Linoleic acid enrichment of cheese by okara flour and Geotrichum candidum overexpressing Δ12 fatty acid desaturase.

BACKGROUND: Mold-ripened cheeses have low levels of unsaturated fatty acids (UFAs). Geotrichum candidum is an adjunct culture for the development of Geotrichum-ripened cheese but has a low ability to produce high levels of UFAs. Δ12 fatty acid desaturase (FADS12) is a pivotal enzyme that converts oleic acid (OA) to linoleic acid (LA) and plays a vital role in UFA biosynthesis. By investigating FADS12 catalytic activity from various species with OA substrates, we found that FADS12 from Mucor circinelloides (McFADS12) had the highest catalytic activity for OA. RESULTS: In the current study, a plasmid harboring McFADS12 was constructed and overexpressed in G. candidum. Our results showed that LA production increased to 31.1 ± 1.4% in engineered G. candidum - three times higher than that in wild-type G. candidum. To enhance LA production, an exogenous substrate (OA) was supplemented, and the yield of LA was increased to 154 ± 6 mg L-1 in engineered G. candidum. Engineered G. candidum was used as an adjunct culture for Geotrichum-ripened cheese production. The LA level reached 74.3 ± 5.4 g kg-1 cheese, whereas the level of saturated fatty acids (SFAs) decreased by 9.9 ± 0.5%. In addition, the soybean byproduct (okara) was introduced into the engineered G. candidum growth and the level of LA increased to 126 ± 4 g kg-1 cheese and the percentage of UFAs:SFAs increased from 0.8:1 to 1.3:1. CONCLUSION: This study offers a suitable technology for converting SFAs to UFAs in Geotrichum-ripened cheeses and provides a novel trend for converting soybean waste into a value-added product. © 2022 Society of Chemical Industry.

Imaging separation of neuronal from vascular effects of cocaine on rat cortical brain in vivo.

MRI techniques to study brain function assume coupling between neuronal activity, metabolism and flow. However, recent evidence of physiological uncoupling between neuronal and cerebrovascular events highlights the need for methods to simultaneously measure these three properties. We report a multimodality optical approach that integrates dual-wavelength laser speckle imaging (measures changes in blood flow, blood volume and hemoglobin oxygenation), digital-frequency-ramping optical coherence tomography (images quantitative 3D vascular network) and Rhod(2) fluorescence (images intracellular calcium for measure of neuronal activity) at high spatiotemporal resolutions (30 µm, 10 Hz) and over a large field of view (3×5 mm(2)). We apply it to assess cocaine's effects in rat cortical brain and show an immediate decrease (3.5±0.9 min, phase 1) in the oxygen content of hemoglobin and the cerebral blood flow followed by an overshoot (7.1±0.2 min, phase 2) lasting over 20 min whereas Ca(2+) increased immediately (peaked at t=4.1±0.4 min) and remained elevated. This enabled us to identify a delay (2.9±0.5 min) between peak neuronal and vascular responses in phase 2. The ability of this multimodality optical approach for simultaneous imaging at high spatiotemporal resolutions permits us to distinguish the vascular versus cellular changes of the brain, thus complimenting other neuroimaging modalities for brain functional studies (e. g., PET, fMRI).

Enhancing detection of bladder carcinoma in situ by 3-dimensional optical coherence tomography.

PURPOSE: We examined the usefulness of 3-dimensional optical coherence tomography to enhance the diagnosis of urothelial carcinoma in situ. MATERIALS AND METHODS: By expressing SV40T antigen with uroplakin II promoter, carcinoma in situ readily develops in SV40T transgenic mice at about ages 8 to 20 weeks and then frank high grade papillary carcinoma develops in bladder epithelium. We examined 10 control and 40 SV40T mice during weeks 8 to 20 after birth by parallel en face white light imaging and 3-dimensional optical coherence tomography, and compared results with histology findings. We applied quantitative analysis of computer aided detection to 3-dimensional tomography images to enhance the diagnosis of carcinoma in situ, including 3-dimensional segmentation, speckle reduction, fast Fourier transform analysis, and standard deviation and histogram evaluation. RESULTS: We identified carcinoma in situ in 23 SV40T mice by histology. Most carcinoma could not be detected by en face imaging and 2-dimensional optical coherence tomography but was well differentiated by 3-dimensional optical coherence tomography. The 56.5% sensitivity and 61.5% specificity of 2-dimensional optical coherence tomography for carcinoma in situ diagnosis were significantly enhanced by 3-dimensional optical coherence tomography to 95.7% and 92.3%, respectively (p ≤0.031). CONCLUSIONS: On quantitative analysis of increased urothelial heterogeneity induced by carcinogenesis we noted that 3-dimensional optical coherence tomography enabled accurate differentiation of carcinoma in situ from normal bladder and benign lesions. Results reveal the potential of cystoscopic 3-dimensional optical coherence tomography to significantly enhance the clinical diagnosis of nonmuscle invasive bladder cancer, particularly carcinoma in situ.

Noninvasive and high-resolution optical monitoring of healing of diabetic dermal excisional wounds implanted with biodegradable in situ gelable hydrogels.

Closure of diabetic dermal chronic wounds remains a clinical challenge. Implant-assisted healing is emerging as a potential class of therapy for dermal wound closure; this advancement has not been paralleled by the development in complementary diagnostic techniques to objectively monitor the wound-healing process in conjunction with assessing/monitoring of implant efficacy. Biopsies provide the most objective morphological assessments of wound healing; however, they not only perpetuate the wound presence but also increase the risk of infection. A noninvasive and high-resolution imaging technique is highly desirable to provide objective longitudinal diagnosis of implant-assisted wound healing. We investigated the feasibility of deploying optical coherence tomography (OCT) for noninvasive monitoring of the healing of full-thickness excisional dermal wounds implanted with a novel in situ gelable hydrogel composed of N-carboxyethyl chitosan, oxidized dextran, and hyaluronan, in both normal and db/db mice. The results showed that OCT was able to differentiate the morphological differences (e.g., thickness of dermis) between normal and diabetic mice as validated by their corresponding histological evaluations (p < 0.05). OCT could detect essential morphological changes during wound healing, including re-epithelization, inflammatory response, and granulation tissue formation as well as impaired wound repair in diabetic mice. Importantly, by tracking specific morphological changes in hydrogel-assisted wound healing (e.g., implants' degradation and resorption, cell-mediated hydrogel degradation, and accelerated re-epithelization), OCT could also be deployed to monitor and evaluate the transformation of implanted biomaterials, thus holding the promise for noninvasive and objective monitoring of wound healing longitudinally and for objective efficacy assessment of implantable therapeutics in tissue engineering.

On the possibility of time-lapse ultrahigh-resolution optical coherence tomography for bladder cancer grading.

It has been recently demonstrated that the cellular details of bladder epithelium embedded in speckle noise can be uncovered with time-lapse ultrahigh-resolution optical coherence tomography (TL-uOCT) by proper time-lapse frame averaging that takes advantage of cellular micromotion in fresh biological tissue ex vivo. Here, spectral-domain 3-D TL-uOCT is reported to further improve the image fidelity, and new experimental evidence is presented to differentiate normal and cancerous nuclei of rodent bladder epithelia. Results of animal cancer study reveal that despite a slight overestimation (e.g., <10%) of nuclear size (D(N)) to histological evaluation, TL-uOCT is capable of distinguishing normal (D(N) approximately 7 microm) and cancerous (e.g., high-grade D(N(") ) approximately 13 microm) urothelia, which may potentially be very useful for enhancing the diagnosis of nonpapillary bladder cancer. More animal study is being conducted to examine the utility to differentiate hyperplasia, dysplasia, and carcinoma in situ.

Diagnosis of bladder cancer with microelectromechanical systems-based cystoscopic optical coherence tomography.

OBJECTIVES: To examine the utility and potential limitations of microelectromechanical systems-based spectral-domain cystoscopic optical coherence tomography (COCT) so as to improve the diagnosis of early bladder cancer. METHODS: An optical coherence tomography catheter was integrated into the single instrument channel of a 22F cystoscope to permit white-light-guided COCT over a large field of view (4.6 mm wide and 2.1 mm deep per scan at 8 frames/s) and 10-microm resolution. Intraoperative COCT diagnosis was performed in 56 patients, with a total of 110 lesions examined and compared with biopsied histology. RESULTS: The overall sensitivity of COCT (94%) was significantly higher than cystoscopy (75%, P = .02) and voided cytology (59%, P = .005); the major enhancement over cystoscopy was for low-grade pTa-1 cancer and carcinoma in situ (P < .018). The overall specificity of COCT (81%) was comparable to voided cytology (88.9%, P = .49), but significantly higher than cystoscopy (62.5%, P = .02). CONCLUSIONS: The microelectromechanical systems-based COCT, owing to its high resolution and detection sensitivity and large field of view, offers great potential for "optical biopsy" to enhance the diagnosis of nonpapillary bladder tumors and their recurrences and to guide bladder tumor resection.

Simultaneous imaging of cortical hemodynamics and blood oxygenation change during cerebral ischemia using dual-wavelength laser speckle contrast imaging.

A dual-wavelength laser speckle contrast imaging technique (DW-LSCI) is presented for simultaneous imaging of cerebral blood flow and hemoglobin oxygenation changes at high spatiotemporal resolutions. Experimental validation was performed using a rat transient forebrain ischemia model. The results showed that DW-LSCI was able to track detailed hemodynamic and metabolic changes induced by ischemia, i.e., decreased oxy- and total hemoglobin concentrations and blood flow as well as increased deoxy-hemoglobin concentration in the downstream regions, thus allowing us to distinguish cerebral arterial and venous flows. Simultaneous cerebral blood flow and oxygenation imaging at high spatiotemporal resolutions is crucial to the understanding of neural process and brain functions.

Quantification of cocaine-induced cortical blood flow changes using laser speckle contrast imaging and Doppler optical coherence tomography.

We present a dual-imaging technique combining laser speckle contrast imaging and spectral-domain Doppler optical coherence tomography to enable quantitative characterization of local cerebral blood flow (CBF) changes in rat cortex in response to drug stimulus (e.g., cocaine) at high spatiotemporal resolutions. To examine the utility of this new technique, animal experiments were performed to study the influences of anesthetic regimes (e.g., isoflurane, alpha-chloralose) on the pharmadynamic effects of acute cocaine challenge. The results showed that cocaine-evoked CBF patterns (e.g., increases in alpha-chloralose and decreases in isoflurane regimes) were quantitatively characterized, thus rendering it a potentially useful tool for imaging studies of brain functions.

A digital frequency ramping method for enhancing Doppler flow imaging in Fourier-domain optical coherence tomography.

A digital frequency ramping method (DFRM) is proposed to improve the signal-to-noise ratio (SNR) of Doppler flow imaging in Fourier-domain optical coherence tomography (FDOCT). To examine the efficacy of DFRM for enhancing flow detection, computer simulation and tissue phantom study were conducted for phase noise reduction and flow quantification. In addition, the utility of this technique was validated in our in vivo clinical bladder imaging with endoscopic FDOCT. The Doppler flow images reconstructed by DFRM were compared with the counterparts by traditional Doppler FDOCT. The results demonstrate that DFRM enables real-time Doppler FDOCT imaging at significantly enhanced sensitivity without hardware modification, thus rendering it uniquely suitable for endoscopic subsurface blood flow imaging and diagnosis.

High-resolution imaging diagnosis and staging of bladder cancer: comparison between optical coherence tomography and high-frequency ultrasound.

A comparative study between 1.3-microm optical coherence tomography (OCT) and 40-MHz high-frequency ultrasound (HFUS) is presented to enhance imaging of bladder cancers ex vivo. A standard rat bladder cancer model in which transitional cell carcinoma (TCC) was induced by intravesical instillation of AY-27 cells was followed independently with both OCT and HFUS, and the image identifications were compared to histological confirmations. Results indicate that both OCT and HFUS were able to delineate the morphology of rat bladder [e.g., the urothelium (low backscattering/echo) and the underlying lamina propria and muscularis (high backscattering/echo]. OCT differentiated inflammatory lesions (e.g., edema, infiltrates and vasodilatation in lamina propria, hyperplasia) and TCC based on characterization of urothelial thickening and enhanced backscattering or heterogeneity (e.g., papillary features), which HFUS failed due to insufficient image resolution and contrast. On the other hand, HFUS was able to stage large T2 tumors that OCT failed due to limited imaging depth. The results suggest that multimodality cystoscopy combining OCT and HFUS may have the potential to enhance the diagnosis and staging of bladder cancers and to guide tumor resection, in which both high resolution (approximately 10 microm) and enhanced penetration (> 3mm) are desirable.

Optical coherence Doppler tomography quantifies laser speckle contrast imaging for blood flow imaging in the rat cerebral cortex.

A dual-imaging modality is demonstrated for high-resolution quantitative imaging of local cerebral blood flow in the rat cortex by combining simultaneous spectral-domain Doppler optical coherence tomography (SDOCT) and full-field laser speckle contrast imaging (LSCI). Preliminary studies in tissue flow phantom and cocaine-induced cerebral blood flow changes indicated that by correlating coregistered cortical arterial blood flow, the relative measurement of flow changes by LSCI could be accurately calibrated by the absolute flow imaging provided by SDOCT (least square fit, r(2) approximately 0.96). Quantitative LSCI of cerebral blood flow is crucial to the quantitative analyses of the spatiotemporal hemodynamics of functional brain activations and thus improved understanding of neural process.

Assessment of dermal wound repair after collagen implantation with optical coherence tomography.

We present an animal study to examine the utility and potential limitations of optical coherence tomography (OCT) for noninvasive evaluation of biomaterial scaffold-assisted wound healing. The transverse and axial resolutions of the OCT system at the wavelength of 1.3 microm were 12 and 10 microm, respectively. A murine full-thickness transcutaneous wound model was employed, in which a phi 10 mm full-thickness wound was created on the back of each male Balb/cJ mouse and a porous collagen scaffold was implanted in the wound bed followed by coverage with a Tegaderm film. Sequential cross-sectional OCT scans were performed at different time points postsurgical intervention to track morphological changes during wound recovery, and the captured OCT images were validated by their corresponding histological specimens. The results indicated that with removal of the high-scattering skin, OCT was capable of imaging to a depth of over 1.5 mm into the wound bed and differentiating various features evolved during wound healing at a high resolution approaching histopathology. OCT was able to not only delineate the epidermis and dermis of normal mouse skin, but also differentiate collagen implant from the underlying subcutaneous tissue; besides, it could track the wound size changes in both lateral and vertical directions. More importantly, OCT was able to detect inflammation, early re-epithelialization, and resorption of the collagen scaffold. These findings suggested the potential of OCT for noninvasive and high-resolution monitoring of assisted wound healing in vivo, longitudinally, and instantaneously.

In vivo bladder imaging with microelectromechanical-systems-based endoscopic spectral domain optical coherence tomography.

We report the recent technical improvements in our microelectromechanical systems (MEMS)-based spectral-domain endoscopic OCT (SDEOCT) and applications for in vivo bladder imaging diagnosis. With the technical advances in MEMS mirror fabrication and endoscopic light coupling methods, the new SDEOCT system is able to visualize morphological details of the urinary bladder with high image fidelity close to bench-top OCT (e.g., 10 mum12 mum axial/lateral resolutions, >108 dB dynamic range) at a fourfold to eightfold improved frame rate. An in vivo animal study based on a porcine acute inflammation model following protamine sulfate instillation is performed to further evaluate the utility of SDEOCT system to delineate bladder morphology and inflammatory lesions as well as to detect subsurface blood flow. In addition, a preliminary clinical study is performed to identify the morphological features pertinent to bladder cancer diagnosis, including loss of boundary or image contrast between urothelium and the underlying layers, heterogeneous patterns in the cancerous urothelium, and margin between normal and bladder cancers. The results of a human study (91% sensitivity, 80% specificity) suggest that SDEOCT enables a high-resolution cross-sectional image of human bladder structures to detect transitional cell carcinomas (TCC); however, due to reduced imaging depth of SDEOCT in cancerous lesions, staging of bladder cancers may be limited to T1 to T2a (prior to muscle invasion).

Increasing the imaging depth of spectral-domain OCT by using interpixel shift technique.

A simple pixel shift technique is proposed to double the spectral sampling rate and enhance the signal to noise ratio of spectral-domain optical coherence tomography (SDOCT) in the 1.3 um wavelength range. Both theoretical analysis and experimental comparison are presented. The results show that interpixel shifted SDOCT can not only double the depth of field of SDOCT image but also eliminate the artifacts induced by aliasing effect, thus improving image contrast in areas with large depths (e.g., Delta z > or = 1.5 mm). If combined with endoscopic OCT, this technique has the potential to enhance in vivo diagnosis of biological tissues that require a larger field of view in the axial direction, such as cartilage degeneration and bladder tumors with deep asperities or invaginations.