Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse.

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

Biosynthesis of polybrominated aromatic organic compounds by marine bacteria.

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds.

Isolation of methicillin-resistant Staphylococcus spp. from ready-to-eat fish products.

UNLABELLED: A hundred samples from ready-to-eat (RTE) fish products were examined for the presence and antimicrobial susceptibility of Staphylococcus spp. Staphylococci were isolated from 43% of these samples (n = 100). The identified species in the samples were Staphylococcus aureus (7%), Staphylococcus epidermidis (13%), Staphylococcus xylosus (12%), Staphylococcus sciuri (4%), Staphylococcus warneri (3%), Staphylococcus saprophyticus (2%), Staphylococcus schleiferi (1%) and Staphylococcus auricularis (1%). Two Staph. aureus (MRSA) isolates, three Staph. epidermidis (MRSE), five Staph. xylosus, four Staph. sciuri, one Staph. schleiferi and one Staph. saprophyticus isolates were resistant to oxacillin and all of them carried the mecA gene. The two MRSA isolates belonged to the spa types t316 (ST359) and t548 (ST5) and none of them was able to produce enterotoxins. Pulsed field gel electrophoresis for Staph. aureus and Staph. epidermidis isolates revealed 6 and 11 distinct PFGE types, respectively, reflecting diversity. The presence of methicillin-resistant staphylococci, especially MRSA and MRSE, in RTE fish products may constitute a potential health risk for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first data on the occurrence of methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci in salted and smoked fish products in Greece. These results are important and useful for Staphylococcus spp. risk assessment and management programmes for ready-to-eat fish products.

Bilateral Hypotony Maculopathy Associated With Ipilimumab and Nivolumab Therapy Unresponsive to Corticosteroid Treatment.

In rare instances, immunotherapy associated with hypotony and uveitis has been documented. We report the case of a 72-year-old man treated with 2 months of ipilimumab and nivolumab for metastatic melanoma who developed bilateral hypotony maculopathy and serous choroidal detachments without prominent initial uveitis. Despite treatment with topical, periocular, and intraocular corticosteroid injection, hypotony persisted 18 months after immunotherapy cessation. The patient's unresponsiveness to corticosteroids indicates the need to further explore the mechanism behind immune checkpoint inhibitor-associated hypotony. We hypothesize that immunotherapy significantly decreased aqueous humor production through ciliary body inflammation, disruption, or shutdown. [Ophthalmic Surg Lasers Imaging Retina 2023;54:301-304.].

Effects of Chilling and Anoxia on the Irradiation Dose-Response in Adult Aedes Mosquitoes.

The success of the sterile insect technique (SIT) relies on the achievement of high levels of sterility and mating success of the factory-reared sterile males and thus their biological quality, which can be enhanced by the reduction of stress factors encountered during rearing, handling, and irradiation procedures. The achievement of consistent sterility levels requires reliable and standard irradiation protocols. Additionally, mosquito adults require immobilization prior to, and during irradiation to increase processing efficiency and to avoid physical damage caused by movement in restricted space. Common methods for immobilization include chilling and anesthetics such as nitrogen. Here we assessed the effects of chilling and exposure to nitrogen on the irradiation dose-response of Aedes mosquitoes, and their downstream effects on some male quality parameters including longevity and flight ability. We found that chilling does not incur damage in the insects in terms of longevity and flight ability when chilling duration and temperature are carefully controlled, and a recovery phase is provided. Irradiation in nitrogen shows high radioprotective effects during irradiation, resulting in reduced induction of sterility. Overall, longevity of males can be improved by irradiating in anoxia, however the exposure to nitrogen itself comes with negative impacts on flight ability. The results reported here will assist in the standardization and optimization of irradiation protocols for the SIT to control mosquito populations of medical relevance.

Comprehensive Quantitative Evaluation of Variability in Magnetic Resonance-Guided Delineation of Oropharyngeal Gross Tumor Volumes and High-Risk Clinical Target Volumes: An R-IDEAL Stage 0 Prospective Study.

PURPOSE: Tumor and target volume manual delineation remains a challenging task in head and neck cancer radiation therapy. The purpose of this study was to conduct a multi-institutional evaluation of manual delineations of gross tumor volume (GTV), high-risk clinical target volume (CTV), parotids, and submandibular glands on treatment simulation magnetic resonance scans of patients with oropharyngeal cancer. METHODS AND MATERIALS: We retrospectively collected pretreatment T1-weighted, T1-weighted with gadolinium contrast, and T2-weighted magnetic resonance imaging scans for 4 patients with oropharyngeal cancer under an institution review board-approved protocol. We provided the scans to 26 radiation oncologists from 7 international cancer centers that participated in this delineation study. We also provide the patients' clinical history and physical examination findings, along with a medical photographic image and radiologic results. We used both the Simultaneous Truth and Performance Level Estimation algorithm and pair-wise comparisons of the contours, using overlap/distance metrics. Lastly, to assess experience and CTV delineation institutional practices, we had participants complete a brief questionnaire. RESULTS: Large variability was measured between observers' delineations for GTVs and CTVs. The mean Dice similarity coefficient values across all physicians' delineations for GTVp, GTVn, CTVp, and CTVn were 0.77, 0.67, 0.77, and 0.69, respectively, for Simultaneous Truth and Performance Level Estimation algorithm comparison, and 0.67, 0.60, 0.67, and 0.58, respectively, for pair-wise analysis. Normal tissue contours were defined more consistently when considering overlap/distance metrics. The median radiation oncology clinical experience was 7 years. The median experience delineating on magnetic resonance imaging was 3.5 years. The GTV-to-CTV margin used was 10 mm for 6 of 7 participant institutions. One institution used 8 mm, and 3 participants (from 3 different institutions) used a margin of 5 mm. CONCLUSIONS: The data from this study suggests that appropriate guidelines, contouring quality assurance sessions, and training are still needed for the adoption of magnetic resonance-based treatment planning for head and neck cancers. Such efforts should play a critical role in reducing delineation variation and ensure standardization of target design across clinical practices.

The third-generation P-glycoprotein inhibitor tariquidar may overcome bacterial multidrug resistance by increasing intracellular drug concentration.

OBJECTIVES: The use of efflux pump inhibitors may be a powerful strategy to overcome transporter-mediated bacterial multidrug resistance. In the present study, we set out to investigate the potency of tariquidar, a third-generation P-glycoprotein inhibitor in clinical development, for overcoming bacterial resistance towards ciprofloxacin. METHODS: Staphylococcus aureus 29213 (SA29213) and S. aureus 1199B (SA1199B), which overexpresses the multidrug transporter NorA, as well as Pseudomonas aeruginosa 27853 and Stenotrophomonas maltophilia BAA-85, which expresses SmeDEF, were exposed to ciprofloxacin in the presence and absence of tariquidar or, for comparative reasons, elacridar. Activity of both P-glycoprotein inhibitors was evaluated by determination of MICs and time-kill curves, and by quantification of uptake of ciprofloxacin into bacterial cells. RESULTS: Activity of tariquidar and elacridar was comparable for S. aureus strains, and both dose-dependently increased susceptibility towards ciprofloxacin. Highest effects were observed for SA1199B, where the addition of tariquidar resulted in a 10-fold reduction of the ciprofloxacin MIC, while no effect was observed for P. aeruginosa. For S. maltophilia, elacridar but not tariquidar improved susceptibility. Uptake of [14C]ciprofloxacin and modification of susceptibility showed significant correlations (r=0.89, P<0.0001). Tariquidar had no intrinsic activity against any strain tested. CONCLUSIONS: We conclude that tariquidar has potent inhibitory effect against certain bacterial efflux pumps in vitro. Their high activity at clinically achievable concentrations might yield this class of drugs promising for future applications in infectious diseases.

Chitosan effects on quality properties of Greek style fresh pork sausages.

The effect of chitosan (0.5% and 1%) added individually or in combination with nitrites (150ppm) on microbiological (Total Viable Counts, Lactic acid bacteria, Pseudomonas spp., Brochothrix thermosphacta, Enterobacteriaceae, yeasts and moulds), physicochemical-chemical (pH, chemical composition, lipid oxidation) and sensory properties of fresh pork sausages stored at 4°C for 28 days was investigated. Chitosan addition resulted in significant (p<0.05) inhibition of microbial growth, while nitrites did not seem to protect sausages from microbial spoilage. A gradual reduction of nitrites was observed till the end of storage, when nitrites were almost depleted in all nitrite containing samples. The rate of lipid oxidation in fresh pork sausages was significantly decreased (p<0.05) by addition of increasing levels of chitosan, while samples containing both chitosan and nitrites showed the lowest malondialdehyde (MDA) values, indicating a synergistic antioxidative effect. Consequently, the samples containing the combination of nitrites and chitosan at any level deteriorated less rapidly and were judged as more acceptable than all the other samples.

Temperature distribution and prevalence of Listeria spp. in domestic, retail and industrial refrigerators in Greece.

The present paper examined the presence of Listeria spp. in the environment of domestic, retail and industrial refrigerators. From 136 household refrigerators, 136 surface samples were taken from the walls or shelves, and 125 from cheese compartments. Only two refrigerators harboured L. monocytogenes. From 228 food store refrigerators, 335 samples were taken. Of these, 118 were in in contact with cheeses, 69 with sausages, 21 with cheese and sausages, 20 with miscellaneous products and 107 from refrigerator handles. Listeria spp. and L. monocytogenes were found in 3.1% and 1.7%, of the samples respectively. Listeria spp. was not detected in any of the nine dairy plant refrigerators examined. Listeria monocytogenes and L. innocua were found in 4.5 and 36.4%, respectively, of the 22 refrigerators inside meat processing plants, with only one of 22 refrigerators handles being positive for L. monocytogenes. Temperature distribution in the refrigerators was also investigated. Fifty five per cent of the 136 domestic and 32% of the 228 retail store refrigerators had temperatures of greater than or equal to 9 degrees C. The range of refrigeration temperatures of the industrial refrigerators was 0-2 degrees C for meat plants and 2-7 degrees C for dairy plants. No correlation of any kind could be established between the prevalence of Listeria spp. and the temperature of the various refrigerators due to the low number of positive samples.

Antibiotic resistance of Salmonella spp. and Listeria spp. isolates from traditionally made fresh sausages in Greece.

Sixty-five samples of traditionally made fresh sausages obtained from retail shops and butcher shops in northern Greece were screened for the presence of Salmonella spp. and Listeria spp. Salmonella spp. were found in 20% of the samples tested (54% Salmonella typhimurium and 46% Salmonella enteritidis). The prevalence of Listeria spp. in the samples was 26% (12% Listeria monocytogenes, 76% Listeria innocua, and 12% Listeria welshimeri). Nine of 13 Salmonella isolates were found to be resistant to ampicillin and 4 of 13 showed intermediate sensitivity; 1 of 13 was found to be resistant to chloramphenicol and 1 of 13 to tetracycline. Two strains of Salmonella typhimurum were multiresistant (resistant to ampicillin, chloramphenicol, and norfloxacin). All Listeria isolates were sensitive to the antibacterial agents tested that are commonly used for the treatment of human listeriosis.

Chemical and bacteriological characteristics of Pichtogalo Chanion cheese and mesophilic starter cultures for its production.

Sixty-two samples of Pichtogalo Chanion cheese traditionally produced in Crete, a few (3 to 6) days old, were analyzed for some chemical and bacteriological characteristics. The results of physicochemical analyses were as follows: (1) moisture content 61.63% (standard deviation 4.67); (2) fat in dry matter 54.03% (SD 7.73); (3) protein content 14.23% (SD 1.72); (4) salt content 1.02% (SD 0.38); (5) water activity (aw) 0.990 (SD 0.003); and (6) pH 4.36 (SD 0.25). None of the samples yielded Salmonella spp. Listeria monocytogenes and coagulase-positive staphylococci were present in 6.45% of the samples. Bacillus cereus and sulfite-reducing clostridia were isolated from 14.51% and 40.32% of the samples, respectively. High populations of coliforms were determined in the cheese samples. In 11.3% of the samples, Escherichia coli was not detected, while 88.7% of the samples yielded E. coli most probable number levels from 1.32 to 5.66 log10/g. The log10 CFU/g counts of enterococci were 6.89 (SD 0.84), of yeasts 6.79 (SD 0.61), of molds 4.68 (SD 0.69), and of psychrotrophic bacteria 7.63 (SD 0.62). The log10 CFU/g counts of lactic acid streptococci and lactococci were 7.91 (SD 0.68) and of lactobacilli 8.11 (SD 0.65). Lactic acid bacteria, mainly mesophilic, were isolated and confirmed using API 50 CH test trips. A pasteurized mixture of ewe's and goat's milk was made into Pichtogalo Chanion cheese according to standard procedure at 23 degrees C, after the addition of 4% commercial mesophilic starter culture or 2%, 3%, and 4% starter culture of the isolated and confirmed lactic acid bacteria and the addition of rennet. Results of this work indicated that high quality of Pichtogalo Chanion cheese can be produced using a pasteurized mixture of ewe's and goat's milk and 4% (vol/vol) of mesophilic starter culture.

Physicochemical, microbiological and sensory attributes for the characterization of Greek traditional sausages.

Physicochemical, microbiological and sensory analyses were performed on 67 samples of Greek traditional sausages. The following physicochemical attributes were recorded: moisture 49.17%±7.05, protein 17.62%±2.67, fat 29.74%±8.02 and ash content 2.99%±0.55, moisture/protein ratio 2.83±0.5. pH value 5.48±0.49, water activity (a(w)) 0.959±0.015, total grill losses 12.81%±5.27 and fat grill losses 9.64%±4.36. The microbial counts, expressed as log(10)cfu/g, were for aerobic plate count 8.22±0.5, lactic acid bacteria 7.45±0.66, Brochothrix thermosphacta 7.02±1.21, pseudomonads 6.88±1.33 and yeasts 5.39±1.03. Mean sensory scores, on a five-point hedonic scale, were 4.46±0.63 for appearance, 4.14±0.63 for firmness, 3.80±0.97 for flavour and 4.12±0.52 for overall quality. The discriminant analysis have shown that, based on their pH and a(w) values, 74.6% of sausages were classified as easily perishable, 19.4% as perishable and 5.9% as shelf-stable. Also, 4.4% of sausages had fat content less than 15%, 23.8% from 15 to 25%, 46.2% from 25 to 35% and 25.3% more than 35%. Principal component analysis has shown that the first two components (PC1 PC2) account for 44.1% of the total variance. PC1 was related to water activity, ash, moisture and fat content, flavour, Br. thermosphacta and pseudomonads count, and to a lesser extent to cross section quality. PC2 was related to aerobic plate count, lactic acid bacteria and moisture content.

Assessment of fruit juice authenticity using UPLC-QToF MS: a metabolomics approach.

In recent years, with the growing complexity of global food supply chains and trade, food fraud, including adulteration of high value foods with cheaper substitutes, has become an increasingly important issue. A metabolomics approach can be applied to discover biomarkers that can be used to trace food adulteration. A study was undertaken to discover novel, potential biomarkers for the rapid detection of the adulteration of fruit juices with cheaper alternatives. Pineapple, orange, grapefruit, apple, clementine, and pomelo were investigated. Untargeted metabolite fingerprinting was performed by UPLC-QToF MS with multivariate data analysis. Twenty-one differential metabolites were selected, contributing to the separation between pineapple, orange and grapefruit juices, and their admixtures down to 1% adulteration level. A targeted metabolomics method was then optimised and adulteration could be detected at 1%. The results demonstrate that metabolomics has potential as a screening tool for the rapid detection of food adulteration.

Corneal icterus resulting from stromal bilirubin deposition.

Three pairs of donor corneas were noted to be stained yellow after dissection from the whole globe. The peripheral cornea was stained more intensely than the central cornea in each case. All three of the donors had had jaundice with elevated serum bilirubin levels for at least 1 month before death. The corneas were finely minced and eluted in chloroform for a period of at least 1 week. Scleras from two of the three donors were similarly treated. Spectrophotometric analysis of the corneal and scleral eluates demonstrated the absorbance spectrum of bilirubin (400-490 nm). Identically prepared eluates of normal corneas and scleras from three donors did not show the absorbance spectrum of bilirubin. Results of slit-lamp examination of both eyes of a severely jaundiced patient disclosed yellow pigmentation of the corneal stroma with the peripheral cornea showing more pigmentation than the central cornea. Corneal bilirubin deposition, a previously undescribed finding, may occur in patients with jaundice.