Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

Garvicin Q: characterization of biosynthesis and mode of action.

Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTSMan) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain.Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts.

Need for speed: evaluation of dilute and shoot-mass spectrometry for accelerated metabolic phenotyping in bioprocess development.

With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with 13C15N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7-443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing L-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.

Immunohistochemical evaluation of suspected oral malignant melanoma in cats.

Oral malignant melanoma (OMM) is considered the third most common oral malignant neoplasm in cats, but its variable morphology and frequent lack of melanin pigment make it a diagnostic challenge. Twenty-two cases of cats with malignant oral neoplasms that were diagnosed as OMM or listed OMM as a suspected differential diagnosis on the biopsy report were examined using an immunohistochemistry (IHC) panel for S100, melan-A, PNL2, laminin, CD34, and pan-cytokeratin. Although OMM was suspected (n = 14) or previously diagnosed (n = 8), only 2 cases were immunohistochemically confirmed as OMM. Seven cases were classified as soft tissue sarcoma based on positive expression of CD34 or laminin, and one was classified as carcinoma based on positive expression of pan-cytokeratin. The majority of cases (n = 12) were categorized as unclassified malignant neoplasms because they did not express melan-A, PNL2, laminin, CD34, or pan-cytokeratin; however, a proportion of these did express S100 (n = 7). Long-term prognosis of all 22 cats was poor, with a median survival time of 87 days (range = 2-249 days). Cases with longer survival times (>100 days) were treated with surgery, radiation therapy, or a combination. For feline oral malignant neoplasms thought to be OMM, routine use of IHC is required for an accurate diagnosis.

Management and Outcome of Maxillofacial Trauma in a 9-Week-Old Dog.

This case report describes diagnosis, treatment, and outcome of maxillofacial trauma in a 9-week-old shih tzu. In addition to an open left mandibular body fracture and a right condylar process fracture, there were several relatively stable and minimally displaced right maxillary fractures. The torn soft tissues associated with the left mandibular body fracture were sutured closed, and a tape muzzle was placed. Due to sufficient fracture healing at the 2-week recheck examination, the tape muzzle was removed. The 6-week reexamination confirmed continued healing and recovery. Anesthesia was performed at the 5.5-month reexamination when the dog was 8 months old, revealing the presence of persistent deciduous teeth, linguoverted, malformed, and partially erupted permanent teeth, and asymmetric skeletal malocclusion (in addition to the breed-specific mandibular mesioclusion). Persistent deciduous teeth and linguoverted and malformed permanent teeth were extracted to allow for normal opening and closing of the mouth without traumatic occlusion. Long-term follow-up is recommended in juvenile dogs with maxillofacial injuries in order to prevent, recognize, and treat dental complications resulting from the trauma.

Assessment of Temporary Crown Extensions to Correct Linguoverted Mandibular Canine Teeth in 72 Client-Owned Dogs (2012-2016).

A search of the medical and dental records at Evidensia Lørenskog Dyreklinikk, in Lørenskog, Norway, was conducted to identify dogs that received temporary crown extensions (TCEs) to correct linguoverted mandibular canine (LMC) teeth over a 54-month investigation period (2012-2016). Criteria for inclusion into the study were the presence of complete medical and dental records, pre- and postoperative clinical photographs and intraoral radiographs of the affected canine teeth, adequate information pertaining to the procedures performed, and at least 1 follow-up >3 months after appliance removal. Seventy-two dogs with LMC teeth were treated with TCE. Thirty-nine breeds were represented in this study. Mean age at the time of appliance installation was 6.4 (range, 4.7-13.4 months [median, 5.9 months] months). Fifty-three (73.6%) dogs presented with class I malocclusion, 14 (19.5%) dogs with class II malocclusion, and 5 (6.9%) dogs with class III malocclusion. Twenty-five (34.7%) dogs were considered to have mild, 32 (44.4%) dogs to have moderate, and 15 (20.8%) dogs to have severe mandibular canine malocclusion. The TCE was combined with other treatment modalities (active orthodontics, extraction of nonstrategic teeth, gingivectomy, and inclined bite plane and ball therapy) to correct mandibular canine tooth malocclusions in 19 (26.4%) dogs. Fractured or detached crown extensions were seen in 9 (12.5%) dogs. Soft tissue ulceration or inflammation was seen in 7 (9.7%) dogs. The mandibular canine teeth occlusion resolved completely with self-retaining, functional, nontraumatic occlusion in 56 (77.8%) dogs. Fifteen dogs (20.8%) resolved with functional, nontraumatic occlusion, but the mandibular canine teeth were too short to be perfectly self-retained, thus left with 1- to 2-mm crown extensions for permanent retention. In 1 (1.4%) dog, both mandibular canine teeth relapsed almost back to original position. The results show that TCE is a viable treatment option to correct LMC teeth in young dogs.

Morphologic and Morphometric Description of the Temporomandibular Joint in the Domestic Dog Using Computed Tomography.

The temporomandibular joint (TMJ) in the domestic dog is a synovial joint with 2 articular surfaces, the mandibular fossa of the squamous portion of the temporal bone and the articular head of the condylar process of the mandible. Although different diagnostic imaging techniques have been used to study the TMJ in dogs, morphologic and morphometric studies based on computed tomography (CT) are scarce. The purpose of the present study was to describe the morphologic and morphometric features of the TMJ in domestic dogs using CT. Width and depth of the mandibular fossa and 2 different angles between the mandibular fossa and the condylar process were measured in 96 TMJs of 48 dogs of different breeds (Labrador retriever, German shepherd, cocker spaniel, boxer, English bulldog, pug, shih tzu, and Cavalier King Charles spaniel). Temporomandibular joint conformation differed between breeds. Mid- and small-sized dogs had mandibular fossae that were more shallow, less developed retroarticular processes, and irregularly shaped condylar processes. The TMJs were more congruent in large dogs, presenting with deeper mandibular fossae, prominent retroarticular processes, and more uniform condylar processes. The measurements proposed in this study demonstrated 3 different morphologic conformations for the TMJ in the dogs of this study.

Protein tyrosine phosphatase 1B is a key regulator of IFNAR1 endocytosis and a target for antiviral therapies.

Type 1 interferons (IFN1) elicit antiviral defenses by activating the cognate receptor composed of IFN-α/ß receptor chain 1 (IFNAR1) and IFNAR2. Down-regulation of this receptor occurs through IFN1-stimulated IFNAR1 ubiquitination, which exposes a Y466-based linear endocytic motif within IFNAR1 to recruitment of the adaptin protein-2 complex (AP2) and ensuing receptor endocytosis. Paradoxically, IFN1-induced Janus kinase-mediated phosphorylation of Y466 is expected to decrease its affinity for AP2 and to inhibit the endocytic rate. To explain how IFN1 promotes Y466 phosphorylation yet stimulates IFNAR1 internalization, we proposed that the activity of a protein tyrosine phosphatase (PTP) is required to enable both events by dephosphorylating Y466. An RNAi-based screen identified PTP1B as a specific regulator of IFNAR1 endocytosis stimulated by IFN1, but not by ligand-independent inducers of IFNAR1 ubiquitination. PTP1B is a promising target for treatment of obesity and diabetes; numerous research programs are aimed at identification and characterization of clinically relevant inhibitors of PTP1B. PTP1B is capable of binding and dephosphorylating IFNAR1. Genetic or pharmacologic modulation of PTP1B activity regulated IFN1 signaling in a manner dependent on the integrity of Y466 within IFNAR1 in human cells. These effects were less evident in mouse cells whose IFNAR1 lacks an analogous motif. PTP1B inhibitors robustly augmented the antiviral effects of IFN1 against vesicular stomatitis and hepatitis C viruses in human cells and proved beneficial in feline stomatitis patients. The clinical significance of these findings in the context of using PTP1B inhibitors to increase the therapeutic efficacy of IFN against viral infections is discussed.

Assessment of 68 Prosthodontic Crowns in 41 Pet and Working Dogs (2000-2012).

A search of the medical and dental records at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania was conducted to identify dogs that received full or partial prosthodontic crowns over a 13-year period (2000-2012). Forty-one dogs with a total of 68 prosthodontic crowns were identified. Further criteria for acceptance into the study included presence of complete medical records containing adequate information pertaining to the procedures performed, and current follow-up either by telephone, electronic mail, or via in-person examination. Treatment was considered to be successful when the prosthodontic crown was in place and there was no further structural injury to the tooth upon making contact with the client or at the time of death of the dog confirmed by the client. The mean number of days the prosthodontic crowns remained in place without further injury to the tooth was 1,598 (range, 161-4, 464 days [median, 1,414 days], standard deviation 1,093 days). Bond failure between the cement and the tooth or the cement and the prosthodontic crown occurred in 3 cases (4.4%). Fracture of the prosthodontically treated tooth occurred in an additional 7 cases (10.3%). Treatment was classified as successful in 58 cases (85.3%). The results suggest that prosthodontic crown therapy is a successful, practical, and durable treatment option for protection of previously injured teeth in pet and working dogs.

Computed tomographic characteristics of odontogenic neoplasms in dogs.

Odontogenic neoplasms are locally invasive oral tumors in dogs. The purpose of this retrospective study was to describe CT characteristics for varying histopathologic types of canine odontogenic neoplasms. A board-certified veterinary radiologist who was unaware of histologic findings reviewed and scored imaging studies. A total of 29 dogs were included in the study. Twenty-three of these dogs had concurrent dental radiographs. The most common CT characteristics for all tumor types were a direct association with or in the region of multiple teeth in 96.4% (27/28), contrast enhancement in 96.3% (26/27), alveolar bone lysis in 93.1% (27/29), and mass-associated tooth displacement in 85.2% (23/27). Mass-associated cyst-like structures were identified in 53.6% (15/28) and were only present in tumors containing odontogenic epithelium. Canine acanthomatous ameloblastomas (n = 15) appeared as extra-osseous (10/15) or intra-osseous (5/15) masses. Intra-osseous canine acanthomatous ameloblastomas were more likely to have mass-associated cyst-like structures and were subjectively more aggressive when compared with extra-osseous canine acanthomatous ameloblastomas. Amyloid-producing odontogenic tumors (n = 3) had subjectively uniform CT imaging characteristics and consisted of round soft tissue and mineral attenuating masses with multiple associated cyst-like structures. Fibromatous epulides of periodontal ligament origin (n = 4) were contrast enhancing extra-osseous masses that were rarely referred for CT examinations and 25% (1/4) were not visible with CT. Other odontogenic tumors were less represented or had more variable CT imaging characteristics. Mass-associated tooth destruction was appreciated more often with dental radiographs and extra-oral tumor extension was identified more often with CT.

Assessment of apical periodontitis in dogs and humans: a review.

Veterinary dentists commonly encounter apical periodontitis in dogs. An overview of the disease is presented, concentrating on pathogenesis and mechanisms of healing. Assessment modalities are reviewed and interpretations of treatment success and failure are discussed. The limitations of readily available diagnostic equipment are illustrated. The benefits of identifying the causative agent and resultant pathosis should not be overlooked. Well-designed clinical studies evaluating various methods of long-term follow-up for apical periodontitis in dogs are needed.

Use of a barrier membrane to repair congenital hard palate defects and to close oronasal fistulae remaining after cleft palate repair: seven dogs (2019-2022).

OBJECTIVE: To describe the use of a barrier membrane in dogs for repair of congenital hard palate defects and closure of oronasal fistulae (ONF) remaining after previous cleft palate (CFP) repair. ANIMALS: 7 client-owned dogs. METHODS: The hard palate defect was closed with medially positioned flaps (Von Langenbeck technique) or pedicle flaps (2-flap palatoplasty) and a membrane composed of autologous auricular cartilage from the pinna or allogenous fascia lata underlying the mucoperiosteal flaps. RESULTS: All palate defects were considered to have a high risk of dehiscence based on their type and size and the characteristics of the surrounding tissue. The barrier membrane was used in 5 dogs for repair of congenital hard palate defects and in 2 dogs for closure of ONF remaining after previous CFP repair. Resolution of clinical signs occurred in all cases. Complete success (ie, complete closure of the palate defect and absence of clinical signs) was achieved in 5 dogs (4 with congenital hard palate defects and 1 with an ONF remaining after previous CFP repair). The persistent ONF in 1 dog with functional success (incomplete closure, but no clinical signs) was smaller than prior to surgery. CLINICAL RELEVANCE: Barrier membranes underlying mucoperiosteal flaps may constitute an alternative technique in dogs for repair of congenital hard palate defects and closure of ONF remaining after previous CFP repair.

Polyamine inhibitors for treatment of feline oral squamous cell carcinoma: a proof-of-concept study.

This study assessed proof-of-concept for use of polyamine inhibitor 2-diluoromethylornithine (DFMO) as a treatment for oral squamous cell carcinoma (SCC) in client-owned cats. Polyamine levels in tumor tissue and normal oral mucosa were quantified before and after treatment. DFMO was administered orally to 14 client-owned cats with histologically confirmed oral SCC. Patients were monitored for gastrointestinal, dermatologic, auditory, hematological, and biochemical abnormalities. Total polyamine levels in tumor tissue decreased after treatment, as did the specific polyamine putrescine in both tumor tissue and normal mucosa. Ototoxicity was observed in 5 of 6 cats receiving pre- and post-treatment brainstem auditory evoked potential tests. Subclinical thrombocytopenia was observed in 6 of 14 cats. One cat showed mild post-anesthetic tremors that resolved without treatment. Oral administration of DFMO at doses used in this study resulted in significantly decreased tumor polyamine levels without life-threatening clinical or hematological toxicities. Further studies are warranted to explore pathophysiology of polyamine biochemistry and use of polyamine inhibitors in treatment of cats with oral SCC.

Surgical treatment for cleft palate in dogs yields excellent outcomes despite high rates of oronasal fistula formation: a narrative review.

Cleft palate is the most common congenital orofacial defect in dogs. Although this topic has been extensively studied in experimental dogs, current literature on clinical patients is limited to case reports and small case series studies with many individual opinions based on experience. This narrative review summarizes the literature of the last 10 years with application to clinical practice, focusing on the preparation of the patient until surgery, options for surgical management, and perioperative complications. Surgical treatment must be performed to obtain separation between the nasal/nasopharyngeal and oral/oropharyngeal passages. This will allow the dog to have independent function and reduce clinical signs associated with food, water, and other foreign material entering the respiratory tract. Surgical treatment has good to excellent outcomes when the procedure is carefully planned and appropriately executed despite the high rates of postoperative oronasal fistula reported.

Etiology, clinical presentation, and outcome of temporomandibular joint luxation in cats: 21 cases (2000-2018).

This study was performed to report etiology, clinical presentation, treatment, and outcome of temporomandibular joint (TMJ) luxation in 21 cats (2000-2018). TMJ luxation was diagnosed by either dental radiography or computed tomography (CT). Causes for TMJ luxation included altercation with a dog (38%), hit-by-car (19%), unknown (19%), fall (19%), and ran into inanimate object (5%). The most common complication was development of permanent malocclusion (24%), followed by reduced vertical mandibular range of motion (5%). Reduction of TMJ luxation was successful in 94.7% of the cases. Reduction of the TMJ luxation was significantly associated with time from injury to treatment. No other significant associations were observed between cause of injury, type of TMJ stabilization, and outcome. TMJ luxation in cats carries an excellent prognosis with early joint reduction and supportive care. Concurrent dental trauma and maxillofacial injuries are common, and all patients with head trauma must be stabilized and evaluated with a thorough examination.

Identification and Characterization of Corynaridin, a Novel Linaridin from Corynebacterium lactis.

Genome analysis of Corynebacterium lactis revealed a bacteriocin gene cluster encoding a putative bacteriocin of the linaridin family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The locus harbors typical linaridin modification enzymes but lacks genes for a decarboxylase and methyltransferase, which is unusual for type B linaridins. Supernatants of Corynebacterium lactis RW3-42 showed antimicrobial activity against Corynebacterium glutamicum. Deletion of the precursor gene crdA clearly linked the antimicrobial activity of the producer strain to the identified gene cluster. Following purification, we observed potent activity of the peptide against Actinobacteria, mainly other members of the genus Corynebacterium, including the pathogenic species Corynebacterium striatum and Corynebacterium amycolatum. Also, low activity against some Firmicutes was observed, but there was no activity against Gram-negative species. The peptide is resilient towards heat but sensitive to proteolytic degradation by trypsin and proteinase K. Analysis by mass spectrometry indicates that corynaridin is processed by cleaving off the leader sequence at a conserved motif and posttranslationally modified by dehydration of all threonine and serin residues, resulting in a monoisotopic mass of 3,961.19 Da. Notably, time-kill kinetics and experiments using live biosensors to monitor membrane integrity suggest bactericidal activity that does not involve formation of pores in the cytoplasmic membrane. As Corynebacterium species are ubiquitous in nature and include important commensals and pathogens of mammalian organisms, secretion of bacteriocins by species of this genus could be a hitherto neglected trait with high relevance for intra- and interspecies competition and infection. IMPORTANCE Bacteriocins are antimicrobial peptides produced by bacteria to fend off competitors in ecological niches and are considered to be important factors influencing the composition of microbial communities. However, bacteriocin production by bacteria of the genus Corynebacterium has been a hitherto neglected trait, although its species are ubiquitous in nature and make up large parts of the microbiome of humans and animals. In this study, we describe and characterize a novel linaridin family bacteriocin from Corynebacterium lactis and show its narrow-spectrum activity, mainly against other actinobacteria. Moreover, we were able to extend the limited knowledge on linaridin bioactivity in general and for the first time describe the bactericidal activity of such a bacteriocin. Interestingly, the peptide, which was named corynaridin, appears bactericidal, but without formation of pores in the bacterial membrane.

Morphometry and Morphology of the Articular Surfaces of the Medial Region of the Temporomandibular Joint in the Felis Catus (Domestic cat) - A Cone Beam Computed Tomography Study.

The articular surfaces of the temporomandibular joint (TMJ) in the cat consist of the articular head of the condylar process of the ramus of the mandible and the mandibular fossa of the petrous part of the temporal bone. Anatomic conformation of the TMJ articular surfaces can vary; however, this has not been studied in the cat. Thus, the aim of this study was to characterize the conformation of the medial region of the TMJ in mesocephalic adult cats as well as to determine the articular relationship by measurement of the degree of coverage of the mandibular fossa over the articular head. This was accomplished by assessing 60 TMJs from 30 mesocephalic adult cats by means of cone-beam computed tomography (CBCT). The width and depth of the mandibular fossa, and the degree of coverage of the mandibular fossa were evaluated using parasagittal reconstructions of the medial aspect of the TMJ by two observers. No statistically significant difference was observed during the intra and interobserver evaluation of the mandibular fossa width and depth and the degree of coverage of the mandibular fossa over the articular head. P-values for the studied variables were greater than 0.05. The resulting measurements demonstrated a concave mandibular fossa with a prominent retroarticular process and a poorly developed articular eminence; a well-defined rounded articular surface, and a high degree of coverage (171°) of the mandibular fossa over the articular head. In conclusion, the articular surfaces at the medial aspect of the TMJ displayed elevated geometric similarity, with an articular head of the condylar process deeply seated in the mandibular fossa.

Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L-1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L-1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L-1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L-1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.

Orofacial manifestations of high-rise syndrome in cats: a retrospective study of 84 cases.

Medical records of cats with high-rise trauma were reviewed to document the prevalence and clinical manifestations of orofacial injury. Cats were presented over a 10-year period from January 2000 to December 2009. Signalment, weight, number of stories fallen, and survival data were recorded in 84 cats and physical examination findings were obtained from 83 cats. Fourteen of these cats were examined by veterinarians of the Dentistry and Oral Surgery Service. Mean age was 37-months. Mean distance fallen was 2.65 stories, and in the majority of cases the substrate the cat fell on was not recorded Overall, survival was 94.0% when including euthanasia as a cause of death and 98.8% when excluding euthanized patients. Orofacial findings included bilateral epistaxis, hard palate fracture +/- tear of palatal soft tissue, palatal soft tissue bruising, mandibular fracture, mandibular symphyseal separation, tongue injury, facial soft tissue injury, dental trauma, and other oral soft tissue injury. Sixty-six percent of cats suffered some degree of orofacial injury. The population was analyzed for the prevalence of each type of injury. An oronasal fistula was seen in one cat as a complication of an untreated hard palate fracture. Possible etiology of the injuries and treatment options are discussed.

Metabolic Footprinting of Microbial Systems Based on Comprehensive In Silico Predictions of MS/MS Relevant Data.

Metabolic footprinting represents a holistic approach to gathering large-scale metabolomic information of a given biological system and is, therefore, a driving force for systems biology and bioprocess development. The ongoing development of automated cultivation platforms increases the need for a comprehensive and rapid profiling tool to cope with the cultivation throughput. In this study, we implemented a workflow to provide and select relevant metabolite information from a genome-scale model to automatically build an organism-specific comprehensive metabolome analysis method. Based on in-house literature and predicted metabolite information, the deduced metabolite set was distributed in stackable methods for a chromatography-free dilute and shoot flow-injection analysis multiple-reaction monitoring profiling approach. The workflow was used to create a method specific for Saccharomyces cerevisiae, covering 252 metabolites with 7 min/sample. The method was validated with a commercially available yeast metabolome standard, identifying up to 74.2% of the listed metabolites. As a first case study, three commercially available yeast extracts were screened with 118 metabolites passing quality control thresholds for statistical analysis, allowing to identify discriminating metabolites. The presented methodology provides metabolite screening in a time-optimised way by scaling analysis time to metabolite coverage and is open to other microbial systems simply starting from genome-scale model information.