Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

INTRAOCULAR EMULSION OF SILICONE OIL (ITEMS) GRADING SYSTEM: An Evidence-Based Expert-Led Consensus.

PURPOSE: To propose the InTraocular EMulsion of Silicone oil (ITEMS) grading system for the assessment of silicone oil (SiO) emulsion, applicable in a routine clinical setting and validated through an expert-led consensus procedure. METHODS: Seven experts on intraocular liquid tamponades, led by a facilitator, performed a literature review on the detection of SiO emulsion. Based on the proposed ideas, a questionnaire was developed and submitted to the experts on the methods to detect SiO emulsion and the items to grade. After 2 rounds of individual ranking using a 9-point scale and related discussion, the final grading system was developed including items that reached consensus (score ≥7 from ≥75% of members). RESULTS: The agreed ITEMS grading system includes the identification of SiO microbubbles and large SiO bubbles through slit-lamp biomicroscopy, gonioscopy, fundus examination under mydriasis, or ultra-wide-field fundus photography. Moreover, macular and disk optical coherence tomography are used to detect SiO-associated hyperreflective dots. CONCLUSION: An evidence-based expert-led consensus was conducted to develop grading system of SiO emulsion, allowing, for the first time, homogenous collection of data on SiO emulsion. This has the potential to improve the understanding of the role and clinical relevance of SiO emulsion, allowing comparisons between different studies.

Oncological monitoring after transanal total mesorectal excision (TaTME) for rectal neoplasia.

BACKGROUND: The surgical treatment of choice for rectal neoplasia is total mesorectal excision (TME). The transanal approach enables a better approach in male and obese patients and/or those with a narrow pelvis and in patients with small tumors. Short-term results are comparable with those for laparoscopy or the open approach, but the medium- and long-term oncological data are sparse. The aim of the present study was to evaluate our early experience with transanal TME (TaTME). METHODS: This was a retrospective study conducted on patients who underwent TaTME at our center between August 2013 and April 2017 with a follow-up ≥ 3 years. Histopathology, complications, mortality, neoplastic recurrence and disease-free survival were analyzed. RESULTS: One hundred patients (68 men and 32 women,, median age 66.8 years [range 29.6-91.2 years]) were included. There were 67 T3 cases (67%) with 74 N0 cases (74%), the mesorectal quality was graded optimal for 87.6% and only 2 cases of radial margin involvement were detected (2%). The median follow-up period was 47.6 months (range 11.8-78.9 months). Eighteen cases of recurrence were diagnosed, of which 3 (3%) recurred locally with an average disease-free period of 43.1 months. Overall survival was 80% and mortality due to progression of disease was 13%. CONCLUSIONS: TaTME is a safe surgical procedure with surgical, anatomopathological and oncological results at 3 years (medium-term) comparable with those for the laparoscopic and open approaches. Better monitoring is required with studies of the long-term functional and quality of life outcomes, i.e., at 5 or 10 years.

Hair cortisol level as a molecular biomarker in retinitis pigmentosa patients.

PURPOSE: Retinitis pigmentosa (RP) patients commonly experience negative psychological states due to their progressive and unpredictable loss of vision and visual variations related to stress. The aim of this study was to examine hair cortisol concentrations (HCCs), which is usually associated with chronic stress, pretending to unveil possible associations between underlying psychological factors and disease severity in RP patients. METHODS: Seventy-eight RP patients and 148 healthy controls were included in this study. A complete ophthalmological exam was performed in all patients to grade into severity disease groups. Perceived stress and trait-anxiety were measured by the State-Trait Anxiety Inventory (STAI) questionnaire. RESULTS: Fifty-two (67%) patients had severe RP and 26 (33%) mild-moderate RP. Fifty-eight (58,9%) patients reported severely levels of stress and 18 (23.,1%) highly levels assessed by STAI questionnaire. RP patients exhibited higher HCCs (500.04 ± 120.99 pg/mg) than in controls (136.17 ± 60.51 pg/mg; p < 0.001). Severe RP patients had significant higher HCCs than mild-moderate patients differing in 274.27 pg/mg (p < 0.001). RP severity grade and perceived anxiety levels in the questionaries were not associated. Group differences were not affected by relevant covariates (age, grade of severity, stress status, and gender). CONCLUSIONS: HCC seems an effective biomarker associated with chronic stress in RP patients. This study shows that HCC in patients with RP are elevated compared to population-based controls, and association between HCC and RP severity was found. Future research is needed to characterize the effect of untreated negative psychological states on progression of the disease if any.

Nanoplastics are bioaccumulated in fish liver and muscle and cause DNA damage after a chronic exposure.

The extent of the widespread, planetary contamination by plastic waste is difficult to fully capture. Nanoplastics (NPs) are currently in the center of research concerning plastic litter, both for the analytical challenges they pose and for their potential to provoke hazardous effects in organisms. However, there are still many unanswered questions in this multidisciplinary field, with a crucial missing piece being the quantification of NPs in fish tissues after in vivo exposures. Another relevant question that is still greatly unexplored is how a chronic exposure to NPs will affect fish health. This study aims to provide answers to both of these relevant knowledge gaps. To this end, goldfish (Carassius auratus) were exposed to 44 nm polystyrene (PS)-NPs via water for 30 days. Following the exposure, gastrointestinal tract, liver and muscle were sampled for PS-NPs analysis by means of size exclusion chromatography coupled to high resolution mass spectrometry. PS-NPs were detected in all liver and muscle samples of exposed fish, with higher concentrations in liver than in muscle, whereas no PS-NPs were detected in the gastrointestinal tract. Nevertheless, exposure to PS-NPs did not induce changes in hematology parameters nor in cortisol and glucose levels in plasma. On the other hand, even a relatively low concentration of PS-NPs was able to cause DNA damage, measured by an increase in erythrocyte nuclear abnormalities, suggesting that PS-NPs can reach the cell nucleus and cause genotoxicity. These results show for the first time that PS-NPs find their way to fish muscle after chronic exposure, where they bioaccumulate, but do not alter fish survival nor hematological or physiological stress indicators. The accumulation of PS-NPs in fish muscle can represent a threat to human health as a possible route of exposure to small-sized plastics. The present results in a model fish species open windows for future studies in edible fish species.

Simulation of feed restriction and fasting: Effects on animal recovery and gastrointestinal permeability in unweaned Angus-Holstein calves.

Feed restriction and fasting experienced during commercial production negatively affect unweaned calves' behavior and health status. Transportation and stays at assembly centers are the main factors generating these disorders. For this study, 20 unweaned Angus-Holstein bull calves [44.1 ± 2.04 kg of body weight (BW) and 14.7 ± 0.63 d of age (± standard error)] were used to evaluate the effects of feed restriction and fasting on performance, energy status [serum concentration of glucose, ß-hydroxybutyrate (BHB), and nonesterified fatty acids], and gastrointestinal permeability [serum concentration of citrulline, chromium (Cr)-EDTA, lactulose, and d-mannitol]. Calves were randomly assigned to 1 of 4 treatments that simulated the feed restrictions of an assembly center situation on one hand, and the fasting hours during transportation on the other. Treatments were as follows. Control (CT): from d -4 to -1, calves were fed 2.5 L of milk replacer (MR) twice daily; concentrate and straw were offered ad libitum. Mild (MD): calves were fed only MR (d -4 to -1) as described for CT, and on d -1 calves were subjected to a 9-h feed withdrawal. Moderate (MO): calves were fed only MR (d -4 to -1) as described for CT and on d -1 subjected to a 19-h feed withdrawal. Severe (SV): calves were fed only 2.5 L of a rehydrating solution twice daily (d -4 to -1) and on d -1 subjected to a 19-h feed withdrawal. From d 0 to d 42 (weaning) all calves were fed the same feeding program (MR, concentrate, and straw ad libitum). Results showed that BW was greater for the CT treatment compared with the others from d 0 to d 7, whereas BW of SV was lesser compared with the others from d -1 to d 7. No differences among treatments were observed at weaning. At d 2 concentrate intakes of MD, MO, and SV were lesser compared with CT. By d 4, concentrate intake of SV was similar to that for CT and greater than MD and MO. Similarly to BW, no differences in concentrate intake among treatments were observed at weaning on d 42 of the study. At d -1 for SV and d 0 in all restricted calves, serum glucose concentration was lesser compared with CT. At d -1 and 0, nonesterified fatty acids and BHB serum concentrations were greater in the SV calves compared with the other treatments. By d 2, serum concentrations of nonesterified fatty acids, BHB, and glucose were restored to CT levels. At d -1 serum citrulline concentration was lesser in SV and greater in MD calves. The CT calves had lower serum concentrations of Cr-EDTA (d -1 and d 0), lactulose (d 0), and d-mannitol (d 0) compared with the other restricted calves. Results showed that degree of dietary restriction, type of liquid diet (MR or rehydrating solution), and fasting hours (9 vs. 19 h) affected calves' BW, concentrate intake, and serum concentration of markers indicative of energy status and gastrointestinal permeability.

Usefulness of histology for predicting infection at the time of hip and knee revision in patients with rheumatoid arthritis.

INTRODUCTION: It remains unclear whether rheumatoid arthritis might be a cause of false positive of the histology for the diagnosis of prosthetic joint infection. Our aim was to evaluate the usefulness of the histology for the diagnosis of infection during hip and knee prosthesis revision in patients with rheumatoid arthritis. MATERIALS AND METHODS: All patients with the diagnosis of rheumatoid arthritis (RA) undergoing hip or knee revision surgery (total or partial) were retrospectively reviewed. Positive histology was considered when ≥ 5 neutrophils per high-power field (400×) were found in at least five separate microscopic fields. Patients who presented ≥ 2 positive cultures for the same microorganism or the presence of fistula were considered as "true positives". RESULTS: Thirty-two hip (n = 12) and knee (n = 20) revision procedures were performed. Sensitivity, specificity, positive and negative predictive value of the histology were 50%, 78.6%, 25% and 91.7%, respectively. Six out of the eight patients presenting with positive histology had negative cultures (75.0% of false positives). CONCLUSIONS: Our results suggest that, in the context of RA, negative histological results have a very high negative predictive value. RA poses false positive histology results for the diagnosis of infection during hip and knee revision when conventional cultures are used for diagnosis of infection.

Inter-annual and spatial climatic variability have led to a balance between local fluctuating selection and wide-range directional selection in a perennial grass species.

BACKGROUND AND AIMS: The persistence of a plant population under a specific local climatic regime requires phenotypic adaptation with underlying particular combinations of alleles at adaptive loci. The level of allele diversity at adaptive loci within a natural plant population conditions its potential to evolve, notably towards adaptation to a change in climate. Investigating the environmental factors that contribute to the maintenance of adaptive diversity in populations is thus worthwhile. Within-population allele diversity at adaptive loci can be partly driven by the mean climate at the population site but also by its temporal variability. METHODS: The effects of climate temporal mean and variability on within-population allele diversity at putatively adaptive quantitative trait loci (QTLs) were evaluated using 385 natural populations of Lolium perenne (perennial ryegrass) collected right across Europe. For seven adaptive traits related to reproductive phenology and vegetative potential growth seasonality, the average within-population allele diversity at major QTLs (HeA) was computed. KEY RESULTS: Significant relationships were found between HeA of these traits and the temporal mean and variability of the local climate. These relationships were consistent with functional ecology theory. CONCLUSIONS: Results indicated that temporal variability of local climate has likely led to fluctuating directional selection, which has contributed to the maintenance of allele diversity at adaptive loci and thus potential for further adaptation.

Chemical compounds causing severe acute toxicity in heavy liquids used for intraocular surgery.

Perfluorocarbon liquids (PFCLs) have been considered safe for intraocular manipulation of the retina, but since 2013 many cases of acute eye toxicity cousing blindness have been reported in various countries when using various commercial PFCLs. All these PFCLs were CE marked (Conformité Européenne), which meant they had been subjected to evaluation complying with the International Organization for Standardization (ISO) guidelines. These dramatic events raised questions about the safety of PFCLs and the validity of some cytotoxicity tests performed under ISO guidelines. Samples from toxic batches were analyzed by gas chromatography-mass spectrometry combined with Raman and infrared spectrometry. Perfluorooctanoic acid, dodecafluoro-1-heptanol, ethylbenzene and tributyltin bromide were identified and evaluated by a direct contact cytotoxicity test using ARPE-19 cell line, patented by our group (EP 3467118 A1). Perfluorooctanoic acid at a concentration of >0.06 mM and tributyltin bromide at a concentration of ≥0.016 mM were shown to be toxic, whereas the concentration found in the toxic samples reached 0.48 mM, and 0.111 mM, respectively. These finding emphasized the idea that determination of partially fluorinated compounds are not enough to guarantee the safety of these medical devices.

Matrix Metalloproteinases in Age-Related Macular Degeneration (AMD).

Age-related macular degeneration (AMD) is a complex, multifactorial and progressive retinal disease affecting millions of people worldwide. In developed countries, it is the leading cause of vision loss and legal blindness among the elderly. Although the pathogenesis of AMD is still barely understood, recent studies have reported that disorders in the regulation of the extracellular matrix (ECM) play an important role in its etiopathogenesis. The dynamic metabolism of the ECM is closely regulated by matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). The present review focuses on the crucial processes that occur at the level of the Bruch's membrane, with special emphasis on MMPs, TIMPs, and the polymorphisms associated with increased susceptibility to AMD development. A systematic literature search was performed, covering the years 1990-2020, using the following keywords: AMD, extracellular matrix, Bruch's membrane, MMPs, TIMPs, and MMPs polymorphisms in AMD. In both early and advanced AMD, the pathological dynamic changes of ECM structural components are caused by the dysfunction of specific regulators and by the influence of other regulatory systems connected with both genetic and environmental factors. Better insight into the pathological role of MMP/TIMP complexes may lead to the development of new strategies for AMD treatment and prevention.

History of thyroid surgery.

The authors present an outline of the development of thyroid surgery from the ancient times to the beginning of the 20th century, when the defini-tive surgical technique have been developed and the physiologic and pathopfysiologic consequences of thyroid resections have been described. The key representatives, as well as the contribution of the most influential czech surgeons are mentioned.

Structural characterization of bulk and nanoparticle lead halide perovskite thin films by (S)TEM techniques.

Lead halide (APbX3) perovskites, in polycrystalline thin films but also perovskite nanoparticles (NPs) has demonstrated excellent performance to implement a new generation of photovoltaic and photonic devices. The structural characterization of APbX3 thin films using (scanning) transmission electron microscopy ((S)TEM) techniques can provide valuable information that can be used to understand and model their optoelectronic performance and device properties. However, since APbX3 perovskites are soft materials, their characterization using (S)TEM is challenging. Here, we study and compare the structural properties of two different metal halide APbX3 perovskite thin films: bulk CH3NH3PbI3 prepared by spin-coating of the precursors in solution and CsPbBr3 colloidal NPs synthetized and deposited by doctor blading. Both specimen preparation methods and working conditions for analysis by (S)TEM are properly optimized. We show that CH3NH3PbI3 thin films grown by a one-step method are composed of independent grains with random orientations. The growth method results in the formation of tetragonal perovskite thin films with good adherence to an underlying TiO2 layer, which is characterized by a photoluminescence (PL) emission band centered at 775 nm. The perovskite thin films based on CsPbBr3 colloidal NPs, which are used as the building blocks of the film, are preserved by the deposition process, even if small gaps are observed between adjacent NPs. The crystal structure of CsPbBr3 NPs is cubic, which is beneficial for optical properties due to its optimal band gap. The absorption and PL spectra measured in both the thin film and the colloidal solution of CsPbBr3 NPs are very similar, indicating a good homogeneity of the thin films and the absence of aggregation of NPs. However, a particular care was required to avoid long electron irradiation times during our structural studies, even at a low voltage of 80 kV, as the material was observed to decompose through Pb segregation.

Influence of physical activity on tibial bone material properties in laying hens.

Laying hens develop a type of osteoporosis that arises from a loss of structural bone, resulting in high incidence of fractures. In this study, a comparison of bone material properties was made for lines of hens created by divergent selection to have high and low bone strength and housed in either individual cages, with restricted mobility, or in an aviary system, with opportunity for increased mobility. Improvement of bone biomechanics in the high line hens and in aviary housing was mainly due to increased bone mass, thicker cortical bone and more medullary bone. However, bone material properties such as cortical and medullary bone mineral composition and crystallinity as well as collagen maturity did not differ between lines. However, bone material properties of birds from the different type of housing were markedly different. The cortical bone in aviary birds had a lower degree of mineralization and bone mineral was less mature and less organized than in caged birds. These differences can be explained by increased bone turnover rates due to the higher physical activity of aviary birds that stimulates bone formation and bone remodeling. Multivariate statistical analyses shows that both cortical and medullary bone contribute to breaking strengthThe cortical thickness was the single most important contributor while its degree of mineralization and porosity had a smaller contribution. Bone properties had poorer correlations with mechanical properties in cage birds than in aviary birds presumably due to the greater number of structural defects of cortical bone in cage birds.

Structural and molecular study of the supraspinatus muscle of modern humans (Homo sapiens) and common chimpanzees (Pan troglodytes).

OBJECTIVES: To analyze the muscle architecture and the expression pattern of the myosin heavy chain (MyHC) isoforms in the supraspinatus of Pan troglodytes and Homo sapiens in order to identify differences related to their different types of locomotion. MATERIALS AND METHODS: We have analyzed nine supraspinatus muscles of Pan troglodytes and ten of Homo sapiens. For each sample, we have recorded the muscle fascicle length (MFL), the pennation angle, and the physiological cross-sectional area (PCSA). In the same samples, by real-time quantitative polymerase chain reaction, we have assessed the percentages of expression of the MyHC-I, MyHC-IIa, and MyHC-IIx isoforms. RESULTS: The mean MFL of the supraspinatus was longer (p = 0.001) and the PCSA was lower (p < 0.001) in Homo sapiens than in Pan troglodytes. Although the percentage of expression of MyHC-IIa was lower in Homo sapiens than in Pan troglodytes (p = 0.035), the combination of MyHC-IIa and MyHC-IIx was expressed at a similar percentage in the two species. DISCUSSION: The longer MFL in the human supraspinatus is associated with a faster contractile velocity, which reflects the primary function of the upper limbs in Homo sapiens-the precise manipulation of objects-an adaptation to bipedal locomotion. In contrast, the larger PCSA in Pan troglodytes is related to the important role of the supraspinatus in stabilizing the glenohumeral joint during the support phase of knuckle-walking. These functional differences of the supraspinatus in the two species are not reflected in differences in the expression of the MyHC isoforms.

Retinal Toxicity of Medical Devices Used during Vitreoretinal Surgery: A Critical Overview.

Retinal toxicity/biocompatibility of medical devices in direct contact with the retina is an important subject for clinicians and scientists. As these effects are not very frequent, there is also a relative lack of information for many clinicians. The past has taught us multiple times that there is a significant safety problem associated with severe loss of vision in affected patients. In this review, we want to classify medical products that are used in the back of the eye, describe recent examples of toxicity, critically reflect on the regulations that exist and suggest improvements that can be done to ensure patient safety without hindering innovation. METHODS: Critical review of the recent papers and personal experience of the authors in this issue. Medical devices used in the back of the eye and recent examples of toxicity are described, regulations that exist are critically reflected and improvements suggested that can ensure patient safety without hindering innovation. RESULTS: There is clear evidence of toxicity after intraocular surgery in any category. Some cytotoxic indirect methods have failed in detecting this toxicity. Some ISO rules do not seem appropriate. Postmarketing safety is missing. There is little data on this issue. CONCLUSIONS: The absence of a clear regulation of the production, purification and evaluation of the toxic effects of the medical devices supposes the possibility that products are not sufficiently safe to obtain the CE mark.

A cryptic species produced by autopolyploidy and subsequent introgression involving Medicago prostrata (Fabaceae).

Although hybridisation through genome duplication is well known, hybridisation without genome duplication (homoploid hybrid speciation, HHS) is not. Few well-documented cases have been reported. A possible instance of HHS in Medicago prostrata Jacq. was suggested previously, based on only two genes and one individual. We tested whether this species was formed through HHS by sampling eight nuclear loci and 22 individuals, with additional individuals from related species, using gene capture and Illumina sequencing. Phylogenetic inference and coalescent simulations were performed to infer the causes of gene tree incongruence. We found no evidence that phylogenetic differences among M. prostrata individuals were the result of HHS. Instead, an autopolyploid origin of tetraploids with introgression from tetraploids of the M. sativa complex is likely. We argue that tetraploid M. prostrata individuals constitute a new species, characterised by a partially non-overlapping distribution and distinctive alleles (from the M. sativa complex). No gene flow from tetraploid to diploid M. prostrata is apparent, suggesting partial reproductive isolation. Thus, speciation via autopolyploidy appears to have been reinforced by introgression. This raises the intriguing possibility that introgressed alleles may be responsible for the increased range exploited by tetraploid M. prostrata with respect to that of the diploids.

Optical contrast of 2D InSe on SiO2/Si and transparent substrates using bandpass filters.

The particular optical and electronic properties recently reported for 2D InSe depict this 2D material as being very versatile for future electronic and optoelectronic devices with tunable and optimized functionalities. For its fundamental study and the development of practical applications, rapid and accurate identification methods of atomically thin InSe are essential. Here, we demonstrate an enhancement of the optical contrast between InSe nanosheets and the underlying SiO2/Si substrate by illuminating with a 40 nm wide bandpass filter centered at 500 nm. Moreover, we study the optical contrast of 2D InSe on transparent substrates. Our results suggest that a good optical contrast is achieved for transparent substrates with low real refractive indices such as LiF or a viscoelastic polydimethylsiloxane stamp. In this case, an optimum optical contrast would be achieved by using a bandpass filter centered at 450 nm. These results can be very useful for speeding up the continuously growing research on 2D InSe and its applications.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

Short communication: Promotion of glucagon-like peptide-2 secretion in dairy calves with a bioactive extract from Olea europaea.

Diarrhea episodes in dairy calves involve profound alterations in the mechanism controlling gut barrier function that ultimately compromise intestinal permeability to macromolecules, including pathogenic bacteria. Intestinal dysfunction models suggest that a key element of intestinal adaptation during the neonatal phase is the nutrient-induced secretion of glucagon-like peptide (GLP)-2 and associated effects on mucosal cell proliferation, barrier function, and inflammatory response. Bioactive molecules found in Olea europaea have been shown to induce the release of regulatory peptides from model enteroendocrine cells. The ability to enhance GLP-2 secretion via the feeding of putative GLP-2 secretagogues is untested in newborn calves. The objectives of this study were to determine whether feeding a bioactive extract from Olea europaea (OBE) mixed in the milk replacer (1) can stimulate GLP-2 secretion beyond the response elicited by enteral nutrients and, thereby, (2) improve intestinal permeability and animal growth as well as (3) reduce the incidence of diarrhea in preweaning dairy calves. Holstein heifer calves (n = 60) were purchased, transported to the research facility, and blocked by body weight and total serum protein and assigned to 1 of 3 treatments. Treatments were control (CON), standard milk replacer (MR) and ad libitum starter; CON plus OBE added into MR at 30 mg/kg of body weight (OBE30); and CON plus OBE added into MR at 60 mg/kg of body weight (OBE60). The concentration of GLP-2 was measured at the end of wk 2. Intestinal permeability was measured at the onset of the study and the end of wk 2 and 6, with lactulose and d-mannitol as markers. Treatments did not affect calf growth and starter intake. Compared with CON, administration of OBE60 increased the nutrient-induced response in GLP-2 by about 1 fold and reduced MR intake during the second week of study. Throughout the study, however, all calves had compromised intestinal permeability and a high incidence of diarrhea. The GLP-2 response elicited by OBE60 did not improve intestinal permeability (lactulose-to-d-mannitol ratio) and incidence of diarrhea over the course of the preweaning period. The response in GLP-2 secretion to the administration of OBE reported herein warrants further research efforts to investigate the possibility of improving intestinal integrity through GLP-2 secretion in newborn calves.

A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells.

PURPOSE: To develop and standardize a novel organ culture model using porcine central neuroretina explants and RPE cells separated by a cell culture membrane. METHODS: RPE cells were isolated from porcine eyes, expanded, and seeded on the bottom of cell culture inserts. Neuroretina explants were obtained from the area centralis and cultured alone (controls) on cell culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane. Finally, cellular and tissue specimens were processed for phase contrast, cyto-/histological, and immunochemical evaluation. Neuroretina thickness was also determined. RESULTS: Compared to the neuroretinas cultured alone, the neuroretinas cocultured with RPE cells maintained better tissue structure and cellular organization, displayed better preservation of photoreceptors containing rhodopsin, lower levels of glial fibrillary acidic protein immunoexpression, and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis. Neuroretina thickness was significantly greater in the cocultures. CONCLUSIONS: A coculture model of central porcine neuroretina and RPE cells was successfully developed and standardized. This model mimics a subretinal space and will be useful in studying interactions between the RPE and the neuroretina and to preclinically test potential therapies.