Unexpected and delayed fragmentation dynamics of the organometallic ferrocene induced by ion-collision.

We have investigated the fragmentation dynamics of the organometallic ferrocene molecule after interaction with multiply charged ions using multicoincidence mass spectrometry and quantum chemistry calculations. We observed unexpected fragmentation dynamics of the two-body breakup channels from ferrocene dications revealing a charge screening effect from the iron atom and delayed fragmentation dynamics. These observations are rationalized through the population of a specific long-lived excited state, where one positive charge is located on each cyclopentadienyl ring.

Biomolecular characterization of 3500-year-old ancient Egyptian mummification balms from the Valley of the Kings.

Ancient Egyptian mummification was practiced for nearly 4000 years as a key feature of some of the most complex mortuary practices documented in the archaeological record. Embalming, the preservation of the body and organs of the deceased for the afterlife, was a central component of the Egyptian mummification process. Here, we combine GC-MS, HT-GC-MS, and LC-MS/MS analyses to examine mummification balms excavated more than a century ago by Howard Carter from Tomb KV42 in the Valley of the Kings. Balm residues were scraped from now empty canopic jars that once contained the mummified organs of the noble lady Senetnay, dating to the 18th dynasty, ca. 1450 BCE. Our analysis revealed balms consisting of beeswax, plant oil, fats, bitumen, Pinaceae resins, a balsamic substance, and dammar or Pistacia tree resin. These are the richest, most complex balms yet identified for this early time period and they shed light on balm ingredients for which there is limited information in Egyptian textual sources. They highlight both the exceptional status of Senetnay and the myriad trade connections of the Egyptians in the 2nd millennium BCE. They further illustrate the excellent preservation possible even for organic remains long removed from their original archaeological context.

[The clinical importance of the critical view of safety in laparoscopic cholecystectomy]. / Die klinische Bedeutung des "critical view of safety" in der laparoskopischen Gallenblasenchirurgie.

BACKGROUND: Injury of the bile duct during cholecystectomy (CHE) is a severe complication. The critical view of safety (CVS) can help to reduce the frequency of this complication during laparoscopic CHE. So far, no scoring of CVS images with a grading system is available. METHOD: The CVS images of 534 patients with laparoscopic CHE could be structurally analyzed and assessed with marks from 1 (very good) to 5 (insufficient). The CVS mark was correlated with the perioperative course. Additionally, the perioperative course of patients after laparoscopic CHE with and without a CVS image was investigated. RESULTS: In 534 patients 1 or more CVS images could be analyzed. The average CVS mark was 1.9, whereby 280 patients (52.4%) had a 1, 126 patients (23.6%) a 2, 114 (21.3%) a 3 and 14 patients (2.6%) a 4 or 5. Younger patients with elective laparoscopic CHE had CVS images significantly more frequently (p ≤ 0.04). The statistical examination with Pearson's χ2-test and the F­test (ANOVA) showed a significant correlation between improving CVS marks and reduction of surgery time (p < 0.01) and the hospitalization time (p < 0.01). For senior physicians the quota of CVS images ranged from 71% to 92% and the average marks from 1.5 to 2.2. The marks for the CVS images were significantly better for female than male patients (1.8 vs. 2.1, p < 0.01). DISCUSSION: There was a relatively broad distribution of marks for CVS images. Injuries of the bile duct can be avoided with a high degree of certainty with marks 1­2 for the CVS image. The CVS is not always adequately visualized in laparoscopic CHE.

[The COVID-19 pandemic had significant impact on duration of surgery and hospitalization time for patients after cholecystectomy]. / Die Auswirkungen der COVID-19 Pandemie führten zu signifikanten Veränderungen der Operations- und Liegezeiten bei Patienten nach Cholezystektomie.

BACKGROUND: The COVID-19 pandemic made substantial changes in medical care necessary. The aims of this study were to find out what influence the pandemic had on the perioperative course in patients with cholecystectomy (CHE) and to highlight possible residual consequences. METHOD: From 1 July 2018 to 31 December 2021 a total of 735 patients with CHE were analyzed. Up to 21 March 2020 patients were assigned to the regular patient group (Reg, n = 430), patients after this date (first lockdown 22 March 2020) to the Cov19 patient group (Cov19, n = 305) and the 2 groups were compared. RESULTS: The average age of all patients was 59 years and 63% were women. The average length of hospitalization (KrVD, time period between surgery and discharge) was 4.4 days. The patient groups Reg and Cov19 did not differ with respect to age, gender or KrVD. The total number of CHEs carried out was reduced by 21.4% in the Cov19 group. This affected elective and emergency CHE to the same extent. The length of surgery significantly increased in the Cov19 group from 64 min (SD 34 min) to 71 min (SD 38 min). The number of short and long hospital stays (KrVD 2 or >4 days) significantly increased in the Cov19 group from 4 % to 20 % (short stay, p < 0.01) and from 23 % to 27 % (long stay, p < 0.01). This was particularly observed for patients >70 years old with an increase in long stays from 43 % to 56 % in the Cov19 group. CONCLUSION: The COVID-19 pandemic led to a clear reduction in CHE both for elective and emergency interventions. Furthermore, a significant lengthening of the surgery and hospitalization times could be observed for older patients. The residual consequences of the pandemic could be shortened hospitalization times after uncomplicated CHE and more interventional treatment procedures in complex cases.

[After inconspicuous elective laparoscopic cholecystectomy patients can be discharged without control of laboratory parameters]. / Patienten* nach unauffälliger elektiver laparoskopischer Cholezystektomie können ohne Laborwertkontrollen entlassen werden ­ Ergebnisse einer prospektiven Studie.

BACKGROUND: The importance of postoperative control of laboratory parameters after elective laparoscopic cholecystectomy (lap-CHE) is controversial. The aim of this prospective study was to find out whether patients can be safely discharged following an inconspicuous perioperative course after lap-CHE without control of the laboratory parameters. METHOD: All patients with a lap-CHE from September 2020 to March 2022 were screened and included in the study after providing informed consent. The course was followed in a structured way with a scoring system (value 3-15 points) and questionnaire. A score of ≤ 9 reflected an inconspicuous perioperative course. Approval was obtained from the ethics committee of Heidelberg University (S-026/2020). RESULTS: A total of 275 patients who underwent gall bladder surgery were documented of which 220 (80%) patients underwent an elective lap-CHE and 56 (25%) of the patients were included in the study. Of the patients 51 with a score of ≤ 9 were discharged without providing a blood sample. The average age of the patients was 50.8 years, the average duration of hospital stay was 2.6 days and 40 out of 51 (78.4%) patients could be postoperatively questioned. None of the patients suffered from relevant complications after being discharged. Out of 40 patients 27 (67.5) visited a general practitioner again postoperatively and 4 were readmitted as inpatients due to other operations and an endoscopic intervention. All patients were satisfied with the course of surgery. CONCLUSION: Patients with an inconspicuous course after elective lap-CHE (score ≤ 9 points) can be discharged without a postoperative control of laboratory parameters.

[Which patient needs controls of laboratory values after elective laparoscopic cholecystectomy?-Can a score help?] / Welcher Patient benötigt Laborwertkontrollen nach elektiver laparoskopischer Cholezystektomie? ­ Kann ein Score helfen?

BACKGROUND: Laparoscopic cholecystectomy is nearly exclusively carried out as an inpatient operation in Germany. The aim of the study was to evaluate for which patients postoperative laboratory control values are necessary. METHODS: This retrospective analysis included 100 patients who underwent elective laparoscopic cholecystectomy. A scoring and data collection sheet was developed, which enables a risk stratification. Using the scoring system patients can achieve between 3 and 15 points. RESULTS: In total 100 patients were included in the study. Of the patients 64 (group 1) had between 3 and 8 points, 29 patients (group 2) between 9 and 11 points and 7 patients (group 3) between 12 and 15 points. In comparison to group 1 the C­reactive protein values as well as the duration of hospital stay were significantly increased in group 2 and group 3 (p > 0.05). In group1 a total of 60 patients (93.7%) were discharged regularly on postoperative days 1-3. In group 2 there were 17 patients (58.6%) who could be discharged with unremarkable blood values and in group 3 there were 3 patients (42.8%). In the total collective hospital discharge without a laboratory control of blood values would have been justified in 80% of the patients. CONCLUSION: A postoperative control of laboratory blood values is not routinely necessary for patients after elective laparoscopic cholecystectomy with a score <9 points.

Customised bi-compartmental knee arthroplasty shows encouraging 3-year results: findings of a prospective, multicenter study.

PURPOSE: The purpose of this study was to assess the clinical and patient-reported outcomes of a customised, individually made (CIM) bi-compartmental knee arthroplasty (BKA). METHODS: A prospectively recruited cohort of 79 patients was implanted with a CIM-BKA (patello-femoral plus either medial or lateral tibio-femoral, iDuo G2 system, Conformis, Billerica MA) at eight centres in the US and Germany. Patients were assessed for the 2011 KSS, KOOS, and ROM pre-operatively and at 2 weeks, 6 weeks, 12 weeks, 1 year, and 2 years post-operatively. RESULTS: The objective KSS score significantly improved from 69 at the pre-operative visit, to 94 at the 2-year post-operative time-point. Similar improvements were observed for the KSS function and satisfaction domains. Significant improvements from pre-operative levels were observed across all five domains of the KOOS. Two patients have undergone surgery to revise their CIM-BKA implant to total knees, resulting in a survivorship rate of 97.5% at an average follow-up of 2.6 years. CONCLUSIONS: CIM-BKA compares favourably to published scores as well as revision rates for previously available monolithic OTS-BKA implants. CIM-BKA implants provide surgeons with a viable and patient-specific monolithic implant solution as an option for patients presenting with bi-compartmental disease, who might, otherwise, be treated by performing uni-condylar + patello-femoral joint or bicruciate sparing TKA surgeries. Longer follow-up and higher numbers have to be awaited for further validation of these encouraging early results. LEVEL OF EVIDENCE: 3b (individual case-controlled study).

Single dose escalation studies with inhaled POL6014, a potent novel selective reversible inhibitor of human neutrophil elastase, in healthy volunteers and subjects with cystic fibrosis.

BACKGROUND: POL6014 is a novel, orally inhaled neutrophil elastase (NE) inhibitor in development for cystic fibrosis (CF). METHODS: Two studies, one in healthy volunteers (HVs, doses 20 to 960 mg) and one in subjects with CF (doses 80 to 320 mg) were conducted to evaluate the safety, tolerability and pharmacokinetics (PK) of single ascending doses of inhaled POL6014 with a Pari eFlow® nebuliser. PK was evaluated over a period of 24 h. In addition, NE activity in CF sputum was measured. RESULTS: After single doses, POL6014 was safe and well tolerated up to 480 mg in HVs and at all doses in subjects with CF. POL6014 showed a dose-linear PK profile in both populations with Cmax between 0.2 and 2.5 µM in HVs and between 0.2 and 0.5 µM in subjects with CF. Tmax was reached at approximately 2-3 h. Mean POL6014 levels in CF sputum rapidly reached 1000 µM and were still above 10 µM at 24 h. >1-log reduction of active NE was observed at 3 h after dosing. CONCLUSION: Inhalation of POL6014 can safely lead to high concentrations within the lung and simultaneously low plasma concentrations, allowing for a clear inhibition of NE in the sputum of subjects with CF after single dosing. TRIAL REGISTRATION: European Medicines Agency EudraCT-Nr. 2015-001618-83 and 2016-000493-38.

Treatment of bone loss in proximal femurs of postmenopausal osteoporotic women with AGN1 local osteo-enhancement procedure (LOEP) increases hip bone mineral density and hip strength: a long-term prospective cohort study.

This first-in-human study of AGN1 LOEP demonstrated that this minimally-invasive treatment durably increased aBMD in femurs of osteoporotic postmenopausal women. AGN1 resorption was coupled with new bone formation by 12 weeks and that new bone was maintained for at least 5-7 years resulting in substantially increased FEA-estimated femoral strength. INTRODUCTION: This first-in-human study evaluated feasibility, safety, and in vivo response to treating proximal femurs of postmenopausal osteoporotic women with a minimally-invasive local osteo-enhancement procedure (LOEP) to inject a resorbable triphasic osteoconductive implant material (AGN1). METHODS: This prospective cohort study enrolled 12 postmenopausal osteoporotic (femoral neck T-score ≤ - 2.5) women aged 56 to 89 years. AGN1 LOEP was performed on left femurs; right femurs were untreated controls. Subjects were followed-up for 5-7 years. Outcomes included adverse events, proximal femur areal bone mineral density (aBMD), AGN1 resorption, and replacement with bone by X-ray and CT, and finite element analysis (FEA) estimated hip strength. RESULTS: Baseline treated and control femoral neck aBMD was equivalent. Treated femoral neck aBMD increased by 68 ± 22%, 59 ± 24%, and 58 ± 27% over control at 12 and 24 weeks and 5-7 years, respectively (p < 0.001, all time points). Using conservative assumptions, FEA-estimated femoral strength increased by 41%, 37%, and 22% at 12 and 24 weeks and 5-7 years, respectively (p < 0.01, all time points). Qualitative analysis of X-ray and CT scans demonstrated that AGN1 resorption and replacement with bone was nearly complete by 24 weeks. By 5-7 years, AGN1 appeared to be fully resorbed and replaced with bone integrated with surrounding trabecular and cortical bone. No procedure- or device-related serious adverse events (SAEs) occurred. CONCLUSIONS: Treating femurs of postmenopausal osteoporotic women with AGN1 LOEP results in a rapid, durable increase in aBMD and femoral strength. These results support the use and further clinical study of this approach in osteoporotic patients at high risk of hip fracture.

Incorporation of RG1 epitope into HPV16L1-VLP does not compromise L1-specific immunity.

The candidate pan-Human Papillomavirus (HPV) vaccine RG1-VLP are HPV16 major capsid protein L1 virus-like-particles (VLP) comprising a type-common epitope of HPV16 minor capsid protein L2 (RG1; aa17-36). Vaccinations have previously demonstrated efficacy against genital high-risk (hr), low-risk (lr) and cutaneous HPV. To compare RG1-VLP to licensed vaccines, rabbits (n = 3) were immunized thrice with 1 µg, 5 µg, 25 µg, or 125 µg of RG1-VLP or a 1/4 dose of Cervarix®. 5 µg of RG1-VLP or 16L1-VLP (Cervarix) induced comparable HPV16 capsid-reactive and neutralizing antibodies titers (62,500/12,500-62,500 or 1000/10,000). 25 µg RG1-VLP induced robust cross-neutralization titers (50-1000) against hrHPV18/31/33/45/52/58/26/70. To mimic reduced immunization schedules in adolescents, mice (n = 10) were immunized twice with RG1-VLP (5 µg) plus 18L1-VLP (5 µg). HPV16 neutralization (titers of 10,000) similar to Cervarix and Gardasil and cross-protection against hrHPV58 vaginal challenge was observed. RG1-VLP vaccination induces hrHPV16 neutralization comparable to similar doses of licensed vaccines, plus cross-neutralization to heterologous hrHPV even when combined with HPV18L1-VLP.

Review of data on the dermal penetration of mineral oils and waxes used in cosmetic applications.

Mineral oils and waxes used in cosmetic products, also referred to as "personal care products" outside the European Union, are mixtures of predominantly saturated hydrocarbons consisting of straight-chain, branched and ring structures with carbon chain lengths greater than C16. They are used in skin and lip care cosmetic products due to their excellent skin tolerance as well as their high protecting and cleansing performance and broad viscosity options. Recently, concerns have been raised regarding potential adverse health effects of mineral oils and waxes from dermal application of cosmetics. In order to be able to assess the risk for the consumer the dermal penetration potential of these ingredients has to be evaluated. The scope and objective of this review are to identify and summarize publicly available literature on the dermal penetration of mineral oils and waxes as used in cosmetic products. For this purpose, a comprehensive literature search was conducted. A total of 13 in vivo (human, animal) and in vitro studies investigating the dermal penetration of mineral oils and waxes has been identified and analysed. The majority of the substances were dermally adsorbed to the stratum corneum and only a minor fraction reached deeper skin layers. Overall, there is no evidence from the various studies that mineral oils and waxes are percutaneously absorbed and become systemically available. Thus, given the absence of dermal uptake, mineral oils and waxes as used in cosmetic products do not present a risk to the health of the consumer.

Fragmentation of pure and hydrated clusters of 5Br-uracil by low energy carbon ions: observation of hydrated fragments.

The fragmentation of the isolated 5-bromouracil (5BrU) molecule and pure and nano-hydrated 5BrU clusters induced by low energy 12C4+ ions has been studied. A comparison indicates that the environment, on the one hand, protects the system against the complete break-up into small fragments, but, on the other hand, triggers 'new' pathways for fragmentation, for example the loss of the OH group. The most striking result is the observation of several series of hydrated fragments in the hydrated cluster case, with water molecules bound to hydrophilic sites of 5BrU. This highlights the strong interaction between 5BrU and water molecules and the blocking of specific fragmentation pathways, such as the loss of the BrC2H group for example.

Multiple electron capture from isolated protein poly-anions in collision with slow highly charged ions.

Collisions of 375 keV Xe25+ ions with trapped mass/charge selected poly-anions of the cytochrome C protein (∼12.5 kDa) were studied by coupling a linear quadrupole ion trap with low-energy ion beam facility. Tandem mass spectra were recorded for the protein precursor charge states ranging from -9 to -17. The present work reports the first study of slow highly charged ion collisions with poly-anions. A high signal to noise ratio allowed the study of the intensity of single and multiple electron removal by a projectile, as well as associated neutral losses, as a function of the target charge state. Relative single and double electron detachment cross sections were found to increase with increasing charge state of the precursor anion. The experimental findings are supported by the calculations of the total electron capture cross sections, based on the classical over-the-barrier model, restricted to a simple uniformly charged linear protein structure and a near-end electron capture.

Determination of Energy-Transfer Distributions in Ionizing Ion-Molecule Collisions.

The ionization and fragmentation of the nucleoside thymidine in the gas phase has been investigated by combining ion collision with state-selected photoionization experiments and quantum chemistry calculations. The comparison between the mass spectra measured in both types of experiments allows us to accurately determine the distribution of the energy deposited in the ionized molecule as a result of the collision. The relation of two experimental techniques and theory shows a strong correlation between the excited states of the ionized molecule with the computed dissociation pathways, as well as with charge localization or delocalization.

Soluble Vascular Cell Adhesion Molecule-1 (VCAM-1) as a Biomarker in the Mouse Model of Experimental Autoimmune Myocarditis (EAM).

Vascular cell adhesion molecule-1 (VCAM-1) is strongly upregulated in hearts of mice with coxsackie virus-induced as well as in patients with viral infection-triggered dilated cardiomyopathy. Nevertheless, the role of its soluble form as a biomarker in inflammatory heart diseases remains unclear. Therefore, we investigated whether plasma levels of soluble VCAM-1 (sVCAM-1) directly correlated with disease activity and progression of cardiac dysfunction in the mouse model of experimental autoimmune myocarditis (EAM). EAM was induced by immunization of BALB/c mice with heart-specific myosin-alpha heavy chain peptide together with complete Freund`s adjuvant. ELISA revealed strong expression of cardiac VCAM-1 (cVCAM-1) throughout the course of EAM in immunized mice compared to control animals. Furthermore, sVCAM-1 was elevated in the plasma of immunized compared to control mice at acute and chronic stages of the disease. sVCAM-1 did not correlate with the degree of acute cardiac inflammation analyzed by histology or cardiac cytokine expression investigated by ELISA. Nevertheless, heart to body weight ratio correlated significantly with sVCAM-1 at chronic stages of EAM. Cardiac systolic dysfunction studied with positron emission tomography indicated a weak relationship with sVCAM-1 at the chronic stage of the disease. Our data provide evidence that plasma levels of sVCAM-1 are elevated throughout all stages of the disease but showed no strong correlation with the severity of EAM.

Retrospective analysis of circulatory support with the Impella CP® device in patients with therapy refractory cardiogenic shock.

BACKGROUND: In cardiogenic shock (CS) the Impella CP® device provides a fast available left ventricular circulatory support of up to 4.0L/min. However, the use of the Impella CP® device was not systematically analysed yet. METHODS: We performed a retrospective analysis of 28 consecutive patients suffering from severe therapy refractory CS treated with Impella CP®. Mortality was estimated using the SAPS II-Score. Primary outcome was 30-day survival. We compared the different aetiologies of CS and the effect of additional extracorporeal life support (ECLS). RESULTS: Aetiology of CS was acute coronary syndrome (ACS) in 15 patients, 9 patients received additional therapy with ECLS. SAPS II was 73±14, representing an estimated mortality of 87.1%. 18 patients deceased representing a 30-day survival of 36%. Comparing the different aetiologies, ACS-CS patients show a trend towards better survival. Additional therapy with ECLS did not change 30-day survival. In 3 cases, vascular complication needing surgical treatment occurred. All other patients showed no relevant complications except for the commonly seen haemolysis with consecutive need of transfusion. CONCLUSION: Our data could demonstrate that the Impella CP® application in these severely diseased patients is feasible and safe. Compared to the estimated mortality, the 30-day survival seems to be improved.

Blast exposure causes dynamic microglial/macrophage responses and microdomains of brain microvessel dysfunction.

Exposure to blast overpressure (BOP) is associated with behavioral, cognitive, and neuroimaging abnormalities. We investigated the dynamic responses of cortical vasculature and its relation to microglia/macrophage activation in mice using intravital two-photon microscopy following mild blast exposure. We found that blast caused vascular dysfunction evidenced by microdomains of aberrant vascular permeability. Microglial/macrophage activation was specifically associated with these restricted microdomains, as evidenced by rapid microglial process retraction, increased ameboid morphology, and escape of blood-borne Q-dot tracers that were internalized in microglial/macrophage cell bodies and phagosome-like compartments. Microdomains of cortical vascular disruption and microglial/macrophage activation were also associated with aberrant tight junction morphology that was more prominent after repetitive (3×) blast exposure. Repetitive, but not single, BOPs also caused TNFα elevation two weeks post-blast. In addition, following a single BOP we found that aberrantly phosphorylated tau rapidly accumulated in perivascular domains, but cleared within four hours, suggesting it was removed from the perivascular area, degraded, and/or dephosphorylated. Taken together these findings argue that mild blast exposure causes an evolving CNS insult that is initiated by discrete disturbances of vascular function, thereby setting the stage for more protracted and more widespread neuroinflammatory responses.

Intracellular LINGO-1 negatively regulates Trk neurotrophin receptor signaling.

Neurotrophins, essential regulators of many aspects of neuronal differentiation and function, signal via four receptors, p75, TrkA, TrkB and TrkC. The three Trk paralogs are members of the LIG superfamily of membrane proteins, which share extracellular domains consisting of leucine-rich repeat and C2 Ig domains. Another LIG protein, LINGO-1 has been reported to bind and influence signaling of p75 as well as TrkA, TrkB and TrkC. Here we examine the manner in which LINGO-1 influences the function of TrkA, TrkB and TrkC. We report that Trk activation promotes Trk association with LINGO-1, and that this association promotes Trk degradation by a lysosomal mechanism. This mechanism resembles the mechanism by which another LIG protein, LRIG1, promotes lysosomal degradation of receptor tyrosine kinases such as the EGF receptor. We present evidence indicating that the Trk/LINGO-1 interaction occurs, in part, within recycling endosomes. We show that a mutant form of LINGO-1, with much of the extracellular domain deleted, has the capacity to enhance TrkA signaling in PC12 cells, possibly by acting as an inhibitor of Trk down-regulation by full length LINGO-1. We propose that LINGO-1 functions as a negative feedback regulator of signaling by cognate receptor tyrosine kinases including TrkA, TrkB and TrkC.

Mobilisation of haemopoietic stem cells in teriparatide-treated patients.

Parathyroid hormone (PTH) is the predominant regulator of calcium/phosphate homeostasis in the human body. Beside this classical function, preclinical and clinical studies indicated a relevant role for PTH in mobilisation of bone marrow-derived cells into peripheral blood. In addition, recombinant PTH (teriparatide) was recently approved for the treatment of severe osteoporosis. Therefore, it was the aim of the present study to investigate the dynamics of haemopoietic stem cells and corresponding in peripheral blood of 13 patients with osteoporosis during treatment with teriparatide. We were able to show that administration of teriparatide is sufficient to mobilise haemopoietic stem cells into the bloodstream accompanied by an alteration of mobilising cytokines. In conclusion, teriparatide might be a useful tool in the context of stem cell mobilisation.

The diversification of Heliconius butterflies: what have we learned in 150 years?

Research into Heliconius butterflies has made a significant contribution to evolutionary biology. Here, we review our understanding of the diversification of these butterflies, covering recent advances and a vast foundation of earlier work. Whereas no single group of organisms can be sufficient for understanding life's diversity, after years of intensive study, research into Heliconius has addressed a wide variety of evolutionary questions. We first discuss evidence for widespread gene flow between Heliconius species and what this reveals about the nature of species. We then address the evolution and diversity of warning patterns, both as the target of selection and with respect to their underlying genetic basis. The identification of major genes involved in mimetic shifts, and homology at these loci between distantly related taxa, has revealed a surprising predictability in the genetic basis of evolution. In the final sections, we consider the evolution of warning patterns, and Heliconius diversity more generally, within a broader context of ecological and sexual selection. We consider how different traits and modes of selection can interact and influence the evolution of reproductive isolation.