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Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective: To assess differences in the sputum microbiota of community-acquired pneumonia (CAP) patients with either COPD or asthma, specifically focusing on a patient population in Turkey. Methods: This retrospective study included hospitalized patients > 18 years of age with a diagnosis of pneumonia between January of 2021 and January of 2023. Participants were recruited from two hospitals, and three patient groups were considered: CAP patients with asthma, CAP patients with COPD, and CAP patients without COPD or asthma. Results: A total of 246 patients with CAP were included in the study, 184 (74.8%) and 62 (25.2%) being males and females, with a mean age of 66 ± 14 years. Among the participants, 52.9% had COPD, 14.2% had asthma, and 32.9% had CAP but no COPD or asthma. Upon analysis of sputum cultures, positive sputum culture growth was observed in 52.9% of patients. The most commonly isolated microorganisms were Pseudomonas aeruginosa (n = 40), Acinetobacter baumannii (n = 20), Klebsiella pneumoniae (n = 16), and Moraxella catarrhalis (n = 8). CAP patients with COPD were more likely to have a positive sputum culture (p = 0.038), a history of antibiotic use within the past three months (p = 0.03), utilization of long-term home oxygen therapy (p < 0.001), and use of noninvasive ventilation (p = 0.001) when compared with the other patient groups. Additionally, CAP patients with COPD had a higher CURB-65 score when compared with CAP patients with asthma (p = 0.004). Conclusions: This study demonstrates that CAP patients with COPD tend to have more severe presentations, while CAP patients with asthma show varied microbial profiles, underscoring the need for patient-specific management strategies in CAP. [ABSTRACT FROM AUTHOR]

So far, there are still no specific treatment methods for severe hepatitis and liver failure, resulting in a mortality rate of over 70%, and they are the difficulties in the treatment of critical illness in China and globally. Liver transplantation is currently the most effective treatment method for end-stage liver disease, but only 1%—2% of patients can receive the opportunity for organ transplantation. The bioartificial liver support system utilizes external mechanical, physical, and biological devices to remove various harmful substances accumulated in the patient’s body, compensate for the metabolic functions of the liver, supplement necessary substances, improve internal environment, promote the recovery of liver function, help patients get through the critical period, and save time for liver transplantation, and therefore, it is considered one of the important methods for the treatment of end-stage liver disease. Since hepatocytes are the core element of bioartificial liver, this article summarizes the sources of liver seed cells，3D culture methods, and corresponding bioreactor culture systems and hopes to gradually solve the core issue of large-scale in vitro preparation of hepatocytes to obtain hepatocytes with adequate quantity and quality, which urgently needs to be addressed in clinical application. [ABSTRACT FROM AUTHOR]

ABSTRACT Objective: To assess differences in the sputum microbiota of community-acquired pneumonia (CAP) patients with either COPD or asthma, specifically focusing on a patient population in Turkey. Methods: This retrospective study included hospitalized patients > 18 years of age with a diagnosis of pneumonia between January of 2021 and January of 2023. Participants were recruited from two hospitals, and three patient groups were considered: CAP patients with asthma, CAP patients with COPD, and CAP patients without COPD or asthma. Results: A total of 246 patients with CAP were included in the study, 184 (74.8%) and 62 (25.2%) being males and females, with a mean age of 66 ± 14 years. Among the participants, 52.9% had COPD, 14.2% had asthma, and 32.9% had CAP but no COPD or asthma. Upon analysis of sputum cultures, positive sputum culture growth was observed in 52.9% of patients. The most commonly isolated microorganisms were Pseudomonas aeruginosa (n = 40), Acinetobacter baumannii (n = 20), Klebsiella pneumoniae (n = 16), and Moraxella catarrhalis (n = 8). CAP patients with COPD were more likely to have a positive sputum culture (p = 0.038), a history of antibiotic use within the past three months (p = 0.03), utilization of long-term home oxygen therapy (p < 0.001), and use of noninvasive ventilation (p = 0.001) when compared with the other patient groups. Additionally, CAP patients with COPD had a higher CURB-65 score when compared with CAP patients with asthma (p = 0.004). Conclusions: This study demonstrates that CAP patients with COPD tend to have more severe presentations, while CAP patients with asthma show varied microbial profiles, underscoring the need for patient-specific management strategies in CAP.

ABSTRACT We investigated whether the introduction of polymerase chain reaction (PCR) to replace culture as the primary diagnostic method for Campylobacter species in the Danish Departments of Clinical Microbiology (DCMs) was associated with a systematic change in the number of reported episodes of human campylobacteriosis from 2015 to 2022. We described a hypothetical scenario where PCR was solely used from 2018 to 2021. We analyzed the effect of primary diagnostic methods (culture or PCR) on the number of weekly episodes of human campylobacteriosis in a negative binomial regression adjusting for time, seasonality, COVID-19 restrictions, and DCMs. Furthermore, we applied the estimated PCR effect to the number of episodes that was identified using culture. Overall, PCR was associated with a 43% estimated increase of weekly campylobacteriosis episodes [rate ratio (RR) 1.43, 95% confidence interval (95% CI) 1.34–1.53]. Up to 17%–25% additional episodes would have been reported yearly from 2018 to 2021, had PCR been solely used. Through the lens of laboratory diagnostic methods, we described a systematic change in the number of reported episodes of human campylobacteriosis in Denmark. This is operationally relevant as considerably more episodes would have been identified had PCR been solely used. Changes in diagnostic methods should be considered in the epidemiological analysis of historical data. IMPORTANCE This study is important because it shows the potential epidemiological silence associated with the use of culture as the primary diagnostic method for the laboratory identification of human campylobacteriosis. Also, we show how polymerase chain reaction methods are associated with a systematic increase in the number of human campylobacteriosis episodes as reported by routine disease surveillance. These findings are operationally relevant and have public health implications because they tell how crucial it is to consider changes in diagnostic methods, e.g., in the epidemiological analysis of historical data and in the interpretation of future data in light of the past. We also believe that this study highlights how the synergy between microbiology and epidemiology is essential for disease surveillance.

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Turkiye Klinikleri Journal of Nursing Sciences is the property of Turkiye Klinikleri and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ObjectiveIntradiscal biologic therapy is a promising strategy for managing intervertebral disc degeneration. However, these therapies require a rich nutrient supply, which may be limited by the transport properties of the cartilage endplate (CEP). This study investigated how fluctuations in CEP transport properties impact nutrient diffusion and disc cell survival and function.DesignHuman CEP tissues harvested from six fresh cadaveric lumbar spines (38-66 years old) were placed at the open sides of diffusion chambers. Bovine nucleus pulposus (NP) cells cultured inside the chambers were nourished exclusively by nutrients diffusing through the CEP tissues. After 72 h in culture, depth-dependent NP cell viability and gene expression were measured, and related to CEP transport properties and biochemical composition determined using fluorescence recovery after photobleaching and Fourier transform infrared (FTIR) spectroscopy.ResultsSolute diffusivity varied nearly 4-fold amongst the CEPs studied, and chambers with the least permeable CEPs appeared to have lower aggrecan, collagen-2, and matrix metalloproteinase-2 gene expression, as well as a significantly shorter viable distance from the CEP/nutrient interface. Increasing chamber cell density shortened the viable distance; however, this effect was lost for low-diffusivity CEPs, which suggests that these CEPs may not provide enough nutrient diffusion to satisfy cell demands. Solute diffusivity in the CEP was associated with biochemical composition: low-diffusivity CEPs had greater amounts of collagen and aggrecan, more mineral, and lower cross-link maturity.ConclusionsCEP transport properties dramatically affect NP cell survival/function. Degeneration-related CEP matrix changes could hinder the success of biologic therapies that require increased nutrient supply.

Background and aimsGastrointestinal pathogen panels (GPPs) are increasingly being used for evaluation of diarrhea. The impact of these tests on patients with inflammatory bowel diseases (IBD) is unknown. We performed a time-interrupted cohort study comparing GPPs and conventional stool evaluation in patients with IBD with diarrhea.MethodsWe included 268 consecutive patients with IBD who underwent GPP (BioFire Diagnostics®) (n = 134) or conventional stool culture and Clostridium difficile polymerase chain reaction testing (n = 134) during suspected IBD flare between 2012 and 2016. Primary outcome was composite of 30-day IBD-related hospitalization, surgery, or emergency department visit; secondary outcome was IBD treatment modification.ResultsOverall, 41/134 (30.6%) patients tested positive on GPP (18 C. difficile, 17 other bacterial infections, and 6 viral pathogens) versus 14/134 patients (10.4%, all C. difficile) testing positive on conventional testing. Rate of IBD treatment modification in response to stool testing was lower in GPP group as compared conventional stool testing group (35.1 vs. 64.2%, p

In vitro miniaturized organoids are innovative tools with varying applications in biomedical engineering, such as drug testing, disease modeling, organ development studies, and regenerative medicine. However, conventional organoid development has several hurdles in reproducing and reconstituting organ-level functions in vitro, hampering advanced and impactful studies. In this review, we summarize the emerging microengineering-based organoid development techniques aiming to overcome these hurdles. First, we provide basic information on microengineering techniques, including those for reconstituting organoids with organ-level functions. We then focus on recent advances in microengineered organoids with better morphological, physiological, and functional characteristics than conventionally developed organoids. We believe that microengineered organoids possessing organ-level functions in vitro will enable widespread studies in the field of biological sciences and have clinical applications.

Background and Objectives: Isolating Helicobacter pylori (H. pylori) from wastewater and culturing it using a conventional method has always been a controversial issue because the bacterium converts into a coccoid form when exposed to an unfavourable environment like wastewater. To clarify the cultivability behaviour of the bacterium in fresh wastewater samples, the effect of municipal wastewater dilation on the cultivation of the bacterium using a conventional method was examined. Materials and Methods: Several dilutions of wastewater samples were inoculated with fresh H. pylori suspension (with McFarland's dilution 0.5) to examine the dilution effect of wastewater on the bacterium isolation. Results: The H. pylori growth was found to be possible for a dilution factor from 1/106 to 1/107 of raw wastewater. In higher dilution factors the growth of fungi was dominant and could prevent the isolation of the bacterium. Conclusion: The optimized technique could be applied in future studies for increasing the chance of H. pylori isolation from fresh wastewater environments. [ABSTRACT FROM AUTHOR]

Resumen: Introducción: La infección por Helicobacter pylori (H. pylori) afecta a más del 50% de la población mundial. El aumento en las resistencias antibióticas es la principal causa del fracaso del tratamiento. El objetivo principal fue analizar el éxito erradicador tras la aplicación de las nuevas recomendaciones de tratamiento ESPGHAN e introducción de la PCR como técnica de diagnóstico directo, describir la evolución del patrón local de resistencias antibióticas y valorar el coste-efectividad de la aplicación de la PCR aislada o en conjunto con el cultivo como estrategia diagnóstica. Pacientes y métodos: Estudio descriptivo retrospectivo del total de aislamientos microbiológicos de H. pylori entre 2013-2019 en nuestro centro, mediante comparación del porcentaje de resistencias y éxito erradicador entre los periodos 2013-2016 y 2017-2019. Estudio de coste-efectividad de las pruebas de diagnóstico directo, comparando 3 opciones distintas: cultivo y PCR, solo cultivo, y solo PCR. Resultados: Se incluyó a 192 pacientes, 98 fueron detectados por cultivo (2013-2016) y 94 por cultivo o PCR (2017-2019). Se instauró tratamiento antibiótico en 153 pacientes, 90 en el primer periodo (pautas ESPGHAN 2011: porcentaje erradicación 62,2%), 63 en el segundo (pautas ESPGHAN 2017: porcentaje erradicación: 73%). Se observó un aumento en las resistencias a claritromicina, pasando de un 16,3% (n = 16) en el primer periodo a un 53,2% (n = 48) entre 2017-2019 (98% detectadas por PCR, 60% por cultivo). No hubo diferencias en el resto de resistencias antibióticas. La solicitud aislada de la PCR presentó una ratio de análisis de coste-efectividad (CEAR) de 71,91, en comparación con un 92,16 del cultivo y un 96,35 del cultivo y la PCR de forma conjunta. Conclusiones: La aplicación de las pautas ESPGHAN 2017 consiguió un mayor éxito de erradicación, aunque menor que lo observado en publicaciones previas, sin llegar al objetivo marcado de al menos un 90%. Se observó un incremento en las resistencias a macrólidos, sin poder discriminar si se trata de un aumento real o de una mayor sensibilidad diagnóstica de las técnicas moleculares, siendo la solicitud aislada de la PCR la estrategia más coste efectiva. Abstract: Introduction: Helicobacter pylori (H. pylori) infection affects more than 50% of the world population. Increased antibiotic resistance is the main cause of treatment failure. The main objective was to analyze the eradication success after the application of the new ESPGHAN treatment recommendations and the introduction of PCR as a direct diagnosis technique, describe the evolution of the local pattern of antibiotic resistance, and assess the cost-effectiveness of PCR application, isolated or in conjunction with culture as a diagnostic strategy. Patients and methods: retrospective descriptive study of all microbiological isolates of H. pylori in 2013-2019 in our center, by comparing the percentage of resistance and eradication success between the periods 2013-2016 and 2017-2019. Cost-effectiveness study of direct diagnostic tests, comparing 3 different options: culture and PCR; only culture; PCR only. Results: 192 patients were included, 98 were detected by culture (2013-2016) and 94 by culture and / or PCR (2017-2019). Antibiotic treatment was established in 153 patients, 90 in the first period (2011 ESPGHAN guidelines: eradication percentage 62.2%), 63 in the second (2017 ESPGHAN guidelines: eradication percentage: 73%). An increase in resistance to clarithromycin was observed, going from 16.3% (n = 16) in the first period, to 53.2% (n = 48) in 2017-2019 (98% detected by PCR, 60% by culture). There were no differences in the rest of antibiotic resistances. The isolated PCR application presented a cost-effectiveness analysis ratio (CEAR) of 71.91, compared to 92.16 for the culture and 96.35 for the culture and PCR combined. Conclusions: the application of the ESPGHAN 2017 guidelines achieved greater eradication success, although less than that observed in previous publications, without reaching the target of at least 90%. An increase in resistance to macrolides was observed, without being able to discriminate whether it is a real increase or a greater diagnostic sensitivity of molecular techniques, with the isolated request for PCR being the most cost-effective strategy.

Background and Objectives: Isolating Helicobacter pylori (H. pylori) from wastewater and culturing it using a conventional method has always been a controversial issue because the bacterium converts into a coccoid form when exposed to an unfavourable environment like wastewater. To clarify the cultivability behaviour of the bacterium in fresh wastewater samples, the effect of municipal wastewater dilation on the cultivation of the bacterium using a conventional method was examined. Materials and Methods: Several dilutions of wastewater samples were inoculated with fresh H. pylori suspension (with McFarland's dilution 0.5) to examine the dilution effect of wastewater on the bacterium isolation. Results: The H. pylori growth was found to be possible for a dilution factor from 1/106 to 1/107 of raw wastewater. In higher dilution factors the growth of fungi was dominant and could prevent the isolation of the bacterium. Conclusion: The optimized technique could be applied in future studies for increasing the chance of H. pylori isolation from fresh wastewater environments.

Bacterial cellulose (BC) is a natural polymer secreted as a protective cell covering of certain bacterial species. In contrary to plant cellulose, BC possesses some unique features like high moisture-holding capacity, high durability, high liquid absorbing capabilities, biostability, and biodegradability, makes BC an excellent raw material in wide-ranging areas like biomedical, food, agriculture, paper, textile industries and electronics. The main objective of this review is to discuss various aspects of BC production (different sources for bacterial strain isolation, culture media and, its alternatives also major culture techniques). In addition, various applications of BC are also reviewed.

Introduction: A hospital-based investigation of bacteriological isolates helps to identify common causative bacteria and their antibiotic sensitivity patterns. This helps in formulating presumptive antibiotic therapy and in reducing antibiotic misuse. The aim of this study was to find out the prevalence of positive bacterial culture isolates among suspected orthopaedic infections in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted from the electronic data record of the Department of Microbiology of a tertiary care centre from 1 January 2017 to 31 December 2021. The study was conducted following ethical approval from the Institutional Review Committee (Reference number: IRC-2021-11-09-1). Culture reports of suspected orthopaedic infections were evaluated, and those with missing data were excluded. A convenience sampling method was used. Point estimate and 95% Confidence Interval were calculated. Results: Out of 6201 specimens, positive bacterial culture were found in 2957 (47.69%) (46.45-48.93, 95% Confidence Interval). Among them, 1561 (56.01%) were gram-negative organisms and 677 (24.29%) were gram-positive. A total of 2787 (94.25%) were wound/pus swab cultures and 170 (5.74%) were tissue cultures. Conclusions: The prevalence of positive bacterial culture among suspected orthopaedic infections was lower than in other international studies. Among bacteriological isolates, gram-negative organisms are more than gram-positive organisms.

To ensure graft persistence and thus to permanently restore the epithelial barrier function after grafting, bilayered tissue-engineered skin substitutes must be reconstructed in a way that favors the formation of an epithelial stem cell niche. It is however unclear what culture and production conditions, or combination thereof, are the most conducive to the establishment of a microenvironment that would be able to regulate epithelial stem cell quiescence and activity to appropriately satisfy the lifelong demand for differentiated cells. To address this issue, we undertook a systematic review of original clinical studies or case reports describing autologous tissue-engineered bilayered skin substitutes grafted in humans in the 25 years-span between August 1993 and August 2018. In total, we included 16 articles describing nine types of autologous bilayered skin substitutes. We examined four culture and production factors; keratinocyte seeding density, substitute scaffold type, whether serum or a serum replacement product was used to culture keratinocytes, and whether or not and what type of feeder layer was used to culture keratinocytes. Taken together, the data extracted from these 16 studies suggest that there are multiple combinations of culture and production factors that could result in the formation of a de novo epithelial stem cell niche in a bilamellar skin substitute.

Background: The authors established an in vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons. Methods: SCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination. Results: The cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media. Conclusions: Insulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy. [ABSTRACT FROM AUTHOR]

Platelets are terminally differentiated anucleated cells, but they still have cell-like functions and can even produce progeny platelets. However, the mechanism of platelet sprouting has not been elucidated so far. Here, we show that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed a spore phenomenon. The number of platelets increased when given a specific shear force. It is found that AMP-related signaling pathways, such as PKA and AMPK are activated in platelets in the spore state. Meanwhile, the mRNA expression levels of genes, such as CNN3 , CAPZB , DBNL , KRT19 , and ESPN related to PLS1 skeleton proteins also changed. Moreover, when we use the AMPK activator AICAR(AI) to treat washed platelets, cultured platelets can still appear spore phenomenon. We further demonstrate that washed platelets treated with Forskolin, an activator of PKA, not only platelet sprouting after culture but also the AMPK is activated. Taken together, these data demonstrate that AMPK plays a key role in the process of platelet budding and proliferation, suggesting a novel strategy to solve the problem of clinical platelet shortage.

Archaeal membrane lipids known as glycerol dibiphytanyl glycerol tetraethers (GDGTs) are the basis of the TEX86 paleotemperature proxy. Because GDGTs preserved in marine sediments are thought to originate mainly from planktonic, ammonia-oxidizing Thaumarchaeota, the basis of the correlation between TEX86 and sea surface temperature (SST) remains unresolved: How does TEX86 predict surface temperatures, when maximum thaumarchaeal activity occurs below the surface mixed layer and TEX86 does not covary with in situ growth temperatures? Here we used isothermal studies of the model thaumarchaeon Nitrosopumilus maritimus SCM1 to investigate how GDGT composition changes in response to ammonia oxidation rate. We used continuous culture methods to avoid potential confounding variables that can be associated with experiments in batch cultures. The results show that the ring index scales inversely (R(2) = 0.82) with ammonia oxidation rate (ϕ), indicating that GDGT cyclization depends on available reducing power. Correspondingly, the TEX86 ratio decreases by an equivalent of 5.4 °C of calculated temperature over a 5.5 fmol·cell(-1)·d(-1) increase in ϕ. This finding reconciles other recent experiments that have identified growth stage and oxygen availability as variables affecting TEX86 Depth profiles from the marine water column show minimum TEX86 values at the depth of maximum nitrification rates, consistent with our chemostat results. Our findings suggest that the TEX86 signal exported from the water column is influenced by the dynamics of ammonia oxidation. Thus, the global TEX86-SST calibration potentially represents a composite of regional correlations based on nutrient dynamics and global correlations based on archaeal community composition and temperature.

Background: There is a risk of bacterial contamination of nasal swabs during passage of the narrow nasal vestibule in patients carrying Staphylococcus aureus in their nares. We aimed to test if a newly developed contamination-free bacterial swab (CFS) device for swab introduction could reduce the risk of contamination with Staphylococcus aureus from the nasal vestibule in patients with acute upper respiratory tract infections. Methodology: A single-blinded non-randomized controlled trial that included 64 participants with acute upper respiratory tract infections. The left and right nasal cavities were swabbed using the present-day technique and the CFS device, respectively. Primary outcome was frequency of Staphylococcus aureus positive cultures; secondary outcome was growth of other bacteria. Results: We found a significantly higher frequency of Staphylococcus aureus in cultures taken with the present-day technique (23%) when compared to the new device (8%, p=0.008). Growth of other bacteria did not differ markedly between sample techniques. Conclusions: The newly developed device reduces contamination with Staphylococcus aureus significantly. It has the potential to increase diagnostic accuracy in acute upper respiratory tract infections, decrease the overall use of antibiotics and thereby counteract overuse of antibiotics and emerging antibiotic resistance.

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

ABSTRACT Objectives: to analyze the microbiological profile of leg ulcers of patients treated at outpatient clinics and hospitals regarding the type of microorganism, microbiological selection of antibiotics, and techniques for the collection of culture material. Methods: literature review performed on LILACS, IBECS, MEDLINE, and CINAHL databases, resulting in a descriptive analysis of 27 studies. Results: 35.7% of the studies occurred in an outpatient care scenario; and 64.2% in hospitals. There was a predominance of swab (100%) in outpatient care and biopsy (55.5%) in the hospital. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were more common at both levels of assistance. Methicillin-resistant Staphylococcus aureus was isolated in both. Conclusions: the microbiological profile of infections was similar, with the presence of resistant bacteria in both environments. This fact causes concern and raises the need for research to elucidate it. The studies did not compare the effectiveness between biopsy and swab.

Copyright of Medicina (Buenos Aires) is the property of Medicina (Buenos Aires) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Bacterial cellulose (BC) is a natural polymer secreted as a protective cell covering of certain bacterial species. In contrary to plant cellulose, BC possesses some unique features like high moisture-holding capacity, high durability, high liquid absorbing capabilities, biostability, and biodegradability, makes BC an excellent raw material in wide-ranging areas like biomedical, food, agriculture, paper, textile industries and electronics. The main objective of this review is to discuss various aspects of BC production (different sources for bacterial strain isolation, culture media and, its alternatives also major culture techniques). In addition, various applications of BC are also reviewed. [ABSTRACT FROM AUTHOR]

Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods.

Analysis of ion channel mutants is a widely used approach for dissecting ion channel function and for characterizing the mechanisms of action of channel-directed modulators. Expression of functional potassium channels in potassium-uptake-deficient yeast together with genetic selection approaches offers an unbiased, high-throughput, activity-based readout that can rapidly identify large numbers of active ion channel mutants. Because of the assumption-free nature of the method, detailed biophysical analysis of the functional mutants from such selections can provide new and unexpected insights into both ion channel gating and ion channel modulator mechanisms. Here, we present detailed protocols for generation and identification of functional mutations in potassium channels using yeast selections in the potassium-uptake-deficient strain SGY1528. This approach is applicable for the analysis of structure-function relationships of potassium channels from a wide range of sources including viruses, bacteria, plants, and mammals and can be used as a facile way to probe the interactions between ion channels and small-molecule modulators.

Schisandra henryi is an endemic species of medicinal potential known from traditional Chinese medicine. As part of this study, a complex biotechnological and phytochemical assessment was conducted on S. henryi with a focus on phenolic compounds and antioxidant profiling. The following in vitro cultures were tested: microshoot agar and callus, microshoot agitated, and suspension, along with the microshoot culture in PlantForm bioreactors. Qualitative profiling was performed by ultra-high-performance liquid chromatography with a photodiode array detector coupled with ion-trap mass spectrophotometry with electrospray ionization and then quantitative analysis by high-performance liquid chromatography with a diode array detector using standards. In the extracts, mainly the compounds from procyanidins were identified as well as phenolic acids (neochlorogenic acid, caffeic acid, protocatechuic acid) and catechin. The highest content of phenolic compounds was found for in vitro agar microshoot culture (max. total content 229.87 mg/100 g DW) and agitated culture (max. total content 22.82 mg/100 g DW). The max. TPC measured using the Folin-Ciocalteu assay was equal to 1240.51 mg GAE/100 g DW (agar microshoot culture). The extracts were evaluated for their antioxidant potential by the DPPH, FRAP, and chelate iron ion assays. The highest potential was indicated for agar microshoot culture (90% of inhibition and 59.31 nM/L TEAC, respectively). The research conducted on the polyphenol profiling and antioxidant potential of S. henryi in vitro culture extracts indicates the high therapeutic potential of this species. KEY POINTS: • Different types of S. henryi in vitro cultures were compared for the first time. • The S. henryi in vitro culture strong antioxidant potential was determined for the first time. • The polyphenol profiling of different types of S. henryi in vitro cultures was shown., (© 2024. The Author(s).)

Split-thickness skin autografts (AGs) are the standard surgical treatment for severe burn injuries. However, the treatment of patients with substantial skin loss is limited by the availability of donor sites for skin harvesting. As an alternative to skin autografts, our research group developed autologous self-assembled skin substitutes (SASSs), allowing the replacement of both dermis and epidermis in a single surgical procedure. The aim of the study was to assess the clinical outcome of the SASSs as a permanent coverage for full-thickness burn wounds. Patients were recruited through the Health Canada’s Special Access Program. SASSs were grafted on debrided full-thickness wounds according to similar protocols used for AGs. The graft-take and the persistence of the SASS epithelium over time were evaluated. 14 patients received surgical care with SASSs. The mean percentage of the SASS graft-take was 98 % (standard deviation = 5) at 5 to 7 d after surgery. SASS integrity persisted over time (average follow-up time: 3.2 years), without noticeable deficiency in epidermal regeneration. Assessment of scar quality (skin elasticity, erythema, thickness) was performed on a subset of patients. Non-homogeneous pigmentation was noticed in several patients. These results indicated that the SASS allowed the successful coverage of full-thickness burns given its high graft-take, aesthetic outcome equivalent to autografting and the promotion of long-term tissue regeneration. When skin donor sites are in short supply, SASSs could be a valuable alternative to treat patients with full-thickness burns covering more than 50 % of their total body surface area.

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Objective: Diagnosis of Leishmaniasis is based on culture, microscopic examination, serological tests, molecular methods. Novy-MacNeal-Nicolle (NNN) medium is mostly used for culture all over the world. The use of molecular methods for diagnostic purposes led to the questioning of the sensitivity of NNN medium. In this study, it is aimed to compare the performance of a new culture medium with those of NNN medium and the molecular method. Methods: Samples of 22 patients with suspected cutaneous leishmaniasis (CL) and 4 patients with suspected visceral leishmaniasis (VL) were sent to Manisa Celal Bayar University Faculty of Medicine Department of Medical Parasitology for diagnosis or confirmation from Kilis, Osmaniye, Bitlis, Gaziantep and Manisa provinces. Pre-prepared media were sent to the health centers in these provinces and inoculation of these media with clinical samples was requested. Needle aspiration fluid for CL and bone marrow samples for VL were cultured simultaneously on NNN medium and newly developed liver extracted medium (LEM), and were sent to our institution with their smears. Smears were examined by staining with Giemsa method. In addition, parasite DNA was identified with real-time polymerase chain reaction method in these materials. Results: Leishmania amastigotes were detected in 14 of 26 samples. Promastigote proliferation was observed in 8 and 24 of the samples cultured in NNN and LEM respectively. No proliferation was detected by either method in samples of 2 patients with suspected CL. L. tropica, L. major and L. infantum/donovani were detected among isolates showing dermotropic location, and L. tropica and L. infantum/donovani were detected among isolates with viscerotropic location by genotyping. Conclusions: NNN medium, which is widely used in leishmaniasis diagnosis, is not sensitive enough for the diagnosis of some Leishmania strains in Turkey. Therefore, an enrichment medium such as liver extract medium which we have presented in this study should be used for diagnosis. However our results should be confirmed with more studies. [ABSTRACT FROM AUTHOR]

• Samples obtained by aspiration and scraping of the edge of the lesion were analyzed. • Direct parasitological exam and three methods of axenic culture in two media were assessed. • The TSTB culture of scraping lesions had the highest diagnostic sensitivity (98 %). • Diagnosis by axenic culture should be implemented whenever possible. Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. In Bolivia, leishmaniasis occurs mainly in the cutaneous form (CL) followed by the mucosal or mucocutaneous form (ML or MCL), grouped as tegumentary leishmaniosis (TL), while cases of visceral leishmaniasis (VL) are rare. The cases of TL are routinely diagnosed by parasitological methods: Direct Parasitological Exam (DPE) and axenic culture, the latter being performed only by specialized laboratories. The aim of the present study was to optimize the parasitological diagnosis of TL in Bolivia, using two sampling methods. Samples from 117 patients with suspected TL, obtained by aspiration (n = 121) and scraping (n = 121) of the edge of the lesion were tested by: direct parasitological exam, culture in TSTB medium, and miniculture and microculture in Schneider's medium. A positive laboratory result by any of the four techniques evaluated using either of the two sampling methods was considered the gold standard. Of the 117 suspected patients included, TL was confirmed in 96 (82 %), corresponding 79 of the confirmed cases (82.3 %) to CL and 16 (16.7 %) to ML. Parasitological techniques specificity was 100 % and their analytical sensitivity was greater with scraping samples in TSTB culture (98 %). Scraping samples in TSTB and miniculture correlated well with the reference (Cohen's kappa coefficient=0.88) and showed good reliability (Cronbach's alpha coefficient ≥0.91). Microculture provided positive results earlier than the other culture methods (mean day 4.5). By day 14, 98 % of positive cultures had been detected. Scraping sampling and miniculture were associated with higher culture contamination (6 % and 17 %, respectively). Bacterial contamination predominated, regardless of the sampling and culture method, while filamentous fungi and mixed contamination were more frequently observed in cultures from scraping samples. In conclusion: (i) scraping samples proved more suitable for the diagnosis of TL as they increased analytical sensitivity, are less traumatic for the patient and are safer for laboratory personnel than aspirates; (ii) culture, mainly in TSBT medium, should be used for the diagnosis of TL due to its high sensitivity (doubling the number of cases diagnosed by DPE) and its low cost compared to other culture media. [Display omitted] [ABSTRACT FROM AUTHOR]

Bacterial cholangitis is a common complication in patients with ischemic type biliary lesions and/or anastomotic strictures after liver transplantation (LTX). Patients frequently need antibiotics and endoscopic retrograde cholangiography (ERC) to improve the bile flow. Antibiotic treatment is based on findings in standard microbiological cultivation (SMC) of bile. However, the cultivation techniques are limited to a subset of bacteria easy-to-cultivate. Therefore, the aim of our study was to evaluate the value of next generation sequencing as an additional diagnostic tool to SMC in ischemic type biliary lesions and/or anastomotic strictures. We sequenced the V1–V2 region of the 16S rRNA gene in 242 stored bile samples in patients after LTX and compared the results with findings of SMC. SMC was performed in n = 135 (56%) fresh bile samples in addition to NGS. SMC was part of the clinical routine in these patients. NGS detected bacterial genera in bile samples more often than SMC (P = 5.42 × 10−74). SMC showed insufficient discovery of bacterial genera compared to NGS with better performance in patients receiving antibiotics prior to ERC. SMC missed many bacterial genera detected by NGS. NGS was more sensitive in detecting bacteria in bile than SMC, no clinical parameters could be used to improve discovery rates in SMC and many genera were missed by SMC. Therefore, NGS should be used in a combined approach with SMC for improved diagnostics to achieve more specific and targeted antibiotic treatments. [ABSTRACT FROM AUTHOR]

Conventional polarization-sensitive optical coherence tomography (PS-OCT) can provide depth-resolved Stokes parameter measurements of light reflected from turbid media. A new algorithm that takes into account changes in the optical axis is introduced to provide depth-resolved birefringence and differential optical axis orientation images by use of fiber-based PS-OCT. Quaternion, a convenient mathematical tool, is used to represent an optical element and simplify the algorithm. Experimental results with beef tendon and rabbit tendon and muscle show that this technique has promising potential for imaging the birefringent structure of multiple-layer samples with varying optical axes.

The neurodegeneration induced by manganese has been attributed to its ability to undergo redox cycling, and catalysis of reactive oxygen species (ROS) formation, as with other transition metals. However, the characterization of manganese as a pro-oxidant is confounded by increasing evidence that the metal may scavenge superoxide anions and protect cells from oxidative damage. The current study was designed to address conflicting reports pertaining to the oxidative capacity of manganese. We found that the metal has distinctive redox dynamics in which the divalent reduced form, unlike iron, possessed no intrinsic oxidative capacity. The apparent ability of Mn(2+) to promote the formation of ROS within a cortical mitochondrial-synaptosomal fraction was quenched by the depletion of contaminating nanomolar concentrations of trivalent metals. The addition of manganic ions at trace concentrations dose-dependently restored the oxidative capacity attributed to divalent manganese, whereas the presence of the ferric ion retarded the rate of ROS generation. This result was paralleled by the spectrophotometric demonstration that the kinetics of iron oxidation is accelerated by trivalent but not divalent manganese. The markedly different capacities of the lower and higher valence states of manganese to promote free-radical formation in cortical fractions and to modulate the process of iron oxidation may account for earlier contradictory reports of anti- and pro-oxidant properties of manganese.

Genetic malleability and amenability to behavioral assays make Drosophila an attractive model for dissecting the molecular mechanisms of complex behaviors, such as learning and memory. At a cellular level, Drosophila has contributed a wealth of information on the mechanisms regulating membrane excitability and synapse formation, function, and plasticity. Until recently, however, these studies have relied almost exclusively on analyses of the peripheral neuromuscular junction, with a smaller body of work on neurons grown in primary culture. These experimental systems are, by themselves, clearly inadequate for assessing neuronal function at the many levels necessary for an understanding of behavioral regulation. The pressing need is for access to physiologically relevant neuronal circuits as they develop and are modified throughout life. In the past few years, progress has been made in developing experimental approaches to examine functional properties of identified populations of Drosophila central neurons, both in cell culture and in vivo. This review focuses on these exciting developments, which promise to rapidly expand the frontiers of functional cellular neurobiology studies in Drosophila. We discuss here the technical advances that have begun to reveal the excitability and synaptic transmission properties of central neurons in flies, and discuss how these studies promise to substantially increase our understanding of neuronal mechanisms underlying behavioral plasticity.

Long-term potentiation (LTP) was elicited by high frequency stimulation in hippocampal slices cultured on multi-electrode arrays. LTP lasting more than 1 h was recorded in 75% of slices, and a significant number of slices exhibited a non-decaying LTP that lasted more than 48 h. LTP induction was completely and reversibly blocked by an antagonist of the NMDA receptor, APV. Our results suggest the possibility of using chronic recording in hippocampal slices cultured on multi-electrode arrays to study the mechanisms underlying LTP maintenance and stabilization.

Efstathia Kiatos,1 James J Armstrong,2,3 Cindy ML Hutnik,3,4 Stephen M Tsioros,5 Monali S Malvankar-Mehta,1,4 William G Hodge1,4 1Department of Epidemiology and Biostatistics, 2Department of Pathology, 3Department of Ophthalmology, Schulich School of Medicine and Dentistry, Western University, 4Department of Ophthalmology, Ivey Eye Institute, St Joseph’s Health Care London, 5Department of Kinesiology, Western University, London, ON, Canada Purpose: This study evaluated the performance of donor corneoscleral rim cultures for predicting infection after corneal transplantation, and determines if there is a correlation between positive corneoscleral rim cultures and postkeratoplasty infection.Design and data sources: This was a systematic review, prognostic accuracy analysis, and cost-effectiveness analysis. Databases searched were: Medline (Ovid), Embase (Ovid), CINAHL, Cochrane Library, Web of Science, and BioSis Previews. Grey literature was also explored.Materials and methods: A systematic review was conducted to locate published and unpublished studies. All studies examining corneal button contamination and its association with endophthalmitis and keratitis posttransplantation were included. Extracted data were used to calculate sensitivity, specificity, positive predictive value, and negative predictive value. Cost data from the London Laboratory Services Group in London, ON were used to calculate the cost-effectiveness of culturing donor rim cultures.Results: Of 7,870 grafts, 954 had a positive rim culture (12.1%), with 12 patients going on to develop keratitis or endophthalmitis (1.3%). The prevalence of keratitis and endophthalmitis in this study was 0.15%, and the positive predictive value 1.5%. Of the 12 infections, nine were fungal and three bacterial. The estimated cost of a positive and negative test result was CAD$45.99 and $14.15, respectively. The cost to run all 7,870 tests was estimated to be $141,735.86, with an incremental cost-effectiveness ratio of $40,215.70.Conclusion: There was a significant divergence between bacterial and fungal rim-culture results. Bacterial cultures predicted clinical infection poorly, did not change management, and were expensive. Fungal cultures predicted clinical infection in over 10% of patients, had the potential to change management, and were 40% less expensive than full rim culturing (bacterial and fungal tests). Fungal rim cultures may be considered in areas where fungal infection rates are high. Keywords: corneoscleral rim cultures, eye infection, keratoplasty, culture techniques

Cultured hippocampal slices prepared from apolipoprotein E-deficient mice were exposed to an inhibitor of cathepsins B and L and then processed for immunocytochemistry using antibodies against human paired helical filaments. Dense, AT8-immunopositive deposits were found in the subiculum, stratum oriens of hippocampal field CA1, and the hilus of the dentate gyrus. This distribution agrees with that described for tangles in Alzheimer's disease. The appearance of the labeled structures fell into categories that correspond to previously proposed stages in the progression of intraneuronal neurofibrillary tangles in human hippocampus. Electron microscopic analyses confirmed that microtubule disruption and twisted bundles of filaments were present in neurons in the affected areas. These results support the hypothesis that partial lysosomal dysfunction is a contributor to Alzheimer's disease and suggest a simple model for studying an important component of the disease.

This study reports on the development of an original, ex-vivo wounded skin culture protocol using autologous Platelet Rich Plasma (PRP) and enriched Dulbecco's Modified Eagle's Medium (DMEM). Human skin samples obtained from specimens harvested during reduction mammoplasty procedures, were injured in their central portion—to create a standard wound—and cultured under three different conditions:– enriched DMEM with saline solution in the central wound (control)– enriched DMEM with the same medium in the central wound– enriched DMEM plus 2.5% autologous PRP, with the same PRP added medium in the central wound.Morphological analysis was carried out at 0 h (T0) and on days 1, 3, 5 and 10 (T1-T3-T5-T10) using Hematoxylin and Eosin; Masson's trichrome staining; Weigert staining and Ki-67 staining to identify the skin histological features in the different experimental conditions. The combination of DMEM and PRP allowed a favorable modulation of the epithelial cells and fibroblasts proliferation, and a relevant anti-inflammatory action. PRP also demonstrated an inhibitory effect on both the collagen and elastic fibers' de-structuration and a favorable modulation of the re-organization of these fibers. The step by step histological and immune-histo-chemical regenerative effects of PRP on human skin wound repair and regeneration process was observed over a period of 10 days.

Importance: This study is important because it shows the potential epidemiological silence associated with the use of culture as the primary diagnostic method for the laboratory identification of human campylobacteriosis. Also, we show how polymerase chain reaction methods are associated with a systematic increase in the number of human campylobacteriosis episodes as reported by routine disease surveillance. These findings are operationally relevant and have public health implications because they tell how crucial it is to consider changes in diagnostic methods, e.g., in the epidemiological analysis of historical data and in the interpretation of future data in light of the past. We also believe that this study highlights how the synergy between microbiology and epidemiology is essential for disease surveillance., Competing Interests: The authors declare no conflict of interest.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Organotypic slice cultures provide several advantages over primary and cell line culture models. Unlike monolayer cultures, organotypic slice cultures retain the tissue's 3-D architecture, multi-lineage cell populations, and many complex functional characteristics. Organotypic slice cultures can be exposed to biological, pharmacological, or environmental agents and analyzed to predict in vivo responses. Herein, we describe a method for preparing organotypic slice cultures generated from rat placenta., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gastric mucosal microvessels were isolated after arterial perfusion of the rat stomach with magnetized iron oxide suspension. After homogenization of scrapped gastric mucosa, microvessels were initially separated with a high power magnet and further separated and purified by using a nylon sieve. Aliquots of purified microvessels were assessed for viability, histologic appearance, ultrastructure and generation of prostacyclin. Microvessels were plated on Matrigel and cultured in DMEM with high glucose and 10% FBS for 1, 3 or 5 days. After 1, 3 and 5 days of culturing, endothelial viability was assessed with Fast green exclusion, and the basal and stimulated (with calcium ionophore) generation of prostacyclin was determined by assaying aliquots of the incubating medium for 6-keto PGF(1alpha). At 1 and 3 hrs after isolation, microvessels demonstrated intact morphologic structures as reflected by transmission EM and 92+/-4% of viable endothelial cells. The microvessels plated on Matrigel maintained good viability for at least 5 days and generated prostacyclin at the baseline and following ionophore stimulation. These data demonstrate that isolated microvessels cultured under optimal conditions are fully viable and functional.