Evaluation of Oral Tebipenem as a Step-Down Therapy following Intravenous Ertapenem against Extended-Spectrum ß-Lactamase-Producing Escherichia coli in a Hollow-Fiber In Vitro Infection Model.

Tebipenem is an orally bioavailable carbapenem in development for the treatment of patients with complicated urinary tract infections. Herein, we describe the results of studies designed to evaluate tebipenem's potential as an oral (p.o.) transition therapy from intravenous (i.v.) ertapenem therapy for the most common uropathogen, Escherichia coli. These studies utilized a 7-day hollow-fiber in vitro infection model and 5 extended-spectrum ß-lactamase-producing E. coli challenge isolates. Human free-drug serum concentration-time profiles for tebipenem 600 mg p.o. every 8 h and ertapenem 1 g i.v. every 24 h were simulated in the hollow-fiber in vitro infection model. Samples were collected for bacterial density and drug concentration determination over the 7-day study period. Generally, ertapenem monotherapy resulted in a greater reduction in bacterial density than did tebipenem monotherapy. In the treatment arms in which ertapenem dosing was stopped following dosing for 1 or 3 days, immediate bacterial regrowth occurred and matched that of the growth control. Finally, in the treatment arms in which ertapenem dosing was stopped following dosing for 1 or 3 days and tebipenem dosing was initiated for the remainder of the 7-day study, the intravenous-to-oral transition regimen reduced bacterial burdens and prevented regrowth. Given that transition from intravenous to oral antibiotic therapy has been shown to reduce hospital length of stay, nosocomial infection risk, and cost, and improve patient satisfaction, these data demonstrate tebipenem's potential role as an oral transition agent from intravenous antibiotic regimens within the antibiotic stewardship paradigm.

Evaluation of the Impact of Comorbidities on Omadacycline Pharmacokinetics.

Omadacycline is approved in the United States for the treatment of patients with community-acquired bacterial pneumonia or acute bacterial skin and skin structure infections. Analyses were undertaken to evaluate pharmacokinetic differences among subjects or patients stratified by comorbidities. Differences in clearance by smoking status, history of diabetes mellitus, chronic lung disease, hypertension, heart failure, or coronary artery disease were evaluated using a Welch two-sample t test. Smoking was the only significant comorbidity after correction for sex, with a clinically insignificant difference of 13%. Omadacycline dose adjustments based on these comorbidities do not appear to be warranted.

Efficient secretory production of proline/alanine/serine (PAS) biopolymers in Corynebacterium glutamicum yielding a monodisperse biological alternative to polyethylene glycol (PEG).

BACKGROUND: PAS biopolymers are recombinant polypeptides comprising the small uncharged L-amino acids Pro, Ala and/or Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behaviour in physiological solution results in a strongly expanded hydrodynamic volume. Thus, apart from their use as fusion partner for biopharmaceuticals to achieve prolonged half-life in vivo, PAS biopolymers appear attractive as substitute for PEG-or other poorly degradable chemical polymers-in many areas. As a prerequisite for the wide application of PAS biopolymers at affordable cost, we have established their highly efficient biotechnological production in Corynebacterium glutamicum serving as a well characterized bacterial host organism. RESULTS: Using the CspA signal sequence, we have secreted two representative PAS biopolymers as polypeptides with ~ 600 and ~ 1200 amino acid residues, respectively. Both PAS biopolymers were purified from the culture supernatant by means of a simple downstream process in a truly monodisperse state as evidenced by ESI-MS. Yields after purification were up to ≥ 4 g per liter culture, with potential for further increase by strain optimization as well as fermentation and bioprocess development. Beyond direct application as hydrocolloids or to exploit their rheological properties, such PAS biopolymers are suitable for site-specific chemical conjugation with pharmacologically active molecules via their unique terminal amino or carboxyl groups. To enable the specific activation of the carboxylate, without interference by the free amino group, we generated a blocked N-terminus for the PAS(1200) polypeptide simply by introducing an N-terminal Gln residue which, after processing of the signal peptide, was cyclised to a chemically inert pyroglutamyl group upon acid treatment. The fact that PAS biopolymers are genetically encoded offers further conjugation strategies via incorporation of amino acids with reactive side chains (e.g., Cys, Lys, Glu/Asp) at defined positions. CONCLUSIONS: Our new PAS expression platform using Corynex® technology opens the way to applications of PASylation® technology in multiple areas such as the pharmaceutical industry, cosmetics and food technology.

[Complications of body piercing]. / Complications liées aux piercings.

The trend of body piercing has grown in popularity in the past decade within the general population and especially among young adults. Complications of body piercing include local inflammation and infections, but severe complications are also possible and largely underestimated. People are usually not aware of the risks before making a piercing, and their medical history, medication and comorbidities are largely neglected by the people who realise the piercing. This article presents a review of the complications that a primary care physician may observe, for a patient who wishes to make a piercing, or presents complications due to the implementation of such a device.

Assessment of the potential risk of oteseconazole and two other tetrazole antifungals to inhibit adrenal steroidogenesis and peripheral metabolism of corticosteroids.

The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia, edema, and gynecomastia by inhibiting steroid synthesis and metabolism. Mechanisms underlying pseudohyperaldosteronism include inhibition of adrenal 11ß-hydroxylase cytochrome-P450 (CYP) 11B1 and 17α-hydroxylase (CYP17A1) as well as peripherally expressed 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). To enhance specificity for fungal CYP51, tetrazoles have been developed. This study employed H295R adrenocortical cells and enzyme activity assays to assess the potential risk of oteseconazole and two other tetrazoles, VT-1598 and quilseconazole, to inhibit adrenal steroidogenesis or 11ß-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted liquid-chromatography-high-resolution mass-spectrometry (LC-HRMS) indicated overall patterns common to oteseconazole, quilseconazole and itraconazole, as well as similarities between VT-1598 and isavuconazole. Additionally, more specific features of the steroid signatures were observed. Targeted quantification of nine adrenal steroids in supernatants from treated H295R cells revealed an overall inhibition of adrenal steroidogenesis by the three tetrazoles, itraconazole and isavuconazole, providing an explanation for their similar steroidomic pattern. Applying recombinant enzymes indicated that this effect is not due to direct inhibition of steroidogenic enzymes because no or only weak inhibition could be observed. Moreover, oteseconazole and the two other tetrazoles did not inhibit 11ß-HSD2, suggesting that they do not pose a risk of pseudohyperaldosteronism. Furthermore, oteseconazole did not alter steroid concentrations in a recent clinical study. Nevertheless, follow-up studies should assess the mechanism underlying the observed overall steroidogenesis inhibition by tetrazoles, itraconazole and isavuconazole, and whether concentrations achievable in a subgroup of susceptible patients might cause adrenal insufficiency and hyperplasia.

Textural heterogeneity of liver lesions in CT imaging - comparison of colorectal and pancreatic metastases.

PURPOSE: Tumoral heterogeneity poses a challenge for personalized cancer treatments. Especially in metastasized cancer, it remains a major limitation for successful targeted therapy, often leading to drug resistance due to tumoral escape mechanisms. This work explores a non-invasive radiomics-based approach to capture textural heterogeneity in liver lesions and compare it between colorectal cancer (CRC) and pancreatic cancer (PDAC). MATERIALS AND METHODS: In this retrospective single-center study 73 subjects (42 CRC, 31 PDAC) with 1291 liver metastases (430 CRC, 861 PDAC) were segmented fully automated on contrast-enhanced CT images by a UNet for medical images. Radiomics features were extracted using the Python package Pyradiomics. The mean coefficient of variation (CV) was calculated patient-wise for each feature to quantify the heterogeneity. An unpaired t-test identified features with significant differences in feature variability between CRC and PDAC metastases. RESULTS: In both colorectal and pancreatic liver metastases, interlesional heterogeneity in imaging can be observed using quantitative imaging features. 75 second-order features were extracted to compare the varying textural characteristics. In total, 18 radiomics features showed a significant difference (p < 0.05) in their expression between the two malignancies. Out of these, 16 features showed higher levels of variability within the cohort of pancreatic metastases, which, as illustrated in a radar plot, suggests greater textural heterogeneity for this entity. CONCLUSIONS: Radiomics has the potential to identify the interlesional heterogeneity of CT texture among individual liver metastases. In this proof-of-concept study for the quantification and comparison of imaging-related heterogeneity in liver metastases a variation in the extent of heterogeneity levels in CRC and PDAC liver metastases was shown.

[Longitudinal model in pain medicine (LoMoS). Needs assessment and learning developement of learning goals]. / Das longitudinale Modul Schmerzmedizin (LoMoS). Bedarfsanalyse und Lernzielformulierung.

BACKGROUND: Pain medicine as an interdisciplinary, multifaceted field has not yet been assigned the status of a separate medical subject in the curriculum of medical schools in Germany. Pain medicine is often taught by anesthesiologists, neurologists, orthopedic or neurological surgeons either by assignment by the Dean's office or because of their own enthusiasm. In the near future pain medicine as an interdisciplinary course will be mandatory in undergraduate medical education. The authors were interested to investigate the needs and demands of both students and instructors from theoretical and clinical fields in order to develop a longitudinal pain medicine curriculum. METHODS: Based on Kern's curriculum development model, the opinions of students and instructors were investigated: quantitative items were analyzed using Student's t-test for independent variables and heterogenic variance and the content of free text answers was analyzed by forming subsets of similar or identical answers. A concise curriculum was developed. RESULTS: Students from advanced classes noted a bigger discrepancy between the needs formulated and what was actually offered as compared to younger students. Instructors from different theoretical and clinical specialties were unaware of the topics of colleagues from other departments. The analysis of written answers revealed a different understanding of the term pain medicine. CONCLUSION: At the Hannover Medical School, a standardized needs assessment helped to develop LoMoS, the longitudinal pain medicine curriculum, which may also serve as a model for other medical faculties. Students required more practical instruction and teachers were interested in improving networking and discussion among specialists.

MEK inhibition induces expression of differentiation marker Keratin 10 in human keratinocytes.

BRAF mutant metastatic melanoma was regularly treated in the past with a BRAF inhibitor (BRAFi) alone or in combination with inhibitors of the mitogen-activated protein kinase kinase (MEKi), which is still a common treatment. This combination therapy strongly reduced the occurrence of keratoacanthomas and squamous cell carcinoma, which was frequently seen when BRAFi was used as monotherapy. Here we addressed the question whether MEK inhibition counteracts squamous cell carcinoma development in part by promoting keratinocyte differentiation. Exposure of human immortalized keratinocytes to different concentrations of MEKi revealed a significant increase in the expression of differentiation-associated keratins K10 and K1 as determined by qRT-PCR and immunofluorescence staining. Taken together, the present study suggests that in a combined treatment of melanoma with BRAFi/MEKi, MEKi reduces the incidence of squamous cell carcinomas by promoting keratinocyte differentiation under combined BRAFi/MEKi treatment in melanoma. This might open further treatment perspectives for skin cancer treatment.

Field trial: disinfection of contaminated anesthetic masks for piglets.

This paper aimed to assess the success of cleaning and disinfection on microbiological contamination of anesthetic masks, which were used for automated isoflurane anesthesia for surgical castration of male piglets. Data collection took place on 11 farms in Southern Germany between September 2020 and June 2022. Each farm was visited three times (one farm having two different anesthesia devices was visited six times), and microbiological assessments took place at four sample points (SP): after unpacking the masks (SP0), after disinfection before anesthesia (SP1), after anesthesia of all piglets to be castrated in this run (SP2), and after disinfection after anesthesia (SP3). The microbiological assessment included the determination of total bacteria count, total count of hemolytic and non-hemolytic mesophilic aerotolerant bacteria and a qualitative detection of indicator bacteria Escherichia (E.) coli, extended-spectrum beta-lactamase-producing E. coli (ESBL) and methicillin-resistant Staphylococcus aureus (MRSA). For analysis, a generalized linear mixed model was applied using farms and farm visits as random effects and sampling points nested in farm visits as fixed effect. The fixed effect was highly significant for all three variables (total bacteria count, total count of hemolytic and non-hemolytic mesophilic aerotolerant bacteria) (p < 0.001). The bacterial counts at SP0 were about the same as at SP3. Concerning indicator bacteria, their presence was highest at SP2 and lowest at SP3. No indicator bacteria were present at SP1. It can be concluded that disinfection of anesthetic masks, especially before performing anesthesia, may effectively protect piglets of the following batch against unwanted transmission of pathogens. These findings will help farmers plan cleaning and disinfection activities.

Characterization of the interferences of systemic azole antifungal drugs with adrenal steroid biosynthesis using H295R cells and enzyme activity assays.

Azole antifungals, designed to inhibit fungal CYP51, have a liability to inhibit human CYP enzymes. Whilst drug-metabolizing CYPs are covered in preclinical safety assessment, those metabolizing endogenous bioactive molecules are usually not. Posaconazole and itraconazole were recently found to cause pseudohyperaldosteronism with hypokalemia and hypertension by inhibiting CYP11B1-dependent adrenal cortisol biosynthesis. Because this was overlooked in preclinical safety assessment, the present study tested whether applying adrenal carcinoma H295R cells could have predicted this liability and whether other systemic triazole antifungals interfere with adrenal steroidogenesis. Forskolin-stimulated H295R cells were exposed to systemic triazole antifungals that are currently used, and key adrenal steroids were quantified by UHPLC-MS/MS. To support the findings from the H295R model, activity assays for steroidogenic enzymes were performed. The analysis of the steroid profiles and product/substrate ratios predicted the CYP11B1 and CYP11B2 inhibition by posaconazole and itraconazole. Comparison of their steroid profiles allowed distinguishing their effects and suggested inhibition of adrenal androgen synthesis by posaconazole but not itraconazole, which was confirmed by CYP17A1 17,20-lyase activity measurements. In line with clinical observations, there was no evidence from these experiments for an inhibition of either CYP11B1/2 or CYP17A1 by voriconazole, fluconazole or isavuconazole. However, itraconazole and isavuconazole exerted an overall inhibition of steroidogenesis by a mechanism warranting further investigations. In conclusion, analyses of steroid profiles from the H295R assay and product/substrate ratios provide important information on the interference of a chemical with adrenal steroidogenesis and the underlying mechanism. This approach facilitates prioritization of further investigations, including enzyme expression and activity studies.

Molecular recognition of structurally disordered Pro/Ala-rich sequences (PAS) by antibodies involves an Ala residue at the hot spot of the epitope.

Pro/Ala-rich sequences (PAS) are polypeptides that were developed as a biological alternative to poly-ethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-life. Like PEG, PAS polypeptides are conformationally disordered and show high solubility in water. Devoid of any charged or prominent hydrophobic side chains, these biosynthetic polymers represent an extreme case of intrinsically disordered proteins. Despite lack of immunogenicity of PAS tags in numerous animal studies we now succeeded in generating monoclonal antibodies (MAbs) against three different PAS versions. To this end, mice were immunized with a PAS#1, P/A#1 or APSA 40mer peptide conjugated to keyhole limpet hemocyanin as highly immunogenic carrier protein. In each case, one MAb with high binding activity and specificity towards a particular PAS motif was obtained. The apparent affinity was strongly dependent on the avidity effect and most pronounced for the bivalent MAb when interacting with a long PAS repeat. X-ray structural analysis of four representative anti-PAS Fab fragments in complex with their cognate PAS epitope peptides revealed interactions dominated by hydrogen bond networks involving the peptide backbone as well as multiple Van der Waals contacts arising from intimate shape complementarity. Surprisingly, Ala, the L-amino acid with the smallest side chain, emerged as a crucial feature for epitope recognition, contributing specific contacts at the center of the paratope in several anti-PAS complexes. Apart from these insights into how antibodies can recognize feature-less peptides without secondary structure, the MAbs characterized in this study offer valuable reagents for the preclinical and clinical development of PASylated biologics.

Short communication: Evaluation of the sample size of individual indicators in gestating sows concerning the Welfare Quality® protocol applied to sows and piglets.

Sample sizes of welfare assessment protocols must warrant to reflect prevalences on-farm properly - regardless of farm size. Still, solely a fixed sample size was specified for the Welfare Quality® protocol for sows and piglets. The present study investigated whether animals may be assessed from only one body side as applied in the protocol and whether the pre-set sample size of 30 animals mirrors the prevalences of the animal-based indicators on-farm in the gestation unit considering different farm sizes. All indicators were assessed for both sides of an animal's body by one observer on 13 farms in Germany, which were visited five times within 10 months. The farm visits were treated as independent since different animals were housed in the gestation units. The number of sows in the gestation units varied between 18 and 549 animals. The comparison of sides was carried out calculating exact agreement between animals' sides and a Wilcoxon signed-rank test (W). The results signified that it is sufficient to assess the animal from one side (exact agreement: 88.3% to 99.5%, except for bursitis (70.0%); W: P-values 0.14 to 0.92). However, if side preferences existed in the indicator bursitis a potential bias must be considered. In the following, the sample size was evaluated by comparing samples' prevalences against true prevalence, that is, the prevalence of all observed animals in the gestation unit in each farm visit. Therefore, subsets of data were generated by applying simple random sampling without replacement. The samples randomly included the animals' right or left sides. Linear regression was rated as appropriate provided: coefficient of determination R2 ≥ 0.90, slope = 1 and intercept = 0 signifying exact agreement. The results revealed that the sample size required by the protocol and the application of calculation formulas are solely appropriate to mirror the prevalences of frequent indicators in the gestation unit, for example, bursitis (mean prevalence 34.4%). Using a proportion of animals, for example, a sample of 30% of all observed animals in a farm visit, pointed out that proportions must increase with indicators' underlying prevalence narrowing 0.00%. Local infections (mean prevalence 13.3%) needed samples including 60% of all observed animals in each farm visit, whereas vulva lesions (mean prevalence 7.28%) only reached accuracy with the inclusion of 70% of the animals. Indicators with a mean prevalence of <1% were not analysed but can most likely only be ascertained by the assessment of all animals.

Requirement of salicylic Acid for the induction of systemic acquired resistance.

It has been proposed that salicylic acid acts as an endogenous signal responsible for inducing systemic acquired resistance in plants. The contribution of salicylic acid to systemic acquired resistance was investigated in transgenic tobacco plants harboring a bacterial gene encoding salicylate hydroxylase, which converts salicylic acid to catechol. Transgenic plants that express salicylate hydroxylase accumulated little or no salicylic acid and were defective in their ability to induce acquired resistance against tobacco mosaic virus. Thus, salicylic acid is essential for the development of systemic acquired resistance in tobacco.

A central role of salicylic Acid in plant disease resistance.

Transgenic tobacco and Arabidopsis thaliana expressing the bacterial enzyme salicylate hydroxylase cannot accumulate salicylic acid (SA). This defect not only makes the plants unable to induce systemic acquired resistance, but also leads to increased susceptibility to viral, fungal, and bacterial pathogens. The enhanced susceptibility extends even to host-pathogen combinations that would normally result in genetic resistance. Therefore, SA accumulation is essential for expression of multiple modes of plant disease resistance.

Retrospective review of the use of daptomycin for diabetic foot infections.

The increase in antibiotic resistance has led to a search for alternative treatments for diabetic foot infections. This retrospective review outlines the clinical outcomes reported for a lipopetide for these infections.

Influence of various preservatives on the quality of minced beef under modified atmosphere at chilled storage.

The effect of preservatives on microbial quality, pH, drip-loss, roasting-loss, colour, and sensorial properties of modified atmosphere packaged (70% O(2) and 30% CO(2)) minced beef (M. semimembranosus) stored at (2±0.5°C) for 12days was investigated. Beef cubes (approx. 20×20×20mm size) were immersed in solutions of 2% and 5% lactic acid, 2% lactic acid combined with 0.5% sodium ascorbate, 20% potassium lactate and 20% potassium sorbate before mincing. Addition of lactic acid was associated with pH drop, which increased drip-loss and roasting-loss. Application of all additives inhibited aerobic micro-organisms (10(3)-10(4)CFUg(-1) on day 12) compared to reference sample (9×10(5)CFUg(-1) on day 12). Lactic acid discoloured samples, while sodium ascorbate seemed to improve colour stability. Despite good visual colour characteristics, potassium sorbate treated samples were organoleptically unacceptable with massive off-flavour.

Salicylic Acid Is Not the Translocated Signal Responsible for Inducing Systemic Acquired Resistance but Is Required in Signal Transduction.

Infection of plants by necrotizing pathogens can induce broad-spectrum resistance to subsequent pathogen infection. This systemic acquired resistance (SAR) is thought to be triggered by a vascular-mobile signal that moves throughout the plant from the infected leaves. A considerable amount of evidence suggests that salicylic acid (SA) is involved in the induction of SAR. Because SA is found in phloem exudate of infected cucumber and tobacco plants, it has been proposed as a candidate for the translocated signal. To determine if SA is the mobile signal, grafting experiments were performed using transgenic plants that express a bacterial SA-degrading enzyme. We show that transgenic tobacco root-stocks, although unable to accumulate SA, were fully capable of delivering a signal that renders nontransgenic scions resistant to further pathogen infection. This result indicated that the translocating, SAR-inducing signal is not SA. Reciprocal grafts demonstrated that the signal requires the presence of SA in tissues distant from the infection site to induce systemic resistance.

Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene.

Systemic acquired resistance (SAR) is an inducible plant response to infection by a necrotizing pathogen. In the induced plant, SAR provides broad-spectrum protection against not only the inducing pathogen, but also against other, unrelated pathogens. Both salicylic acid (SA) and SAR-gene expression have been implicated as playing important roles in the initiation and maintenance of SAR. Here, we describe the characterization of transgenic Arabidopsis plants that express the bacterial nahG gene encoding salicylate hydroxylase, an enzyme that can metabolize SA. Strong, constitutive expression of this gene prevents pathogen-induced accumulation of SA and the activation of SAR by exogenous SA. We show that SAR in Arabidopsis can be induced by inoculation with Pseudomonas syringe pv. tomato against infection by a challenge inoculation with Peronospora parasitica. This response is abolished in transgenic, nahG-expressing Arabidopsis, but not in ethylene-insensitive mutants. These experiments support the critical role of SA in SAR and show that ethylene sensitivity is not required for SAR induction. The NahG Arabidopsis plants will be important for future studies aimed at understanding the role of SA in plant disease resistance mechanisms.

NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides.

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.