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The presence of bacteria carrying antimicrobial resistance (AMR) genes in wildlife is an indicator that resistant bacteria of human or livestock origin are widespread in the environment. In addition, it could represent an additional challenge for human health, since wild animals could act as efficient AMR reservoirs and epidemiological links between human, livestock and natural environments. The aim of this study was to investigate the occurrence and the antibiotic resistance patterns of several bacterial species in certain wild animals in Germany, including wild boars ( Sus scrofa ), roe deer ( Capreolus capreolus ) and wild ducks (family Anatidae, subfamily Anatinae) and geese (family Anatidae, subfamily Anserinae). In the framework of the German National Zoonoses Monitoring Program, samples from hunted wild boars, roe deer and wild ducks and geese were collected nationwide in 2016, 2017, and 2019, respectively. Fecal samples were tested for the presence of Salmonella spp. (in wild boars and wild ducks and geese), Campylobacter spp. (in roe deer and wild ducks and geese), Shiga toxin-producing Escherichia ( E .) coli (STEC), commensal E. coli and extended-spectrum beta-lactamase- (ESBL) or ampicillinase class C (AmpC) beta-lactamase-producing E. coli (in wild boars, roe deer and wild ducks and geese). In addition, the presence of methicillin-resistant Staphylococcus aureus (MRSA) was investigated in nasal swabs from wild boars. Isolates obtained in the accredited regional state laboratories were submitted to the National Reference Laboratories (NRLs) for confirmation, characterization and phenotypic resistance testing using broth microdilution according to CLSI. AMR was assessed according to epidemiological cut-offs provided by EUCAST. Salmonella spp. were isolated from 13 of 552 (2.4%) tested wild boar fecal samples, but absent in all 101 samples from wild ducks and geese. Nine of the 11 isolates that were submitted to the NRL Salmonella were susceptible to all tested antimicrobial substances. Campylobacter spp. were isolated from four out of 504 (0.8%) roe deer fecal samples, but not from any of the samples from wild ducks and geese. Of the two isolates received in the NRL Campylobacter , neither showed resistance to any of the substances tested. From roe deer, 40.2% of the fecal samples (144 of 358) yielded STEC compared to 6.9% (37 of 536) from wild boars. In wild ducks and geese, no STEC isolates were found. Of 150 STEC isolates received in the NRL (24 from wild boars and 126 from roe deer), only one from each animal species showed resistance. Of the 219 isolates of commensal E. coli from wild boars tested for AMR, 210 were susceptible to all 14 tested substances (95.9%). In roe deer this proportion was even higher (263 of 269, 97.8%), whereas in wild ducks and geese this proportion was lower (41 of 49, 83.7%). Nevertheless, selective isolation of ESBL-/AmpC-producing E. coli yielded 6.5% (36 of 551) positive samples from wild boars, 2.3% (13 of 573) from roe deer and 9.8% (10 of 102) from wild ducks and geese. Among the 25 confirmed ESBL-/AmpC-producing isolates from wild boars, 14 (56.0%) showed resistance up to five classes of substances. This proportion was lower in roe deer (3 of 12, 25%) and higher in wild ducks and geese (7 of 10, 70%). None of the 577 nasal swabs from wild boars yielded MRSA. Results indicate that overall, the prevalence of resistant bacteria from certain wild animals in Germany is low, which may reflect not only the low level of exposure to antimicrobials but also the low level of resistant bacteria in the areas where these animals live and feed. However, despite this low prevalence, the patterns observed in bacteria from the wild animals included in this study are an indicator for specific resistance traits in the environment, including those to highest priority substances such as 3rd generation cephalosporins, fluoroquinolones and colistin. Therefore, also continuous monitoring of the occurrence of such bacteria in wildlife by selective isolation is advisable. Furthermore, the possible role of wildlife as reservoir and disperser of resistant bacteria would need to be assessed, as wild animals, and in particular wild ducks and geese could become spreaders of resistant bacteria given their capacity for long-range movements., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Plaza-Rodríguez, Alt, Grobbel, Hammerl, Irrgang, Szabo, Stingl, Schuh, Wiehle, Pfefferkorn, Naumann, Kaesbohrer and Tenhagen.)

The objective of this study was to compare the prevalence and antimicrobial resistance (AMR) rates of Campylobacter spp. isolated from conventional and organic turkey meat sold at retail in Germany. Samples of conventional ( N  = 527) and organic ( N  = 245) fresh turkey meat without skin were collected at retail markets throughout Germany and tested for Campylobacter spp.. Campylobacter isolates were tested for resistance to six antimicrobials (gentamicin, streptomycin, ciprofloxacin, nalidixic acid, erythromycin, and tetracycline) using broth microdilution. Prevalence of Campylobacter spp. was higher in organic (32.7%) than in conventional (19.4%) turkey meat. The proportion of fully susceptible isolates was lower in Campylobacter coli (6.8%) than in Campylobacter jejuni (33.9%) and higher in isolates from organic (38.4%) than from conventional production (17.4%). Overall, resistance rates were the highest to ciprofloxacin, nalidixic acid, and tetracycline. Resistance to erythromycin was only observed in C. coli and resistance to gentamicin was absent. Overall, resistance rates to tetracycline and fluoroquinolones were higher in isolates from conventional (60.9% and 78.9%) than from organic meat (32.9% and 58.9%, respectively). However, this significant difference was only observed for C. jejuni , but not for C. coli . Further studies are needed to identify the reasons for the differences in the association of production type of turkeys with AMR in the different Campylobacter spp. and the critical parameters for the reduction of AMR in Campylobacter from turkey meat.

Mitochondrial dysfunction is known to play a key role in the pathophysiological pathway of neurodegenerative disorders. Nuclear-encoded proteins are involved in mtDNA replication, including DNA polymerase gamma, which is the only known replicative mtDNA polymerase, encoded by nuclear genes Polymerase gamma 1 (POLG) and Polymerase gamma 2 (POLG2). POLG mutations are well-known as a frequent cause of mitochondrial myopathies of nuclear origin. However, only rare descriptions of POLG2 mutations leading to mitochondriopathies exist. Here we describe a 68-year-old woman presenting with a 20-year history of camptocormia, mild proximal weakness, and moderate CK increase. Muscle histology showed COX-negative fibres. Genetic analysis by next generation sequencing revealed an already reported heterozygous c.1192-8_1207dup24 mutation in the POLG2 gene. This is the first report on a POLG2 mutation leading to camptocormia as the main clinical phenotype, extending the phenotypic spectrum of POLG2 associated diseases. This underlines the broad phenotypic spectrum found in mitochondrial diseases, especially in mitochondrial disorders of nuclear origin., Competing Interests: The authors declare no conflict of interest.

Drug carriers typically require both stealth and targeting properties to minimize nonspecific interactions with healthy cells and increase specific interaction with diseased cells. Herein, the assembly of targeted poly(ethylene glycol) (PEG) particles functionalized with cyclic peptides containing Arg-Gly-Asp (RGD) (ligand) using a mesoporous silica templating method is reported. The influence of PEG molecular weight, ligand-to-PEG molecule ratio, and particle size on cancer cell targeting to balance stealth and targeting of the engineered PEG particles is investigated. RGD-functionalized PEG particles (PEG-RGD particles) efficiently target U-87 MG cancer cells under static and flow conditions in vitro, whereas PEG and cyclic peptides containing Arg-Asp-Gly (RDG)-functionalized PEG (PEG-RDG) particles display negligible interaction with the same cells. Increasing the ligand-to-PEG molecule ratio improves cell targeting. In addition, the targeted PEG-RGD particles improve cell uptake via receptor-mediated endocytosis, which is desirable for intracellular drug delivery. The PEG-RGD particles show improved tumor targeting (14% ID g -1 ) when compared with the PEG (3% ID g -1 ) and PEG-RDG (7% ID g -1 ) particles in vivo, although the PEG-RGD particles show comparatively higher spleen and liver accumulation. The targeted PEG particles represent a platform for developing particles aimed at balancing nonspecific and specific interactions in biological systems.

MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L -1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L -1 , pointing towards the potential of miRNA KO for cell line engineering., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for example, in nanomedicine. Here, the synthesis of functional phosphonate-ester-bearing brush polymers based on poly(2-oxazine)s is reported through a combination of cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazine and reversible addition-fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well-defined (Đ ≤ 1.17) poly(oligo(2-ethyl-2-oxazine) methacrylate) P(OEtOzMA) n brushes with tunable lower critical solution temperature (LCST) behavior and negligible cell toxicity is prepared. Upon deprotection, the phosphonic acid end-group of the P(OEtOzMA) n brush enables the successful grafting-onto iron oxide nanoparticles (IONPs). Colloidal stability of the particle suspension in combination with suitable magnetic resonance imaging (MRI) relaxivities demonstrates the potential of these particles for future applications as negative MRI contrast agents., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We assembled genome-wide data from 271 ancient Iberians, of whom 176 are from the largely unsampled period after 2000 BCE, thereby providing a high-resolution time transect of the Iberian Peninsula. We document high genetic substructure between northwestern and southeastern hunter-gatherers before the spread of farming. We reveal sporadic contacts between Iberia and North Africa by ~2500 BCE and, by ~2000 BCE, the replacement of 40% of Iberia's ancestry and nearly 100% of its Y-chromosomes by people with Steppe ancestry. We show that, in the Iron Age, Steppe ancestry had spread not only into Indo-European-speaking regions but also into non-Indo-European-speaking ones, and we reveal that present-day Basques are best described as a typical Iron Age population without the admixture events that later affected the rest of Iberia. Additionally, we document how, beginning at least in the Roman period, the ancestry of the peninsula was transformed by gene flow from North Africa and the eastern Mediterranean., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFv anti-GPIIb/IIIa . Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

Methicillin-resistant Staphylococcus aureus (MRSA) have been described repeatedly in dairy herds. In this study, we compared the prevalence and antimicrobial resistance of MRSA in bulk tank milk from conventional and organic dairy herds in Germany. Samples were collected from 372 conventional and 303 organic dairy herds throughout Germany. Bulk tank milk (25 mL) was tested for MRSA using an established double selective enrichment method. The MRSA isolates were typed using spa typing and tested for resistance to 19 antimicrobials using the broth microdilution method. Methicillin-resistant Staph. aureus was detected more frequently in bulk tank milk from conventional (9.7%) than from organic (1.7%) dairy herds. Herd size and region were associated with differences in prevalence. Most isolates (38/41) were from spa types associated with the livestock-associated clonal complex CC398. Isolates from conventional herds tended to be more resistant to antimicrobials; however, because of the limited number of isolates from organic herds, no statistical tests were performed. In conclusion, prevalence of MRSA in dairy herds in Germany seems to be increasing and is more prevalent in regions with high livestock density. Organic herds are also affected although at a lower level. Therefore, MRSA should be specifically included in biosecurity protocols for dairy herds, and effective control measures need to be investigated., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).)

Protecting the heart after an acute coronary syndrome is a key therapeutic goal to support cardiac recovery and prevent progression to heart failure. A potential strategy is to target cardiac glucose metabolism at the early stages after ischemia when glycolysis is critical for myocyte survival. Building on our discovery that high-density lipoprotein (HDL) modulates skeletal muscle glucose metabolism, we now demonstrate that a single dose of reconstituted HDL (rHDL) delivered after myocardial ischemia increases cardiac glucose uptake, reduces infarct size, and improves cardiac remodeling in association with enhanced functional recovery in mice. These findings applied equally to metabolically normal and insulin-resistant mice. We further establish direct effects of HDL on cardiomyocyte glucose uptake, glycolysis, and glucose oxidation via the Akt signaling pathway within 15 min of reperfusion. These data support the use of infusible HDL preparations for management of acute coronary syndromes in the setting of primary percutaneous interventions., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Therapeutic nanoparticles hold clinical promise for cancer treatment by avoiding limitations of conventional pharmaceuticals. Herein, a facile and rapid method is introduced to assemble poly(ethylene glycol) (PEG)-modified Pt prodrug nanocomplexes through metal-polyphenol complexation and combined with emulsification, which results in ≈100 nm diameter nanoparticles (PtP NPs) that exhibit high drug loading (0.15 fg Pt per nanoparticle) and low fouling properties. The PtP NPs are characterized for potential use as cancer therapeutics. Mass cytometry is used to quantify uptake of the nanoparticles and the drug concentration in individual cells in vitro. The PtP NPs have long circulation times, with an elimination half-life of ≈18 h in healthy mice. The in vivo antitumor activity of the PtP NPs is systematically investigated in a human prostate cancer xenograft mouse model. Mice treated with the PtP NPs demonstrate four times better inhibition of tumor growth than either free prodrug or cisplatin. This study presents a promising strategy to prepare therapeutic nanoparticles for biomedical applications., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Magnetic resonance imaging (MRI) is a powerful and indispensable tool in medical research, clinical diagnosis, and patient care due to its high spatial resolution and non-limited penetration depth. The simultaneous use of positive and negative MRI imaging that employs the same contrast agents will significantly improve detection accuracy. Here we report the development of functional multimodal iron oxide nanoparticles for targeted MRI of atherothrombosis using a combination of chemical and biological conjugation techniques. Monodisperse, water-soluble and biocompatible ultra-small magnetic dual contrast iron oxide nanoparticles (DCIONs) were generated using a high-temperature co-precipitation route and appeared to be efficient positive and negative dual contrast agents for magnetic resonance imaging. Using a unique chemo-enzymatic approach involving copper-free click chemistry and Staphylococcus aureus sortase A enzyme conjugation, DCIONs were functionalized with single-chain antibodies (scFv) directed against activated platelets for targeting purposes. The DCIONs were also labelled with fluorescent molecules to allow for optical imaging. The antigen binding activity of the scFv was retained and resulted in the successful targeting of contrast agents to thrombosis as demonstrated in a range of in vitro and in vivo experiments. T 1 - and T 2 -weighted MRI of thrombi was recorded and demonstrated the great potential of dual T 1 /T 2 contrast iron oxide particles in imaging of cardiovascular disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Thrombolytic therapy for acute thrombosis is limited by life-threatening side effects such as major bleeding and neurotoxicity. New treatment options with enhanced fibrinolytic potential are therefore required. Here, we report the development of a new thrombolytic molecule that exploits key features of thrombosis. We designed a recombinant microplasminogen modified to be activated by the prothrombotic serine-protease thrombin (HtPlg), fused to an activation-specific anti-glycoprotein IIb/IIIa single-chain antibody (SCE5), thereby hijacking the coagulation system to initiate thrombolysis., Methods and Results: The resulting fusion protein named SCE5-HtPlg shows in vitro targeting towards the highly abundant activated form of the fibrinogen receptor glycoprotein IIb/IIIa expressed on activated human platelets. Following thrombin formation, SCE5-HtPlg is activated to contain active microplasmin. We evaluate the effectiveness of our targeted thrombolytic construct in two models of thromboembolic disease. Administration of SCE5-HtPlg (4 μg/g body weight) resulted in effective thrombolysis 20 minutes after injection in a ferric chloride-induced model of mesenteric thrombosis (48±3% versus 92±5% for saline control, P<0.01) and also reduced emboli formation in a model of pulmonary embolism (P<0.01 versus saline). Furthermore, at these effective therapeutic doses, the SCE5-HtPlg did not prolong bleeding time compared with saline (P=0.99)., Conclusions: Our novel fusion molecule is a potent and effective treatment for thrombosis that enables in vivo thrombolysis without bleeding time prolongation. The activation of this construct by thrombin generated within the clot itself rather than by a plasminogen activator, which needs to be delivered systemically, provides a novel targeted approach to improve thrombolysis., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

A reliable method for the diagnosis of minimal cardiac ischemia would meet a strong demand for the sensitive diagnosis of coronary artery disease in cardiac stress testing and risk stratification in patients with chest pain but unremarkable ECGs and biomarkers. We hypothesized that platelets accumulate early on in ischemic myocardium and a newly developed technology of non-invasive molecular PET imaging of activated platelets can thus detect minimal degrees of myocardial ischemia. To induce different degrees of minimal cardiac ischemia, the left anterior descending artery (LAD) was ligated for 10, 20 or 60 min. Mice were injected with a newly generated scFv anti-GPIIb/IIIa - 64 CuMeCOSar radiotracer, composed of a single-chain antibody that only binds to activated integrin GPIIb/IIIa (α IIb β III ) and thus to activated platelets, and a sarcophagine cage MeCOSar complexing the long half-life PET tracer copper-64. A single PET/CT scan was performed. Evans Blue/TTC staining to detect necrosis as well as classical serological biomarkers like Troponin I and heart-type fatty acid-binding protein (H-FABP) were negative, whereas PET imaging of activated platelets was able to detect small degrees of ischemia. Taken together, molecular PET imaging of activated platelets represents a unique and highly sensitive method to detect minimal cardiac ischemia., Competing Interests: K.P. is inventor on patents describing activated platelet-targeting recombinant antibodies. P.S.D. is inventor on patents describing sarcophagine chelators for positron emission tomography.

Targeted polymer capsules can selectively bind to unstable plaques in mice after intravenous injection. Different formulations of the capsules are explored with a synthetic/biopolymer hybrid capsule showing the best stability and small-molecule drug retention. The synthetic polymer is composed of pH-sensitive blocks (PDPA), low-binding blocks (PEG), and click-groups for postfunctionalization with targeting peptides specific to plaques., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Smart poly(2-oxazoline) (POx)-based multifunctional polymer capsules that specifically target glycoprotein (GP) IIb/IIIa on the surface of activated platelets are degraded by the serine protease thrombin and release the urokinase plasminogen activator loaded into the polymer capsules, only in the area of acute thrombosis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

Dual-responsive boronate-phenolic network (BPN) capsules are fabricated by the complexation of phenylborate and phenolic materials. The BPN capsules are stable in the presence of competing carbohydrates, but dissociate at acidic pH or in the presence of competing cis-diols at physiological pH. This engineered capsule system provides a platform for a wide range of biological and biomedical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: In January 2013, the National Reference Centre for Salmonella (NRC) detected a salmonellosis cluster in Saxony-Anhalt, Germany, caused by uncommon O4 non-agglutinating, monophasic Salmonella (S.) Typhimurium DT193. Circulating predominant monophasic S. Typhimurium DT193 clones typically display resistance phenotype ASSuT. We investigated common exposures to control the outbreak, and conducted microbiological investigations to assess the strains' phenotype., Methods: We conducted a case-control study defining cases as persons living or working in Saxony-Anhalt diagnosed with the O4 non-agglutinating strain between January and March 2013. We selected two controls contemporarily reported with norovirus infection, frequency-matched on residence and age group, per case. We interviewed regarding food consumption, especially pork and its place of purchase. We calculated odds ratios (ORs) with 95% confidence intervals (95% CI) using logistic regression. The NRC investigated human and food isolates by PCR, SDS-PAGE, MLST, PFGE, MLVA and susceptibility testing., Results: Altogether, 68 O4 non-agglutinating human isolates were confirmed between January and April 2013. Of those, 61 were assigned to the outbreak (median age 57 years, 44% female); 83% cases ≥ 60 years were hospitalized. Eating raw minced pork from butcheries within 3 days was associated with disease (31 cases, 28 controls; OR adjusted for sex: 3.6; 95% CI: 1.0-13). Phage type DT193 and MLST ST34 were assigned, and isolates' lipopolysaccharide (LPS) matched control strains. Isolates linked to Saxony-Anhalt exhibited PFGE type 5. ASSuT- and ACSSuT phenotype proportions were 34 and 39% respectively; 54% were resistant to chloramphenicol. Three pork isolates matched the outbreak strain., Discussion: Raw minced pork was the most likely infection vehicle in this first reported outbreak caused by O4 non-agglutinating, mostly chloramphenicol-resistant S. Typhimurium DT193. High hospitalization proportions demand awareness on the risk of consumption of raw pork among elderly. LPS analysis indicated O4 expression; therefore, testing with antisera from different lots is recommendable in unexpected agglutination reactions.

We report the engineering of poly(ethylene glycol) (PEG) hydrogel particles using a mesoporous silica (MS) templating method via tuning the PEG molecular weight, particle size, and the presence or absence of the template and investigate the cell association and biodistribution of these particles. An ex vivo assay based on human whole blood that is more sensitive and relevant than traditional cell-line based assays for predicting in vivo circulation behavior is introduced. The association of MS@PEG particles (template present) with granulocytes and monocytes is higher compared with PEG particles (template absent). Increasing the PEG molecular weight (from 10 to 40 kDa) or decreasing the PEG particle size (from 1400 to 150 nm) reduces phagocytic blood cell association of the PEG particles. Mice biodistribution studies show that the PEG particles exhibit extended circulation times (>12 h) compared with the MS@PEG particles and that the retention of smaller PEG particles (150 nm) in blood, when compared with larger PEG particles (>400 nm), is increased at least 4-fold at 12 h after injection. Our findings highlight the influence of unique aspects of polymer hydrogel particles on biological interactions. The reported PEG hydrogel particles represent a new class of polymer carriers with potential biomedical applications.

Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.

Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.

The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metal-organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli-responsiveness. Previous studies have primarily focused on the assembly aspects of metal-coordination capsules; however, the engineering of metal-specific functionality for capsule design has not been explored. A library of functional metal-phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a diverse range of functional materials can be generated., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The prevalence of MRSA in the turkey meat production chain in Germany was estimated within the national monitoring for zoonotic agents in 2010. In total 22/112 (19.6%) dust samples from turkey farms, 235/359 (65.5%) swabs from turkey carcasses after slaughter and 147/460 (32.0%) turkey meat samples at retail were tested positive for MRSA. The specific distributions of spa types, SCCmec types and antimicrobial resistance profiles of MRSA isolated from these three different origins were compared using chi square statistics and the proportional similarity index (Czekanowski index). No significant differences between spa types, SCCmec types and antimicrobial resistance profiles of MRSA from different steps of the German turkey meat production chain were observed using Chi-Square test statistics. The Czekanowski index which can obtain values between 0 (no similarity) and 1 (perfect agreement) was consistently high (0.79-0.86) for the distribution of spa types and SCCmec types between the different processing stages indicating high degrees of similarity. The comparison of antimicrobial resistance profiles between the different process steps revealed the lowest Czekanowski index values (0.42-0.56). However, the Czekanowski index values were substantially higher than the index when isolates from the turkey meat production chain were compared to isolates from wild boar meat (0.13-0.19), an example of a separated population of MRSA used as control group. This result indicates that the proposed statistical method is valid to detect existing differences in the distribution of the tested characteristics of MRSA. The degree of similarity in the distribution of spa types, SCCmec types and antimicrobial resistance profiles between MRSA isolates from different process stages of turkey meat production may reflect MRSA transmission along the chain.

Rationale: Fibrinolysis is a valuable alternative for the treatment of myocardial infarction when percutaneous coronary intervention is not available in a timely fashion. For acute ischemic stroke, fibrinolysis is the only treatment option with a very narrow therapeutic window. Clinically approved thrombolytics have significant drawbacks, including bleeding complications. Thus their use is highly restricted, leaving many patients untreated., Objective: We developed a novel targeted fibrinolytic drug that is directed against activated platelets., Methods and Results: We fused single-chain urokinase plasminogen activator (scuPA) to a small recombinant antibody (scFvSCE5), which targets the activated form of the platelet-integrin glycoprotein IIb/IIIa. Antibody binding and scuPA activity of this recombinant fusion protein were on par with the parent molecules. Prophylactic in vivo administration of scFvSCE5-scuPA (75 U/g body weight) prevented carotid artery occlusion after ferric chloride injury in a plasminogen-dependent process compared with saline (P<0.001), and blood flow recovery was similar to high-dose nontargeted urokinase (500 U/g body weight). Tail bleeding time was significantly prolonged with this high dose of nontargeted urokinase, but not with equally effective targeted scFvSCE5-scuPA at 75 U/g body weight. Real-time in vivo molecular ultrasound imaging demonstrates significant therapeutic reduction of thrombus size after administration of 75 U/g body weight scFvSCE5-scuPA as compared with the same dose of a mutated, nontargeting scFv-scuPA or vehicle. The ability of scFvSCE5-scuPA to lyse thrombi was lost in plasminogen-deficient mice, but could be restored by intravenous injection of plasminogen., Conclusions: Targeting of scuPA to activated glycoprotein IIb/IIIa allows effective thrombolysis and the potential novel use as a fibrinolytic agent for thromboprophylaxis without bleeding complications.

Introduction: Activated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel (18)F PET radiotracer based on this antibody., Methods: ScFv(anti-LIBS) and control antibody mut-scFv were reacted with N-succinimidyl-4-[(18)F]fluorobenzoate (S[(18)F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl(3) induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied., Results: After incubation with increasing concentrations of (18)F-scFv(anti-LIBS) clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled (18)F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6)., Conclusions: Our results show that the novel antibody radiotracer (18)F-scFv(anti-LIBS) is useful for the sensitive detection of activated platelets and thrombosis., Advances in Knowledge and Implications for Patient Care: We describe the first (18)F variant of a scFv(anti-LIBS) against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Campylobacter is a poorly recognized foodborne pathogen, leading the statistics of bacterially caused human diarrhoea in Europe during the last years. In this review, we present qualitative and quantitative German data obtained in the framework of specific monitoring programs and from routine surveillance. These also comprise recent data on antimicrobial resistances of food isolates. Due to the considerable reduction of in vitro growth capabilities of stressed bacteria, there is a clear discrepancy between the detection limit of Campylobacter by cultivation and its infection potential. Moreover, antimicrobial resistances of Campylobacter isolates established during fattening of livestock are alarming, since they constitute an additional threat to human health. The European Food Safety Authority (EFSA) discusses the establishment of a quantitative limit for Campylobacter contamination of broiler carcasses in order to achieve an appropriate level of protection for consumers. Currently, a considerable amount of German broiler carcasses would not comply with this future criterion. We recommend Campylobacter reduction strategies to be focussed on the prevention of fecal contamination during slaughter. Decontamination is only a sparse option, since the reduction efficiency is low and its success depends on the initial contamination concentration.

Background: For redirecting T-lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single-chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T-cell receptor (TCR)-associated CD3 molecule on T-cells., Methods: The PSMA × CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model., Results: Specific binding of the PSMA × CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA × CD3 bsc diabody proved to be a potent agent for retargeting CD4+ and CD8+ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth., Conclusions: The PSMA × CD3 bsc diabody bears a high potential for the immunotherapy of prostate cancer., (Copyright © 2010 Wiley-Liss, Inc.)

Aim: The goal of the present study was to test the (177)Lu-labelled anti-PSMA monoclonal antibody 3/F11 ((177)Lu-DOTA-3/F11) as a new radioimmunotherapeutic agent in a prostate cancer SCID mouse xenograft model., Materials and Methods: The mAb 3/F11 was (177)Lu labelled using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as chelating agent. DOTA-3/F11 was tested for cell binding and serum immunoreactivity by flow cytometry. The biodistribution and the therapeutic efficacy of (177)Lu-DOTA-3/F11 in mice bearing PSMA-positive C4-2 prostate cancer xenografts were evaluated., Results: 3/F11 and DOTA-3/F11 showed high and specific cell binding and similar serum half-lives of approximately seven days. Biodistribution studies revealed an increasing tumour uptake of (177)Lu DOTA-3/F11 over time with maximum tumour-to-muscle and tumour-to-blood ratios after 72 h. A single dose of 1 MBq (177)Lu-DOTA-3/F11 inhibited tumour growth and prolonged survival., Conclusion: This study indicated that (177)Lu-DOTA-3/F11 may be a suitable radioimmunotherapeutic agent for the treatment of prostate cancer.

Background: The prostate specific membrane antigen (PSMA) is expressed by virtually all prostate cancers and represents an ideal target for diagnostic and therapeutic strategies. This article compares the in vivo behavior and tumor uptake of three different radiolabeled anti-PSMA monoclonal antibodies (mAbs) and corresponding F(ab)(2) and Fab fragments thereof., Methods: The mAbs 3/A12, 3/F11, and 3/E7 and fragments of 3/A12 were conjugated with the chelating agent DOTA and radiolabeled with 64Cu. For the microPET imaging studies, SCID mice bearing PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used. Each animal received 20-30 microg radiolabeled mAb or fragment corresponding to an activity of 8-14 MBq. Imaging was performed 3, 24, and 48 hr post-injection. After the last scan, mice were sacrificed and tracer in vivo biodistribution was measured by gamma-counting., Results: Static microPET images of mice with PSMA-positive tumors revealed a high uptake of the mAbs in the C4-2 tumors at 24 and 48 hr after tracer injection and only a minimal distribution in the DU 145 tumors and other organs. In contrast, the F(ab)(2) and Fab fragments of 3/A12 were detected at a high extend in the kidney but not in the C4-2 tumors. These results were confirmed by gamma counting of dissected organs after the final imaging., Conclusions: Due to the high and specific uptake of the 64Cu-labeled mAbs in PSMA-positive tumors, these antibodies represent excellent tools for prostate cancer imaging.

The prostate-specific membrane antigen (PSMA) is abundantly expressed on prostate cancer epithelial cells and its expression correlates with tumor progression. Therefore, a specific immunotherapy against this antigen may be a novel therapeutic option for the management of prostate cancer. We generated an anti-PSMA single-chain antibody fragment (scFv), called D7, by phage display from the monoclonal antibody 3/F11. By C-terminal ligation of the toxic domain of Pseudomonas Exotoxin A (PE40) to the genes of D7, the immunotoxin D7-PE40 was generated. D7 and D7-PE40 specifically bound to PSMA transfectants and to the PSMA expressing prostate cancer cell line C4-2. In addition, D7-PE40 showed a high serum stability and induced a 50% reduction of viability (IC50) in C4-2 cells at a concentration of 140 pM. In vivo, D7-PE40 was well tolerated in SCID mice up to a single dose of 20 microg, whereas higher doses induced severe hepatotoxicity with deaths of the animals. Immunotoxin treatment of mice bearing C4-2 tumor xenografts caused a significant inhibition of tumor growth, whereas mice with PSMA-negative DU 145 tumors remained unaffected. Owing to its high and specific cytotoxicity and its capability to inhibit prostate tumor growth in vivo the immunotoxin D7-PE40 represents a promising candidate for the immunotherapy of prostate cancer.

Background: The prostate specific membrane antigen (PSMA) represents an attractive antigen for antibody-based diagnostic and therapeutic intervention in prostate cancer, since it is highly restricted to the prostate and overexpressed in all tumor stages. The present work describes the in vitro characterization of the three anti-PSMA monoclonal antibodies (mAbs) 3/A12, 3/E7, and 3/F11 in comparison to the mAb J591., Methods: The mAbs were tested for saturation and competitive binding on C4-2 prostate cancer cells by flow cytometry. Immunohistochemical staining was conducted on frozen prostate normal and cancer tissues as well as on lymph node metastases. Similarly, potential crossreactivities were tested on a broad panel of human normal tissues., Results: The anti-PSMA mAbs showed a strong binding to C4-2 cells with mean half-maximal saturation concentrations of about 14 nM for 3/A12, 17 nM for 3/E7, 9 nM for 3/F11, and 16 nM for J591. Competitive binding studies revealed that our three mAbs bind to different extracellular PSMA epitopes. The mAbs showed comparable staining of epithelial cells for all tested normal and tumorous prostate tissues. Extraprostatic staining was observed on secretory cells of the salivary glands and on the brush border of the duodenal columnar epithelium. J591 additionally showed positive staining of the normal breast epithelium., Conclusions: Due to their specific binding characteristics, the anti-PSMA mAbs 3/A12, 3/E7, and 3/F11 show great promise for diagnostic and therapeutic applications in prostate cancer., ((c) 2009 Wiley-Liss, Inc.)

Unlabelled: Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is highly expressed by virtually all prostate cancers and is currently the focus of several diagnostic and therapeutic strategies. We have previously reported on the generation of several monoclonal antibodies (mAb) and antibody fragments that recognize and bind with high affinity to the extracellular domain of cell-adherent PSMA. This article reports the in vivo behavior and tumor uptake of the radiolabeled anti-PSMA mAb 3/A12 and its potential as a tracer for PET., Methods: The mAb 3/A12 was conjugated with the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and radiolabeled with (64)Cu. Severe combined immunodeficient mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were used for small-animal PET imaging. Mice with PSMA-negative DU 145 tumors served as controls. For PET studies, each animal received 20-30 microg of radiolabeled mAb corresponding to an activity of 7.6-11.5 MBq. Imaging was performed 3, 24, and 48 h after injection. After the last scan, the mice were sacrificed and tracer in vivo biodistribution was measured by gamma-counting., Results: Binding of the mAb 3/A12 on PSMA-expressing C4-2 cells was only minimally influenced by DOTA conjugation. The labeling efficiency using (64)Cu and DOTA-3/A12 was 95.3% +/- 0.3%. The specific activity after (64)Cu labeling was between 327 and 567 MBq/mg. After tracer injection, static small-animal PET images of mice with PSMA-positive tumors revealed a tumor-to-background ratio of 3.3 +/- 1.3 at 3 h, 7.8 +/- 1.4 at 24 h, and 9.6 +/- 2.7 at 48 h. In contrast, no significant tracer uptake occurred in the PSMA-negative DU 145 tumors. These results were confirmed by direct counting of tissues after the final imaging., Conclusion: Because of the high and specific uptake of (64)Cu-labeled mAb 3/A12 in PSMA-positive tumors, this ligand represents an excellent candidate for prostate cancer imaging and potentially for radioimmunotherapy.

Infections with extraintestinal avian pathogenic Escherichia coli (APEC) cause significant economic losses in the poultry industry worldwide. In a previous study we applied signature-tagged transposon mutagenesis and identified 28 virulence-associated genes in APEC strain IMT5155 (O2 : H5 : K1). One of them, yjjQ, encodes a putative transcriptional regulator whose function and role in pathogenesis are still unknown. In the present study, this mutant has been characterized. The yjjQ-defective mutant of IMT5155 (M18E10) was out-competed by the wild-type strain in vivo, and infection of chickens with this yjjQ mutant led to strongly reduced bacterial loads in several organs. Expression studies showed that transcription of yjjQ was significantly upregulated in M9 minimal medium. Correspondingly, the yjjQ mutant showed significantly reduced growth in M9 medium. Although the mutation could not be complemented, a yjjQ deletion mutant showed phenotypes similar to the transposon-generated mutant M18E10, whereas deletion and overexpression of the downstream gene bglJ did not cause a growth defect in M9. To identify virulence genes regulated by YjjQ, one- and two-dimensional protein gel electrophoresis was performed. The proteomic analysis revealed that in the yjjQ mutant M18E10 the expression of several genes involved in iron uptake was downregulated and some other genes were upregulated. The regulation of genes involved in iron uptake was shown to occur at the transcription level using real-time RT-PCR. Taking the results together, this functional analysis strongly suggests that YjjQ is a regulator involved in virulence of APEC by affecting iron uptake.

Background: Expression of the prostate specific membrane antigen (PSMA) is highly restricted to prostate epithelial cells. Therefore, toxin-based immunotherapy against this antigen may represent an alternative therapeutic option for prostate cancer. For these purposes, the effects of the recombinant anti-PSMA immunotoxin A5-PE40 on prostate tumor growth were investigated in vitro and in vivo., Methods: The in vitro binding and cytotoxicity of A5-PE40 were tested on the PSMA-expressing prostate cancer cell line C4-2 and on the PSMA-negative cell line DU145 by flow cytometry and WST assays. The binding of the immunotoxin to SCID mouse xenografts and to various mouse organs was examined by Western blot analysis. In vivo, the antitumor activity of the immunotoxin was tested by injecting A5-PE40 in mice bearing C4-2 or DU145 xenografts., Results: In vitro, a specific binding of A5-PE40 to C4-2 cells could be shown with a concentration-dependent cytotoxicity (IC(50) value=220 pM). In the next step, a specific binding of the immunotoxin to C4-2 xenografts could be demonstrated. In contrast, no binding on mouse organs expressing high homologous mouse PSMA was found. The treatment of mice with C4-2 tumors caused a significant inhibition of tumor growth in vivo, whereas DU145 xenografts remained totally unaffected., Conclusions: A5-PE40 represents a recombinant anti-PSMA immunotoxin with potent antitumor activity in mice bearing human prostate cancer xenograft tumors. Therefore, A5-PE40 could be a promising candidate for therapeutic applications in patients with prostate cancer.

We present an extensive ancient DNA analysis of mainly Neolithic cattle bones sampled from archaeological sites along the route of Neolithic expansion, from Turkey to North-Central Europe and Britain. We place this first reasonable population sample of Neolithic cattle mitochondrial DNA sequence diversity in context to illustrate the continuity of haplotype variation patterns from the first European domestic cattle to the present. Interestingly, the dominant Central European pattern, a starburst phylogeny around the modal sequence, T3, has a Neolithic origin, and the reduced diversity within this cluster in the ancient samples accords with their shorter history of post-domestic accumulation of mutation.

In this paper, we present a well-preserved isolated human molar found in 1986 in the Hunas cave ruin, south-east Bavaria. The tooth was located at the bottom of layer F2, which belongs to a long stratigraphic sequence comprising faunal remains as well as archaeological levels (Mousterian). A stalagmite from layer P at the base of the stratigraphic sequence was recently dated to 79.373+/-8.237 ka (base) and 76.872+/-9.686 ka (tip) by TIMS-U/Th (Stanford University). We identified the tooth as a right (possibly third) mandibular molar. Characteristic parameters such as crown and root morphology, fissure pattern, enamel thickness, occlusal and interproximal wear, dental dimensions and indices, and radiological features indicate that the Hunas molar represents the tooth of a Neanderthal. This is corroborated by both the palaeontological and archaeological findings (Mousterian) of layer F2.

A comprehensive classification of midfacial/craniofacial fractures, based on two- and three-dimensional computed tomography (2D and 3D-CT) is presented. We performed a postmortem analysis of 24 patients who had died from trauma with signs of craniofacial fractures, based on 2D and 3D-CT studies with pathoanatomical findings. In addition, CT findings for 100 patients with craniofacial injuries requiring an emergency CT were correlated with surgical findings and follow-up results. On the basis of the analysis of a total of 377 fractures a classification system is proposed. The system is based on the use of the AO/ASIF (Arbeitsgemeinschaft für Osteosynthesefragen/Association for the Study of Internal Fixation) scheme, defining three types (A, B, C), three groups within each type (e.g. A1, A2, A3) and three subgroups within each group (e.g. A1.1, A1.2, A1.3) with increasing severity from A1.1 (lowest) to C3.3 (highest). The craniofacial region is divided into three units: the lower midface (I), the upper midface (II) and the craniobasal-facial unit (III). Lateral and central fractures are also distinguished. Type A fractures are non-displaced fractures, type B are displaced fractures and type C are complex/defect fractures. Groups A1, B1 and C1 comprise fractures of an isolated unit; groups A2, B2 and C2, combined fractures without involvement of the skull base; and groups A3, B3 and C3 are those combined fractures with involvement of the skull base. A correlation between the severity of the fracture and (i). the number of posttraumatic functional limitations (Spearman rank test, correlation coefficient r=0.42), (ii). the need for bone grafting or dural plastic (r=0.39) and (iii). facial asymmetry (r=0.37), was observed. The proposed classification system allows standardised documentation of midfacial and craniofacial fractures, including those not precisely defined by the Le Fort classification scheme.

The origin and subsequent spread of the treponematoses, especially that of venereal syphilis, has been the subject of considerable scientific attention. Various theories were put forth and palaeopathological specimens were used for their validation in recent times. One influential contribution was the paper by Baker & Armelagos in 1988. Numerous new findings and results on both sides of the Atlantic call for a new evaluation of the available osseous material. A review of the recent literature leads to the suggestion of a worldwide distribution of non-venereal treponemal disease since the emergence of Homo and to a first epidemic outbreak of venereal syphilis in Europe of the late 15th and the early 16th century, which was a time of change and enormous sexual liberty. Old World specimens with pathological alterations attributed to venereal syphilis and dated to precolumbian times seem to invalidate the Columbian theory and call for a more differentiated analysis of the phenomenon of syphilis than a theory based on a single factor can provide. With the help of molecular methods which now allow a positive identification of Treponema pallidum pallidum, causative agent of venereal syphilis, in palaeopathological material, it seems possible to elucidate the matter of origin and spread of syphilis further and to evaluate previous diagnoses of treponemal disease.

Thirty-two human mandibles were marked with three typical fracture lines: a low condylar fracture, a fracture of the mandibular angle, and one through the mental foramen on each side. The mandibles were sectioned at the fixation sites of the miniplate screws. The thickness of the cortical layer was measured with a scaled magnifying glass at the points of anthropological interest and at the marked screw holes. The inferior cortical layer turned out to be thickest in the anterior area. In contrast, it was very thin at the mandibular angle, which might explain the higher complication rate in treating fractures in this region. The thickness of the lingual cortex increased up to the symphysis, whereas the buccal cortical layer showed a decline in size from the mandibular angle up to the chin. The cortical bone at the alveolar ridge was porous. The cortical supply for miniplate osteosynthesis at the condylus ranged from 1.1 mm up to 1.74 mm, which seems to be limited, but due to the small diameter of the condylus most common screws obtain additional anchorage in the inner cortex. The thickness of the cortical layer at the mandibular angle increased from 1.47 mm at the ramus up to 1.97 mm at the beginning of the corpus, reaching 2.14-2.38 mm for the lower plate at the mental foramen. The results for the upper plate were slightly lower.

Teeth are one of the most important factors in the identification of unknown cadavers. One of the most efficient methods the police have of publicizing a case is to publish the victim's dental x-rays of treatment records in the dental print media which dentists will often recognize and thus be able to contribute to the identification. To evaluate the efficiency of this procedure, 177 cases published between 1975 and 1995 were reviewed. The results show that only 3% of the cases were identified from recognition in the dental print media. A survey of 500 dentists was carried out to gauge the efficacy of the presentation of identification cases in their dental print media. According to these results a revised method of presentation needs to be developed to improve the efficiency of this odontoforensic publicity medium. New technologies could also open new avenues for forensic identification such as digital x-raying or online data transfer. The Internet itself could be useful regarding international cooperation of forensic odontologists in the identification of unknown corpses.

The length of the dental root and the diameter of the tooth neck are odontometric parameters which to date have rarely been used for the differentiation of the sexes. Teeth of 166 individuals from the early medieval burial ground of Eichstetten (south Germany) were available for examination. A correlation analysis was performed to show the possible dimensional interrelation of teeth which, however, applies only to the dental neck parameters. Using only diameters of the tooth neck and length of dental roots, it was possible to perform a new sex determination for 68 of the 166 individuals. It shows a good coincidence with a traditional sex determination. A traditional sex determination, performed in advance, as well as the archeological assignment of typical sex related grave goods were used for verification. For future sex determination based on teeth, examinations are recommended to be restricted to the six-year molar, the first upper premolar and, in particular, the canine. Also the dental neck diameter proved to be more useful than the crown diameter. Dental root lengths can be neglected. By comparing the results with those of a second Alamannic burial ground it could be established that discriminant analyses performed within the same population are highly congruent.

The purpose of this study was to determine the degree of bony asymmetry between the right (R) and left (L) mandibular condyles and rami. On 25 dry skulls, condylar height, breadth and length, as well as ramus height and breadth were assessed. On each skull the average of the three measurements on the right and left sides, respectively, were calculated with the corresponding reference points being redetermined for each measurement. The raw absolute differences magnitude of R - L as well as the relative absolute differences magnitude of R - L/R + L were computed. The relative absolute differences showed that asymmetries were of similar magnitude (between 1.20% and 2.58%) for all variables except for condylar height (11.06%). After taking three additional measurements with reference points chosen only once, the intra-individual measurement error, consisting of reproducibility of the reference points and accuracy in measuring the distance between the chosen points, was assessed with the help of a statistical variance component technique. Estimates of the 95% range of the absolute differences were computed. Between 60% and 88% of the measured differences were outside the predicted 95% range, thus much too large to be explained by random measurement error; hence, they were due to real individual asymmetries. We concluded that asymmetries of mandibular condyles and rami are part of the biologic variation of humans. It still remains unclear, however, when such asymmetries should be considered "unphysiological."

One hundred fifty years ago, Richard Owen published the first detailed monograph on the comparative anatomy of teeth entitled Odontography; or, A Treatise on the Comparative Anatomy of the Teeth; their Physiological Relations, Mode of Development, and Microscopic Structure, in the Vertebrate Animals. The treatise is considered to be the first fundamental work of odontology. The 150th anniversary of its publication is an appropriate time to rediscover the roots and development of this scientific discipline, as well as the life and work of its author.

In 1986 a paleolithic triple burial was discovered near Dolní Vĕstonice (Czech Republic). The occurrence of anatomic variants in all three skeletons gave rise to speculations that the buried individuals may have been closely related. To test this hypothesis the skeletons were submitted to a systematic kinship analysis based on odontologic and other non-metric traits. Statistical tests showed that the coincident occurrence of several rare traits in the individuals is highly unlikely to occur at random. This and further data included in the analysis therefore suggest that the three individuals buried together were genetically related and actually belonged to one family.

This paper reports PCR-based genotype distribution and allele frequencies of the locus HumFES/FPS (human c-fes/fps proto oncogene). For this study 300 unrelated Caucasoids of German ancestry living in Düsseldorf were analyzed. Seven of the eight alleles previously described and fourteen of the corresponding thirty-six diploid genotypes were detected. The proportions of heterozygotes and homozygotes observed and expected and the parameters of forensic interest, e.g. the mean paternity exclusion chance, were calculated. No deviations from the Hardy-Weinberg equilibrium were observed. A preliminary German data base (3,238 individuals) was created by using the weighted arithmetical mean which included data from Düsseldorf.