findings demonstrate that broadly reactive HIV-2 NAbs can be elicited by using a vaccinia virus vector-prime/rpC2-C3-boost immunization strategy and suggest a potential relationship between escape to neutralization and cell tropism.

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation.

BACKGROUND: Insulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin. RESULTS: Methylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics. CONCLUSIONS: We propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-ß core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.

Neuroprotection or Neurotoxicity of Illicit Drugs on Parkinson's Disease.

Parkinson's Disease (PD) is currently the most rapid growing neurodegenerative disease and over the past generation, its global burden has more than doubled. The onset of PD can arise due to environmental, sporadic or genetic factors. Nevertheless, most PD cases have an unknown etiology. Chemicals, such as the anthropogenic pollutant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and amphetamine-type stimulants, have been associated with the onset of PD. Conversely, cannabinoids have been associated with the treatment of the symptoms'. PD and medical cannabis is currently under the spotlight, and research to find its benefits on PD is on-going worldwide. However, the described clinical applications and safety of pharmacotherapy with cannabis products are yet to be fully supported by scientific evidence. Furthermore, the novel psychoactive substances are currently a popular alternative to classical drugs of abuse, representing an unknown health hazard for young adults who may develop PD later in their lifetime. This review addresses the neurotoxic and neuroprotective impact of illicit substance consumption in PD, presenting clinical evidence and molecular and cellular mechanisms of this association. This research area is utterly important for contemporary society since illicit drugs' legalization is under discussion which may have consequences both for the onset of PD and for the treatment of its symptoms.

hiPSC-Based Model of Prenatal Exposure to Cannabinoids: Effect on Neuronal Differentiation.

Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 µM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.

Validation of the Third Molar Maturation Index (I3M) to assess the legal adult age in the Portuguese population.

Age estimation is a major step in forensic and legal procedures. Its relevance has been increasing due to growing society issues, such as identification of missing people, crimes against minors or lack of valid identification papers from locals or foreigners. Evaluation of the cut-off value of the Third Molar Maturation Index (I3M) = 0.08 for discriminating minors from adults in the Portuguese population. The left lower third molars were analysed by applying a specific cut-off value of 0.08 determined by Cameriere et al. in 2008. A sample of 778 digital panoramic radiographs of a representative Portuguese sample (442 females and 336 males), in the age range of 12-24 years (mean age 17.7 ± 2.98 years in females and 18.1 ± 3.0 years in males), was retrospectively evaluated. I3M decreased as the real age gradually increased in both sexes. The 0.08 cut-off score was valuable in discriminating adults from minors. According to the pooled results, the accuracy, by means of area under the curve, was 92.8% (95% confidence interval (CI) 91.0-94.6%). The proportion of correctly classified subjects (sensitivity) was 90.7% (95% CI 88.7-92.8%) and the specificity was 94.9% (95% CI 93.3-96.4%). The results show that I3M is a valuable method to differentiate minors from adults in the Portuguese population.

Fold-unfold transitions in the selectivity and mechanism of action of the N-terminal fragment of the bactericidal/permeability-increasing protein (rBPI(21)).

Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI(21), a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI(21) bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI(21) loses its conformational stability, as studied by circular dichroism. This interaction of rBPI(21) at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (K(p)), suggesting a preference for bacterial membranes over mammalian membranes. rBPI(21) binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI(21) in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI(21). The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation.