Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcome. The current study presents a simple, fast, and uniform surface tension based technique that can be implemented for lyopreservation of mammalian cells. The technique involves withdrawing cells attached to rigid substrates to be submerged in a solution of lyoprotectant and then withdrawing the samples at a specific rate to an inert environment. This creates a uniform thin film of desiccated lyoprotectant due to sudden change of surface tension. The residual moisture contents at different locations in the desiccated film was quantified using a spatially resolved Raman microspectroscopy technique. Post-desiccation cellular viability and growth are quantified using fluorescent microscopy and dye exclusion assays. Cellular injury following desiccation is evaluated by bioenergetic quantification of metabolic functions using extracellular flux analysis and by a Raman microspectroscopic analysis of change in membrane structure. The technique developed here addresses an important bottleneck of lyoprocessing which requires the fast and uniform desiccation of cellular samples. Storage of biologics and cellular material using lyopreservation has the potential to simplify logistics and transportation by reducing the need for cold-chain logistics. Development of such a technique for mammalian cells can have a significant impact in clinical application of advanced cell-based therapies, particularly in resource limited regions , 2. The Lyopreservation is believed to involve use of glass forming agents, such as trehalose, during acute desiccation to impart stability to the biomolecules. There are two important obstacles related to successful lyopreservation of mammalian cells: first being overcoming the processing injury for the cells, followed by storage in desiccated state. The principal concerns which must be considered for lyopreservation are imparting desiccation tolerance , creatinWhile it is important to explore techniques to increase desiccation tolerance of cells using different chemical strategies, it is important to develop strategies to desiccate mammalian cells that minimize cellular injury . Injury In this study, we have developed a surface-tension mediated fast drying technique that can be used to desiccate mammalian cells attached to a substrate with highly uniform residual moisture content. When cells attached to glass substrate are withdrawn from a solution of lyoprotectant to an inert environment, the sudden change of surface tension creates a uniform thin film of uniformly desiccated lyoprotectant. Commonly known as dip-coating, the technique is widely applied for ultraclean drying of semiconductor substrates in the electronics industry –16.A Raman microspectroscopic technique was used to determine both residual moisture content as well as spatial uniformity of the lyoprocessed samples. Analysis of the desiccated samples indicate significantly low moisture content as well as high spatial uniformity. Viability and bioenergetic assays were to demonstrate post-desiccation cellular health. Finally, a detailed microspectroscopic study of the change in chemical composition of the cell membrane was undertaken to investigate the injury to the cellular membrane structure or composition.2, and 37°C). Cells were maintained at a confluency of 90% using 75 cm2 cell culture flasks . Cells were collected using trypsinization followed by centrifugation. A hemocytometer  was used to count live cells using Trypan Blue exclusion technique. Collagen coated microscope slides  were outfitted with FastWell™ reagent barriers  to create a defined cellular boundary on the glass substrate and prevent any non-standard attachment conditions. The cell suspension was then pipetted into the wells at a final concentration of 150k cells/well.Human hepatocellular carcinoma (HepG2) cells were cultured in Opti-MEM  supplemented with 5% fetal bovine serum (Gibco) and penicillin-streptomycin  under standard cell culture conditions . The stock solution for Syto-13/ethydium bromide staining was prepared by adding 10 mL of 1 mg/mL Syto-13 solution (aq.) and 5 mL of 1.0 mg/mL ethydium bromide solution (aq.) to 8 mL of OptiMEM (Thermofisher Scientific). After rehydration, 100 μL of Syto-13/ethydium bromide solution was added to the cells attached on coverslips, and the samples were incubated at 37°C for 5 min. These samples were then imaged using an inverted microscope  with FITC and PI filters. Cell membrane integrity was determined immediately after rehydration by counting intact membranes (green) and membrane compromised (red) cells in five representative images obtained at different locations on the slide.Long-term viability and growth profiles were determined by incubating the rehydrated cell samples in fully complemented medium for 7 days in 100 mm culture dishes. Parallel samples were used for the various experimental treatments, and the viability of cells after desiccation was measured by counting cells on days 1, 3, and 7. To ensure that only viable cells were counted, the membrane integrity of these cells was determined by trypan blue exclusion.4 cells per well. Both oxygen consumption rate (OCR)  and extracellular acidification rate (ECAR) (representing glycolysis) measurements were taken using standard Seahorse protocol, following optimization. Briefly, XFp cartridge sensors were hydrated and injection ports loaded by the following; mitochondrial test reagents used oligomycin (1 μM), carbonyl-cyanide-4-(trifluoromethoxy) phenylhydrazone , and rotenone/antimycin A (0.5 μM). Glycolysis test reagents used D-glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose . All reagents have corresponding final concentrations in cell chambers immediately after being listed. Oligomycin was used as the F0F1-ATPase inhibitor and oxygen consumption rates measured in presence of this inhibitor indicate proton leak. FCCP acts as an uncoupling agent that collapses the mitochondrial proton gradient and thereby uncouples oxidation from phosphorylation, maximizing oxygen consumption rates. The contribution of energy generation via non-mitochondrial processes was quantified in relation to the overall oxygen flux using rotenone and antimycin A. Rotenone and antimycin A inhibit complex I and III, respectively, of the respiratory system. Glucose was supplied as to fully saturate basal ECAR. Then, since oligomycin inhibits ATP synthase in the electron transport chain, ATP/ADP ratio decreases, which stimulates glycolysis. 2-DG is a glucose analog which competitively inhibits hexokinase, effectively arresting glycolysis.Analysis of metabolic function for both dip-coated and unprocessed cells were carried out on an extracellular flux analyzer . Following dip-coating, the cell samples were rehydrated immediately in growth medium and cultured for 14 days. Then cells were trypsinized and counted using trypan blue exclusion. Cells were seeded in prescribed microwell plates at a seeding density of 2x10Raman microspectroscopic measurements for both residual water content and membrane function were conducted with a customized confocal microscope and Raman spectrometer combination . Raman spectra were collected using an EMCCD camera  at a spatial resolution of 500 nm. A 532 nm solid state laser calibrated to 20 mW was used for excitation and spectra were collected using a custom 50X high resolution objective .A Raman microspectroscopy technique was used to correlate residual moisture content in desiccated samples. The desiccated samples were transferred to a custom made sealed quartz chamber immediately following dip-coating at the four drawing rates using 1.2 M trehalose solution. This was done with a view to minimize the effect of interference from atmospheric humidity. The trehalose films were then scanned at arbitrarily preselected points to acquire average spectrum for each condition. A sample processed at 100 mm/min was analyzed by a linear Raman scanning method across a region of 22 mm, with a linear spectra polling rate every 30 μm. Raman signatures were collected from a location close (2–5 μm) to the glass substrate. The ratio of OH stretching peak (~3430 rel. 1/cm) to a characteristic trehalose peak  was used to quantify residual water. Such spectroscopic data collected from different spatial locations was used to profile uniformity of the residual water content in desiccated samples.3360:I923) and the water content (gH20/gDW), gravimetrically obtained.Solutions of trehalose buffer in water were created on a weight by weight basis ranging from 0 to 55%. A 100% sample was created by drying a 55% droplet in a 210°C oven for 24 hr. Samples were scanned via Raman spectroscopy to generate a calibration curve relating the spectral intensity of water to trehalose  and after 18 hr in a vacuum (-25 in. Hg) oven at 210°C  was used to reduce the dimensionality of the collected spectral data arrays into principal components (PCs). A non-linear iterative partial least squares algorithm was used for related vector analysis to eliminate noise in the spectral baseline . FollowiData were analyzed with a one-way analysis of variance (ANOVA) on ranks of experimental groups. Origin Pro 2017  was used for the analyses. Data sets are presented as mean ± (SD).H20/gDW). The 100 mm/min full film dataset (H20/gDW) across the full film length of the coated substrate, indicative of high spatial uniformity. High uniformity was also observed for the 50 mm/min condition, while the 150 and 200 mm/min conditions displayed greater instability in film evolution.The spatial uniformity of the dry-processed samples was quantified using a Raman microspectroscopic technique as described before. A trehalose concentration of 1.2 M was used in the desiccation solution as lesser molarities produced poorer cellular viability outcomes upon rehydration, while higher molarities produced greater instability in film formation upon drying.  dataset  shows veWhen the desiccation processed samples were placed in a desiccation box, the loss of moisture after two hours was minimal indicating that the surface tension mediated desiccation technique developed here is highly efficient in desiccating the samples . By desiPost processing membrane integrity is quantified by fluorescent microscopy in Metabolic profiles of the dip-coated cells are presented in The effect of desiccation on the molecular components of the cell membrane was investigated using Raman microspectroscopy and hyperspectral imaging techniques. Spatially resolved spectral masks (shown as green pixels) representing signatures of the cell membranes obtained through hierarchal cluster analysis (HCA) were developed  for threFinally, taking the ratio of the CH2 asymmetric peak at 2940 rel. 1/cm and amide III at 1320 rel. 1/cm shows the strong correlation between unprocessed and 100 mm/min processed cell membrane characteristics, which are significantly different to the 50 mm/min processed cells.Desiccated storage of biological samples is an alternative to low temperature preservation. Desiccated state storage is widely observed in nature among the cryptobiotic organisms and development of comparable biopreservation strategies can facilitate easy and economic transport of stored biologics including cells. In recent years, several lyoprocessing techniques have been developed for desiccated-state preservation of cells including freeze-drying , spray dFor successful lyopreservation it is important for the cells to survive the process of desiccation with minimal injury. Fast lyoprocessing techniques that limit the duration of exposure to non-physiological hyperosmotic environments have been shown to improve survival in mammalian cells following desiccation processes when compared with passive slow drying techniques , 13, 26.While fast drying is an important factor in determining post-desiccation survival of cells in lyoprocessing, spatial heterogeneity of the desiccated matrix have also been hypothesized to play a crucial role in preventing loss of viability following desiccation . Local n2>0.99) of residual moisture content from Raman spectral intensity ratio. This allows for easy conversion of sensitive Raman spectral detection into otherwise indeterminate moisture change. Spatially resolved Raman microspectroscopic characterization of the residual moisture content in the desiccated samples here demonstrate high processing uniformity  plane. Such depth scans show that the trehalose films had consistent depth across the stable processed areas, including cellular regions. It follows that cells were fully enveloped in the trehalose film. A representative hyperspectral image  shows thImmediate membrane integrity following cellular processing can indicate the extent of cellular injury caused by this technique. Both the membrane integrity of the cells immediately following desiccation processing  and metaA detailed investigation of cellular energetic characteristics of the cells processed using 100 mm/min indicated no appreciable difference in metabolic properties  when comLong term growth following lyoprocessing indicates the propensity for cellular proliferation. Comparing with an unprocessed control group shows the growth rate to be about halved, but still growing steadily. While the bioenergetics of the cell were found to be unaffected, potential for growth was diminished. This reduced capacity may be due to residual chemical triggers left by the lyoprocessing impetus. This may be improved by intracellular stabilizers introduced into growth or desiccation medium, but is left for future studies.Improvements in the dry processing outcomes may also depend on pre-processing strategies in addition to the cellular density parameters. There is also a significant scope of improvement considering the pre-processing strategies involved that can be used to ameliorate processing stresses in the cells. Here fluid phase endocytosis was used to introduce trehalose in the intracellular space. While it has been demonstrated that trehalose plays an important role in protecting the cells from desiccation stresses when present on both sides of the cell membrane , in receChanges in membrane composition and related injury have a strong impact in survival of the desiccated mammalian cells , 33. DesContinuing development and optimization of the surface tension based drying technique described is a viable technique to lyoprocess mammalian cells. The technique is capable of desiccating cell samples attached to a substrate rapidly and uniformly. The cellular injury during processing can be minimized by changing the processing parameters that are easy to modulate and automate. While the current study focuses on the drying aspect only, significant work needs to be done to store the desiccated sample at non-cryogenic or ambient temperatures. Based on the stability requirement during storage, the processing parameters developed in the technique developed here can be modulated to suit a wide range of biologics for lyoprocessing.S1 FigQuartz plates were processed and immediately scanned by Raman. They were dried by drierite (2 hr) and vacuum oven (18 hr) and scanned after both drying steps. Taking a ratio of the OH stretching (~3300 rel. 1/cm) band to the CH2 stretching (~2940 rel. 1/cm) band gives an estimate of water to dry weight. Comparing spectral ratios of initial and dried conditions shows that dry box had marginal effects on water content and vacuum drying for the 100 and 150 mm/min conditions. The 50 mm/min condition had an apparent increase in moisture content after baking, while the 200 mm/min condition lost residual moisture. A ratio of 1 indicates no moisture change upon drying.(TIF)Click here for additional data file.S2 FigA) Depth scan separated by HCA analysis, directionally, orange being air and brown being glass plate. B) Polarized light micrograph of cells in coating. Line 1–2 shows scanning position of A. C) Raman spectra color matched to A. The cell is predominantly in blue region and the trehalose coating is magenta.(TIF)Click here for additional data file.


Photodynamic therapy (PDT) is a promising modern approach for cancer therapy with low normal tissue toxicity. This study was focused on a vascular-targeting Chlorine E6 mediated PDT. A new angiographic imaging approach known as M-mode-like optical coherence angiography (MML-OCA) was able to sensitively detect PDT-induced microvascular alterations in the mouse ear tumour model CT26. Histological analysis showed that the main mechanisms of vascular PDT was thrombosis of blood vessels and hemorrhage, which agrees with angiographic imaging by MML-OCA. Relationship between MML-OCA-detected early microvascular damage post PDT (within 24 hours) and tumour regression/regrowth was confirmed by histology. The advantages of MML-OCA such as direct image acquisition, fast processing, robust and affordable system opto-electronics, and label-free high contrast 3D visualization of the microvasculature suggest attractive possibilities of this method in practical clinical monitoring of cancer therapies with microvascular involvement. Mechanisms of tumour tissue damage in photodynamic therapy (PDT) are still a subject of scientific research134The temporal dynamics are also complex in that each type of reaction has its own characteristic time. The blood vessels response is typically the earliest, developing within minutes/tens of minutes from the beginning of the treatment, and can in fact be detected even during the PDT process itself36To improve PDT effect on tumours, minimize the tumour regrowth, decrease collateral normal tissue damage and improve cosmetic outcomes, it is important to develop approaches to a real-time noninvasive monitoring of tumour response to optimize treatment delivery parameters. There are ‘explicit’ and ‘implicit’ classes of PDT dosimetry. The complicated interaction between light, photosensitizer and tumour microenvironment in PDT makes ‘explicit’ dosimetry a dynamic and difficult problem28102The vascular-targeted regime is mainly responsible for some of the more successful clinical implementations of PDT today13Blood flow dynamics can be studied with laser speckle interferometry7192021in vivo. The key goal of the study was to find criteria to predict early tumour response to PDT based on MML-OCA visualization of microvascular network within 24 hours after treatment.In this study, we hypothesized that the early tumour response to PDT may manifest itself as the disappearance of microvascular network on MML-OCA images due to vascular damage and blood flow disruption. The study was performed on the ear model of the murine colon carcinoma CT26. Tumour microvascular and growth were assessed during the 7 days following Photoditazin  PDT, and histopathology with hematoxylin-eosin (H&E) staining was performed at the final time point to analyze cellular and vascular damages. Accumulation of the photosensitizer in the tumour and its photobleaching dynamics were also detected by fluorescence imaging 3, both between and within the animal groups. Still, 9 of 11 tumours (#1–9) showed significant volume decrease (#1–7) by 7 days after PDT, with their TGI metric  varying from 147% to 165%  tumour cells. The tumour tissue had a dense structure and consisted of polymorphic cells with diameter varying between 10 and 20 μm and round or oval nuclei. The slightly basophilic cytoplasm formed a thin ring around the nucleus. Microvascular damages were not seen . HistoloIn the PDT treated animals, necrosis was detected after PDT. Tumours #1–7 showed no or minor amounts (2%) of viable tumour cells remaining . The othIn terms of the microvascular compartment, 9 of 11 mice (#1–9) exhibited severe microvascular damage in the tumours , howeverin vivo would be most desirable.Given this complex and heterogeneous spectrum of volumetric changes and histo-morphological alterations in the tumour PDT response, an alternate sensitive and robust early treatment response indicator in vivo confirmed that Photoditazine accumulated in the CT26 tumour within one hour after intravenous administration . One obvious outlier is seen, but its fluorescence response parameters are indeed correct (as confirmed by repeat measurements); this must be due to inherent biological variability/tumour heterogeneity effects. r = 0.052, p = 0.879), with same outlier point as in (a). Overall then, neither fluorescence metric seems to be strongly correlated with, and thus predictive of, PDT response as measured by tumour volume changes.Given the variety of tumour growth trajectories, histological findings, and fluorescence responses, it was not surprising that MML-OCA also showed that not all tumours responded to PDT in the same manner. The tumours with severe microvascular damage , also displayed significant disappearance of blood vessels on MML-OCA within the shorter time interval at t = 1 day. Three of the nine responding tumours (#1–3) exhibited essentially no microvascular network immediately after PDT ; two tumBecause 9 of 11 treated tumours showed severe microvascular damage within 24 hours of PDT on MML-OCA, this change was chosen as the MML-OCA PDT response metric. st line of Table, It is encouraging that the early-response noninvasive MML-OCA findings correspond well with the later-measured histology trends and with gross tumour volume as quantified by TGI. For the seven tumours that responded well to PDT, OCT-detected microvasculature was essentially obliterated, 7-day histology confirmed severe microvascular damage, and the tumours shrank significantly (TGI was 147–163%). In the two cases of moderate PDT responders (#8 and 9), blood vessels around the tumour showed modest alterations on MML-OCA images, tumour volume effects were less pronounced (TGI was 135 and 142%) percent of viable tumour cells was 20 and 60%, and these tumours may probably regrow. When weak microvascular damages were found by histology at 7 days and blood vessels were still visible on MML-OCA during/after 24 hours post-PDT (#10 and 11), the tumour response was the weakest – the tumour continued to grow (TGI = 97 and 81%) and contained a greater number of viable cells (85 and 97%). Note that the photobleaching data  due to our employed scanning pattern being sensitive to flowing blood flow onlyIn this study, we searched for an early microvascular PDT response criterion via M-mode-like OCT, an approach that allows non-invasive Tumour heterogeneity greatly modulates the efficacy of antitumour therapies, often in unpredictable and poorly understood waysIn the present study, PDT was primarily vascularly targeted as the treatment commenced shortly after PS injection when it was still primarily in the blood stream. The histological analysis confirmed the presence of blood vessels damage, as well as signs of cell death (necrosis). Microvascular reactions were divided into severe  and weak . Thrombosis is the most common microvascular reaction to PDT and appears to be a result of photochemically induced endothelial cell damageet al. showed correlation of in vivo photosensitizer fluorescence in a tumour and PDT-induced depth of necrosis3434The fluorescence studies yielded largely negative results: Specifically, we found that photosensitizer photobleaching exhibited a weak positive correlation with inhibition of tumour growth, whereas the correlation with fluorescence intensity was even weaker . AlthougSince biological action of PDT depends on oxygen concentration in biotissue, direct measurement of singlet oxygen yield (‘explicit’ metric) was suggested to be an effective method of PDT efficacy estimation8integrin and neuropilin-13941i.v. injectionIt is known that tumour vasculature is an important target for PDTApplying the new MML-OCA approach to derive a microvascular criteria of PDT efficacy, we found that early tumour reaction can be predicted and is essentially independent on the degree of PS photobleachihg during PDT. Namely, even if photobleaching was only partial in some cases, the blood vessels were not visualized 24 hours after PDT in the responding tumours. We attribute this microvascular disappearance to thrombosis and stasis of blood vessels and interruption of blood flow, likely the main factors determining the success of the vascular targeted PDT. The ability of the OCT-based micro-angiography to sensitively detect these microvascular features and quantify their changes makes this approach especially attractive for monitoring PDT treatment efficacy.Specifically, we show that immediately following and 6–24 hours post PDT, disappearance of the viable  microvascular network on MML-OCA  might pr3744Approaches that visualize blood regardless of whether it is flowing or stationary  require full elimination of the blood in its liquid state before vessel disappearance is seen on corresponding angiographic images. This will likely occur at higher PDT doses that can cause significant damage of the surrounding normal tissues. In other words, stagnant vessels may already be biologically compromised /dysfunctional at more modest PDT levels, yet still be detected by these angiographic approaches thus giving a false indication of the ineffective treatment. Thus MML-OCA’s ability to detect only flowing/functional microvasculature is a definite advantage for this context. Further, MML-OCA outperforms Doppler OCT in the visualization of flows that are transversal to the scanning beam (since Doppler techniques become severely compromised as flow direction approaches perpendicular orientation) and/or are slow . More technically, Doppler OCT can miss transversal flows because it is particularly sensitive to monotonic change of OCT phase in the vessel regions; yet transversal flows demonstrate chaotic phase variations and often these are filtered out as noisevs non-responders17et al. investigated relationship between PDT efficacy and values of blood oxygen saturation using optoacoustics and showed that 24 hours post PDT these measurements predicted treatment outcome quite wellet al. used this method to quantify blood flow, oxygenation and drug photobleaching in PDT-treated cancer patientsIt is worth to mention alternative vasculature-based PDT-monitoring technologies that are not OCT-based. The use of photoacoustics for microvascular PDT-response monitoring has also been examined, suggesting that photoacoustic imaging for PDT may lead to improved efficacy, treatment guidance, and predicting responders 5 cells/20 μl of phosphate buffered saline. The specifics of the ear tumour model have been previously described4849The study was performed on murine colon carcinoma (CT26) transplanted subcutaneously in auricle tissue of mouse strain BALB/c purchased from Nursery for Laboratory Animals, Pushino, Russian Federation (n = 21) at the dose 2 × 10et al.2, exposure time 16 min 40 sec, spot size diameter 5 mm, total irradiance 100 J/cm2). For PDT procedure, the animals were anesthetized with Zoletil 50 and Rometar 2%  intramuscularly, as per REB-approved protocol.PDT was performed 10–14 days after tumour inoculation when the linear dimension of tumours reached 3–3.5 mm and blood vessels were visibly well developed. Animals were randomly divided into 3 groups: PDT (n = 11), light only (n = 5) and untreated controls (n = 5). The animals in the PDT group received intravenous injection of a photosensitizer Photoditazin  into the tail vein at a dose of 5 mg/kg body weight. One hour after PS injection, when the PS primarily accumulated in the blood vessels as shown by Chen In vivo fluorescence imaging was carried out to gauge accumulation of the PS in the tumour and estimate its PDT photobleaching. Epi-fluorescence imaging was performed using IVIS-Spectrum system  with excitation at 640 nm (bandwidth 30 nm) and emission at 720 nm (bandwidth 20 nm). The images were acquired before the injection of PS , 1 hour after PS administration just prior to treatment (control of Ps accumulation in the tumour), and immediately after PDT (photobleaching). The average fluorescence intensity (FI) in the regions of interest was quantified (Living Image 2.5 software). The background signal before the PS injection was subtracted. Fluorescence intensity was measured in the tumour area. Photobleaching was calculated according toWe used a Fourier domain OCT system20Microvasculature visualization before and after PDT treatment was performed with a scanning pattern optimized previously202054V ≈ π/6(a·b·c) (mm3), where c is the tumour depth. To characterize tumour growth inhibition (TGI), a coefficient similar toTumours were measured daily in three dimensions with a caliper. The tumour volume V was calculated as: VT is the mean tumour volume on day 7 after PDT, VTO is the mean tumour volume on the day of PDT; VC and VCO are the same means for the untreated group. This derived metric quantifies volumetric treatment efficacy while accounting for volume changes due to natural disease progression over the monitoring time interval . At 7 days post PDT, animals were sacrificed by cervical dislocation under anesthesia and tissues were harvested for histologic analysis.where Verification of the blood vessels reaction and grading of pathological tumour response were carried out by histological examination at t = 7 days after PDT. The excised tumours were embedded in paraffin. Many cross sections were made through the tumour bulk from one side to another. Histological preparations were stained with hematoxylin and eosin (H&E). 7-μm-thick sections were examined with light microscopy on Leica DM1000 system. Several cross-sections from the borders and the center of the tumour were analyzed. Histopathology examination included evaluation of percentage of viable tumour cells (not damaged by PDT) and degree of microvascular damage. Tumour cells without both visible nuclear  and cytoplasm (irreversible dystrophy) injuries were considered to be viable and thus not damaged by PDT. The quantitative assessment of morphological changes in the tumour CT-26 was undertaken via direct cell counting using the grid plate on a Leica DM1000 microscope. Under 40x magnification, the number of grid squares containing the damaged and not damaged (viable) cells and the number of squares containing thrombosis, stasis and normal vessels were counted. The result was presented as a percentage of the total number of grid squares. From each tumour, 3–4 cross-sections were thus examined. >30% of vessels with wall damage  in the tumour was classified as having severe microvascular damage, <30% of vessel with wall damage was considered to be weak microvascular damage. The 30% categorization value was selected based on preclinical and clinical histo-pathological experience; future work will examine the influence of this particular selection.Statistical analysis was performed in Statistica 6.0 software. Pearson’s correlation coefficient was used for comparison between photobleaching of PS and TGI and between fluorescence intensity and TGI. Statistical analysis of tumour growth was performed using the one-tailed Student’s t-test. P values less than 0.05 were considered statistically significant.How to cite this article: Sirotkina, M. A. et al. Photodynamic therapy monitoring with optical coherence angiography. Sci. Rep.7, 41506; doi: 10.1038/srep41506 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mouse has been extensively used as a tool for investigating the onset and development of human neurological disorders. As a first step to construct a transgenic mouse model of human brain lesions, it is of fundamental importance to clarify the similarity and divergence of genetic background between non-diseased human and mouse brain tissues.We systematically compared, based on large scale integrated microarray data, the transcriptomes of three anatomically distinct brain regions; prefrontal cortex (PFC), hippocampus (HIP) and striatum (STR), across human and mouse. The widely used DAVID web server was used to decipher the biological functions of the highly expressed genes that were identified using a previously reported approach. Venn analysis was used to depict the overlapping ratios of the notably enriched biological process (BP) terms  between two brain tissues. GOSemSim, an R package, was selected to perform GO semantic similarity analysis. Next, we adjusted signal intensities of orthologous genes by the total signals in all samples within species, and used one minus Pearson’s correlation coefficient to assess the expression distance. Hierarchical clustering and principal component analysis (PCA) were selected for expression pattern analysis. Lineage specific expressed orthologous genes were identified by comparison of the most extreme sub-datasets across species and further verified using reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR).COX5B, WIF1, SLC4A10 and PLA2G7) that are species-specific, which have been widely studied and confirmed to be closely linked with neuro- physiological and pathological functions.We found that the number of the significantly enriched BP terms of the highly expressed genes in human brain regions is larger than that in mouse corresponding brain regions. The mainly involved BP terms in human brain tissues associated with protein-membrane targeting and selenium metabolism are species-specific. The overlapping ratios of all the significantly enriched BP terms between any two brain tissues across species are lower than that within species, but the pairwise semantic similarities are very high between any two brain tissues from either human or mouse. Hierarchical clustering analysis shows the biological functions of the highly expressed genes in brain tissues are more consistent within species than interspecies; whereas it shows the expression patterns of orthologous genes are evidently conserved between human and mouse equivalent brain tissues. In addition, we identified four orthologous genes (Our study highlights the similarities and divergences in gene function and expression between human and mouse corresponding brain regions, including PFC, HIP and STR. Neurological disorders have become serious threatens to human health and quality of life, especially in the low-to-middle-income countries ,2. To reAlthough mouse’s life cycle is regarded as human’s life cycle in miniature, there are remarkable variations between human and mouse brains with respect to size, complexity, and cognitive abilities . Howeverde novo protein-coding gene, namely C20orf203, that is most abundantly expressed in brain, whose aberrant expression is involved in human-specific pathogenesis of Alzheimer’s disease (AD) [GLUD2, a hominoid specific brain enzyme that contributes to human cognitive abilities but also appears to confer vulnerability to brain tumors [Cross-species analysis of transcriptomes between healthy human and mouse brains can highlight the similarity and difference, providing a powerful approach to evaluate the effectiveness of the mouse model for human neurological diseases. Paying close attention to the species-specific expressed brain genes may also be conducive to comprehending each species typical nervous system activities or genetically susceptible brain diseases. Janssen et al.  found huase (AD) , and GLUn tumors .Our current study mainly focused on three brain regions, PFC, HIP and STR, because these structures are anatomically distinguishable brain areas that are structurally comparable between human and mouse; each species has specific phenotypes regarding advanced emotion, language, cognitive ability and bipedalism that are closely related to these structures; and these brain structures are vulnerable targeted regions for human brain disorders, such as tumors or neurodegenerative diseases. In this study, we integrated large scale microarray data, systematically compared the biological functions of the highly expressed genes, explored the gene expression pattern and excavated species-specific expressed orthologous genes of the three corresponding brain tissues across human and mouse. We endeavor to explore the similarities and differences across human and mouse brain transcriptomes, and provide a detailed knowledge of gene expression and function for mouse modeling regarding human brain disorders.http://www.ncbi.nlm.nih.gov/geo/). Only the datasets generated from Affymetrix microarray chip platforms were considered in the present study. To reduce the intrinsic errors within Affymetrix microarray platforms, we selected the relevant chip data from two platforms of Affymetrix . All human and mouse samples in the merged datasets were used as control groups in corresponding studies and diagnosed with no mental or neurological diseases. The basic information of these datasets is provided in S1 (human) and S2 (mouse) Tables. Primary analysis of microarray data was performed using Expression Console , a freely available software designed for preprocessing microarray datasets. The robust multichip average (RMA) algorithm was selected for signal intensity normalization [Microarray data of human and mouse PFC, HIP and STR transcriptomes were downloaded from Gene Expression Omnibus  were selected for further analysis. Afterwards, we performed functional enrichment analysis  using DAVID . The stVenn analysis was used to depict which BP terms were specific for one dataset or overlapped across two datasets. The statistical significance of overlap between two datasets was determined using the one-tailed Fisher’s exact test. A p < 0.05 indicated that the number of common BP terms shared by two different datasets was greater than what would be expected by chance. In general, the GO semantic similarity provides the basis for functional comparison of gene products. Here, we used mgoSim function from an R package, GOSemSim , for semhttp://www.informatics.jax.org/, [n is the number of datasets from one species and is 6 in present study. S indicates the antilog-transformed RMA value of gene i in dataset j. R indicates the relative expression level of gene i in dataset j. Third, the expression divergence between any two datasets was measured by one minus Pearson’s Correlation Coefficient (1-P). P was calculated using Eq  or R represents the median value of the relative expression levels of gene i across all samples in dataset j1 or j2. The dendrogram of datasets was derived from the hierarchical clustering algorithm implemented in R  using average linkage method and “1–P” as a distance metric between two clusters. Principal components analysis (PCA) that allows identification of categorical variables  in the data based on observed variables was performed, to verify the result of hierarchical clustering. PCA was implemented using the prcomp function from the stats package in R. The confidence ellipse was drawn using the autoplot function from the ggplot2 package in R with default parameters.Human and mouse orthologous gene-pair information was obtained from Mouse Genome Informatics . A compax.org/, . Therefoax.org/,  to measuusing Eq .P=∑i=1NWe cannot directly compare microarray data across two species. Thus, we adopted a methodology according to Booij et al  and JansS4 Table. Eight-month-old C57BL/6 mice  were purchased from Vital River Laboratories  and acclimatized in cages for 3 days before sacrifice under pentobarbital (50mg/kg) anesthesia by extracting the eyeballs for bleeding accompanied by perfusion with saline. PFC , HIP (both sides) and STR  were precisely dissected according to mouse brain anatomic atlas. The specimens were immediately frozen in liquid nitrogen until use. All mice related studies were approved by the Institutional Animal Care and Use Committee of Institute of Laboratory Animal Science, Peking Union Medical College. Both of the methods for the human and mouse protocols were carried out in accordance with the approved guidelines.Human postmortem brain tissues , frozen in liquid nitrogen, were obtained from the Chinese Brain Bank Center . Informed consent for the use of human tissues for research was obtained in writing from all donors or their next of kin. All procedures of the human brain materials have been approved by the Ethics Committee of Peking Union Medical College . The basic information of these human brain tissues are listed in st Strand cDNA Synthesis Kit . Even though intrinsically shorter and partially degenerated RNA fragments usually occur in human postmortem donor materials, we designed primers near the 3’ end of a given gene in keeping with the microarray probes for RT-PCR. The densitometry values were corrected by the expression of GAPDH gene. For qRT-PCR, gene expression was quantified using an Applied Biosystem 7500 Real-Time PCR System. The assay used gene-specific primers spanning an exon-exon junction and One Step SYBR PrimeScript RT-PCR Kit , according to the manufacturer’s manual. RT-PCR and qRT-PCR were used to verify the species-specific gene expressions at the same time. All the primers used in present study are listed in Tables.Total RNA of each brain tissue was purified using Trizol  and was quantified using a spectrophotometer. The RNA samples were reverse-transcribed using PrimeScript 1n = 228 for human sample, n = 100 for mouse sample), HIP  and STR  (S1 and ). Since the probes targeting the same sequence usually vary across different microarray products, the detection results are often nonlinear between different microarray products [S1 Table), and the lowest ACI value between any two mouse samples from the same microarray platform is 0.868 (S2 Table), indicating a rather small perturbation among samples.We selected and merged a retrospective series of transcriptome profiles using public microarray data from the GEO database  for human brain tissues is observed to be over two times than that for mouse brain tissues (detailed information is provided in S7 Table). However, the number of the evidently enriched neuro-related BP terms (adjusted p < 0.01) appears to show no variation between human and mouse corresponding brain regions . Even so, several neuro-related BP terms, such as “central nervous system development”, “midbrain development”, “brain development”, “neuron projection guidance”, “axon guidance”, “neural nucleus development”, “head development”, “locomotion”, and “learning”, are specific to human, in comparison with mouse (S8 Table). Next, we ranked all BP terms according to Benjamini-corrected p values in descending order. The most significantly enriched (top ten) BP terms in mouse brain tissues are also found in human brain tissues, whereas the top ten BP terms in human brain tissues pertaining to protein-membrane targeting and selenium metabolism are found to be species-specific with respect to mouse (S9 Table).We selected the widely-used DAVID  to perfoFig 2A). However, the smallest overlapping ratio of the evidently enriched BP terms between any two brain tissues within species is 69.5%, which is approximately twofold larger than any one across species . In all cases, the agreement between any two brain regions is greater than expected by chance . Furthermore, we performed semantic similarity analysis of the notably enriched BP terms between any two brain tissues using GOSemSim. Semantic similarity can be, to some extent, used for evaluating functional coherence of different sets of genes [Fig 2B). Based on the overlapping ratios or semantic similarity scores, hierarchical clustering with a Pearson distance metric and average linkage method significantly separates the brain tissues into two clusters corresponding to the two species, human and mouse, implying a more species-dominated similarity  among the four microarray platforms were used. These genes expression is observed similar overall across all brain tissue samples . We normalized the signal intensities of the common orthologous genes using the total signals in all datasets within species. The expression distance between any two datasets was assessed by “1-P”  shows that Affymetrix chip platforms measuring one brain tissue from one species tend to be markedly clustered, indicating a high degree of consistency although some Affymetrix probes with name suffixes _x_at and _s_at are deemed to be prone to cross-hybridization [Fig 3A). This implies a conserved gene expression pattern between comparable human and mouse brain tissues. The PCA, however, is less clear in this regard . We applied the 8659 orthologous genes that were considered as observed variables to PCA and used the categorical variables  to visualize the PCs later. The first two PCs that are the most informative explain ~75.7% and ~12.5%, respectively, of the total observed variances. While the first two PCs clearly distinguish STR from PFC and HIP, they does not distinctly separate PFC from HIP; PFC and HIP tend to cluster close together .After orthologous gene mining, as shown in 1-P” see . The dendization ,30. AnalS2A Fig, we found NSA2 is highly expressed in all three brain tissues of human but almost undetectable in mouse brain tissues; COX5B is highly expressed in human PFC but lowly expressed in mouse PFC; PEG10 and WIF1 are highly expressed in human STR but lowly expressed in mouse STR. In the same way, by comparing mouse “high expression” datasets with human “very low expression” datasets in terms of the analogous brain tissues, as shown in S2B Fig, we found SLC4A10 is highly expressed in all three brain tissues of mouse but lowly expressed in human corresponding brain regions; PLA2G7 is highly expressed in mouse HIP but lowly expressed in human HIP. Since there exist intrinsic demerits for microarray products, such as cross-hybridization and low sensitivity, the species-specific expressed genes will be further verified.Comparing human “high expression” datasets with mouse “very low expression” datasets in terms of the analogous brain tissues, as shown in Fig 4A–4C, COX5B and WIF1 are highly expressed in human PFC and STR, respectively; SLC4A10 is highly expressed in almost all the mouse brain tissues, encompassing PFC, HIP and STR; PLA2G7 is highly expressed in mouse HIP compared to the comparable human HIP. However, NSA2 and PEG10 that are identified as specifically expressed orthologous genes in human brain tissues are also highly expressed in mouse corresponding brain tissues. To further validate these results, we designed intron-spanning primers to perform qRT-PCR. The result is in accordance with that of RT-PCR in terms of the following four genes, COX5B, SLC4A10, PLA2G7 and WIF1 .To confirm the expressions of species-specific expressed orthologous genes in brain tissues predicted by comparing the most extreme microarray sub-datasets across human and mouse, we first performed RT-PCR. As shown in Mouse is a facile and low-cost species for genetic modification studies compared to other laboratory animals –5. BrainIn accordance with some previous studies –12, our NSA2 and PEG10) were confirmed to be false positive by RT-PCR. This may have been caused by significantly different probe affinities for a given gene across species. Brain fractured RNA segments are usually associated with agonal and postmortem factors [Due to different affinities of probes to target RNAs across species, it is difficult to interpret the variations by directly comparing human and mouse microarray data. Thus, we compared the most extreme sub-datasets across species see . The ort factors ,35. Also factors ,27. TherTable 1). COX5B is a component of cytochrome c oxidase (complex IV) that is responsible for electron transport. Complex IV activation can contribute to sustaining a reduced reactive oxygen species production [COX5B will lead to a failure in complex IV assembly [WIF1 acting as a Wnt antagonist and tumor suppressor is involved in the pathogenesis of some brain cancers, such as astrocytomas [WIF1 in glioblastoma inhibits migration, suggesting a role of WIF1 in the regulation of cell cycle and proliferation [WIF1 has also been confirmed to be directly implicated in the myelination process and hippocampal development, abnormal expression of which may be a possible risk for cognitive defect and dementia [SLC4A10, encoding a Na+-dependent Cl-–HCO3- exchanger, is a major contributor to physiological cerebrospinal fluid (CSF) secretion [SLC4A10, with subsequently reduced ability of Aβ, is suspected to be a risk factor for AD onset and progression [SLC4A10, by mediating acid extrusion, can also regulate intracellular pH, while acid-base homeostasis in the central nervous system is vital to neuronal excitability [SLC4A10 is highly expressed in mouse brain but poorly expressed in human brain, which indicates a different basal neuronal excitability and regulation. Disruption of SLC4A10 gene in mice does not exert obvious behavioral abnormalities, but yields compromised regulation of neuronal pH and an ascending seizure threshold [SLC4A10 is widely reported as a candidate risk-related gene for some neuro-related disorders linked with neuronal excitability, such as epilepsy, autism and major depressive disorder (MDD) [PLA2G7, encoding for platelet-activating factor acetylhydrolase (PAF-AH), promotes the degradation of PAF, generating the biologically inactive products lyso-platelet activating factor (lyso-PAF). Abnormally expressed PLA2G7 may give rise to dysregulation of phospholipid metabolism. PLA2G7 gene has been widely studied as a candidate risk factor of coronary heart disease [PLA2G7 in neurophysiological processes and pathological disorders. Meng et al. [PLA2G7 may affect the clinical manifestation of schizophrenia. A meta-analysis based on differentially expressed genes in autism identified PLA2G7 as a genetic marker involved in the development and progression of child autism [The species-specific expressed orthologous genes detected in our study have been extensively studied in previous studies  ,64–66. P disease ,68. Onlyg et al.  found PLd autism . OverallIn summary, we systematically integrated and analyzed large scale microarray data of human and mouse PFC, HIP and STR, and confirmed a more species-dominated similarity in biological function and a conserved expression pattern of orthologous genes between the equivalent brain structures across human and mouse. We also identified a cluster of species-specific expressed orthologous genes by both microarray data and experimental verification. These genes have been widely studied and found to be closely linked with multiple neurophysiological and pathological processes. More attentions should be paid to these four species-specific orthologous genes in future studies.S1 Fig(a) Venn diagram depicts the number of the common orthologous genes among four Affymetrix microarray platforms that are labeled by different colors. GN represents gene number. (b) Heatmap of expression for the common orthologous genes across all the samples. Before heatmap generation, the RMA values of all the genes were ranked in a descending order according to the average values across all the samples. The three bars above the heatmap represent species, brain tissues and microarray platforms, respectively, that are labeled by different colors from top to bottom. Pink = human, light blue = mouse, green = PFC, red = HIP, blue = STR, black = HG-U133A or Mouse430A_2, and white = HG-U133_Plus_2 or Mouse430_2.(TIF)Click here for additional data file.S2 Fig(a) Human- or (b) mouse-specific expressed orthologous genes in brain tissues are located in the overlap of the four datasets. Our focus is limited to the genes located in the overlap shared by the four datasets to obtain more precise results.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.


Protein quality control or proteostasis is an essential determinant of basic cell health and aging. Eukaryotic cells have evolved a number of proteostatic mechanisms to ensure that proteins retain functional conformation, or are rapidly degraded when proteins misfold or self-aggregate. Disruption of proteostasis is now widely recognized as a key feature of aging related illness, specifically neurodegenerative disease. For example, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease and Amyotrophic Lateral Sclerosis (ALS) each target and afflict distinct neuronal cell subtypes, yet this diverse array of human pathologies share the defining feature of aberrant protein aggregation within the affected cell population. Here, we review the use of budding yeast as a robust proxy to study the intersection between proteostasis and neurodegenerative disease. The humanized yeast model has proven to be an amenable platform to identify both, conserved proteostatic mechanisms across eukaryotic phyla and novel disease specific molecular dysfunction. Moreover, we discuss the intriguing concept that yeast specific proteins may be utilized as bona fide therapeutic agents, to correct proteostasis errors across various forms of neurodegeneration. The regulation of cellular protein levels is an indispensable feature that ensures fidelity of fundamental processes within all cell types. Although nascent peptide strands contain the requisite information to fold proteins into functional structures and confer biological activity, the dynamic nature of protein folding as well as the crowded cell environment pose significant barriers in maintaining protein homeostasis or proteostasis Loss of proteostatic control has emerged as a common cellular pathology in seemingly disparate forms of neurodegenerative disease A consensus has arisen supporting the concept that protein misfolding/aggregation and the progression of neurodegenerative disease are inherently linked. This model also suggests that ongoing aberrant protein misfolding and decline in proteostatic machinery function may accelerate the pathogenic cascade Saccharomyces cerevisiae, to elucidate the molecular basis of various pathologies. 9101410In recent years, the field of neurodegeneration has derived significant benefit from the use of baker's yeast, The mislocalized accumulation of the pre-synaptic protein α-synuclein (α-syn) as a result of mutations or gene multiplications is a hallmark feature that defines PD and other disorders collectively termed synucleinopathies 13Drosophila and C. elegans These studies highlight the great complexity in dealing with PD and neurodegenerative disorders in general, yet have confirmed the utility of the yeast system in addressing complex human disease pathology. Yeast models have also been instrumental in identifying other molecular determinants that modify α-syn cytotoxicity. In particular, the functional identification of the GTPase Rab1, as a suppressor of α-syn toxicity, stemmed from initial studies in yeast vps35 gene suggesting that VPS35 is protective against toxicity relating to EIF4G1 upregulation. Likewise, the PD-associated D620N mutation in VPS35 also had a similar effect. This genetic interaction between VPS35 and EIF4G1 was further confirmed in neurons of both C. elegans and transgenic mouse models In addition to Rab GTPases, studies using similar approaches continue to identify novel protein mediators of α-syn related cytotoxicity in PD, including endonuclease G (endoG) and the sorting protein VPS35 Understanding the cellular etiology of ALS, a fatal and intractable neurodegenerative disorder, has also benefitted greatly by use of yeast model systems 99et. al.9The first yeast TDP-43 proteinopathy model by Johnson Pab1) - binding protein, Pbp1 as a specific enhancer of TDP-43 toxicity; overexpression of Pbp1 increased TDP-43 toxicity whereas as Δpbp1 cells showed increased cell survival upon TDP-43 overexpression atxn2), which has been implicated in spinocerebellar ataxia type 2 (SCA2) that is caused by glutamine expansion within the polyQ tract. This interaction between TDP-43 and Pbp1/Atx2 was further validated in Drosophila and humans. Moreover, the physical interaction between TDP-43 and Atx2 was shown to be dependent on RNA binding, as mutations within the RRMs as well as RNase treatment, dissolve the interaction between Atx2 and TDP-43 The success of modifier gene discovery in α-syn yeast models of PD has also propelled the use of similar genome wide screens to elucidate TDP-43 induced toxicity, i.e. yeast overexpression screens have led to the identification of a pool of 40 genes that modify TDP-43 toxicity. This pool of factors contained the Poly (A) binding protein , as having a role in TDP-43 mediated toxicity. Using the fly model, investigators have shown that an Atx2 mutant lacking the PAM1 motif (through which it binds Pab1), cannot interact with TDP-43 and confer toxicity in the retina. Similarly, human PABPC1 was observed to be mislocalized in ALS patients et. al.dbr1 deletion mitigated the toxic effects of wildtype TDP-43 and the mutant TDP-43 Q331K expression. This suppression of TDP-43 toxicity was also validated in the human M17 neuroblastoma cell line and primary neurons using a siRNA-based knockdown approach, suggesting that inhibiting the enzymatic activity of Dbr1 is sufficient to reduce TDP-43 toxicity Act1 gene in the cytoplasm and the suppression of TDP-43 toxicity in Δdbr1 results from interactions of TDP-43 with the accumulated intronic lariats. The postulate derived from these experiments implied that TDP-43 associated toxicity in ALS and FTLD-U arose from a loss of essential TDP-43 RNA interactions 313233TDP-43 toxicity appears to also segregate with modifications in DNA structure. Here, Armakola +1 induced mitochondrial stress, which is a hallmark feature of AD pathology The prior discussion has focused on the role of yeast models as a test bed to explore disease mechanisms in α-syn linked PD and TDP-43 linked ALS, yet yeast has served as an ideal platform to study the cell biology of various aggregation prone proteins that characterize neurodegeneration. Of note, is the recent elucidation that manipulation of the ubiquitin protease system (UPS) in yeast (by enhancing the expression level of Cdc48 and Vms1) can curtail UBBMuch effort has focused on defining genetic susceptibility in neurodegenerative disease, yet this line of investigation has failed to provide new or effective clinical interventions. Indeed, the capacity to actively reverse aggregation and control protein misfolding will be an essential attribute when considering therapeutic solutions for proteinopathies. Provocative evidence now suggests that yeast specific proteins have the capacity to degrade aggregate prone human disease proteins. Although speculative at this point in time, the data does support the conjecture that yeast proteins may be eventually employed to combat human neurodegeneration. In this regard, the yeast specific Hsp104 chaperone disaggregase has shown considerable promise Hsp104 is an AAA+ chaperone of the heat shock protein family which is known for its ability to completely remodel denatured aggregates, conferring “thermotolerance” in yeast. Structurally, Hsp104 is hexameric pore-like structure consisting of an N-terminal domain, a middle domain (MD) flanked by two AAA (AAA-1/2) domains and a C-terminal domain. ATP hydrolysis at the two AAA domains is required for substrate translocation through the pore. The unique M-domain, which is absent in other AAA+ proteins, is suggested to be the molecular switch which mediates the ATPase activity of Hsp104 and co-ordinates interaction with other chaperones of the Hsp70 and Hsp40 families 38in vitro as well as prevent α-syn inclusion formation in vivo37in vitro revealed that the enhanced disaggregase activity was dictated by amino acid identity within the middle domain of Hsp104, and key mutations in this region increased chaperone collaboration leading to disaggregation of a wide range of substrates 3742Metazoans lack the presence of a clear Hsp104 ortholog, yet despite its absence Hsp104 can be stably expressed and can rescue protein aggregation and prevent neurodegeneration The unexpected role of Hsp104 in mitigating human aggregate-sensitive disease proteins raises the prospect that additional unforeseen regulatory mechanisms may exist to manage this critical aspect of cell biology. As such, a novel proteostasis role has been identified for the clan C/D family of proteases, which include metacaspases and caspases. While this protease clan is typically associated with induction of programmed cell death, through broad range protein destruction, a growing body of evidence suggests that discrete activation of these proteases is essential for numerous complex cell behaviors, independent of inducing cell death 4446et al.Most recently, Hill Admittedly, the delivery of yeast proteins as a therapeutic intervention to treat human neurodegeneration is an entirely speculative proposition. However, the emerging data for Hsp104 indicates that measured delivery of this protein in preclinical animal models of PD and Huntington’s disease has no observable off target toxicity 4050The discovery in yeast, that a metacaspase subdues toxic aggregate formation, raises an interesting prospect, i.e. that mammalian/human caspase proteins may have retained similar beneficial proteostatic function(s). Early studies characterizing cellular pathology in neurodegenerative disease have noted that affected and dying neurons display robust elevation in effector caspase activity (caspase 3 and 6), with subcellular distribution to protein inclusions 5255In conclusion, we have demonstrated that deployment of the yeast model has provided exceptional advances in the study of neurodegenerative disorders including PD and ALS. Similar concerted efforts using yeast proteinopathy models have advanced understanding of disease related proteins such as tau, Aβ and prions. Likewise, the concept that yeast specific proteins, such as Hsp104 and/or Yca1, may be engineered to combat toxic human aggregates opens the door to entirely novel avenues of therapeutic intervention. The use of yeast proteins as therapeutic agents needs to be approached with considerable caution, yet the early studies in relevant animal models support the basic premise. Clearly, the ease and flexibility of the yeast model will ensure continued use of this eukaryotic cell as a preferred means to interrogate and define the molecular pathology of neurodegeneration.


The nature and development of cardiorespiratory impairments associated with sickle cell disease are poorly understood. Given that the mechanisms of these impairments cannot be addressed adequately in clinical studies, we characterized cardiorespiratory pathophysiology from birth to maturity in the sickle cell disease SAD mouse model. We identified two critical phases of respiratory dysfunction in SAD mice; the first prior to weaning and the second in adulthood. At postnatal day 3, 43% of SAD mice showed marked apneas, anemia, and pulmonary vascular congestion typical of acute chest syndrome; none of these mice survived to maturity. The remaining SAD mice had mild lung histological changes in room air with an altered respiratory pattern, seizures, and a high rate of death in response to hypoxia. Approximately half the SAD mice that survived to adulthood had an identifiable respiratory phenotype including baseline tachypnea at 7–8 months of age, restrictive lung disease, pulmonary hypertension, cardiac enlargement, lower total lung capacity, and pulmonary vascular congestion. All adult SAD mice demonstrated impairments in exercise capacity and response to hypoxia, with a more severe phenotype in the tachypneic mice. The model revealed distinguishable subgroups of SAD mice with cardiorespiratory pathophysiology mimicking the complications of human sickle cell disease. Beginning in early childhood, SCD results in signficant cardiorespiratory complications including acute chest syndrome (ACS)3, chronic changes in lung function8, impaired exercise capacity10, pulmonary hypertension12, left ventricular dysfunction14, sleep disordered breathing16, and premature death19. ACS, the most severe acute respiratory disease associated with SCD, occurs most commonly in younger children, is the major contributor to mortality in children, and is associated with chronic lung disease22. Despite this, the fundamental mechanisms of acute and chronic SCD associated cardiorespiratory disease are poorly understood.Sickle cell disease (SCD) is an autosomal recessive disorder that results in structural changes to hemoglobin molecules which lead to oxygen release, increased red cell fragility, hemolysis, and mircovascular obstruction. It is a multi-system chronic disease affecting 1 in 365 African-American births25 including gene and cell therapy approaches26. The SAD mouse model is likely the most pertinent for hematological, histopathological, and pathophysiologic analysis and does not have evidence of globin chain imbalance or thalassemic features seen in other transgenic mice models. In comparison to other SCD mouse models, adult SAD mice have milder anemia, and normal to slightly increased platelet counts. The erythrocytes of the SAD mice are characterized by hemoglobin polymerization and sickling in vitro and in vivo. Further, SAD mice display clinical pathologies similar to that of human SCD, including glomerulopathy, acute spontaneous vasoocclusive events and reduced life-span29. Here, we systematically investigate cardiorespiratory disease progression in the SAD-1 transgenic mouse model of SCD31 using physiological measurements from the newborn stage through to adulthood. Data from this study provides a platform of well-defined cardiorespiratory functional measures to evaluate pathogenic mechanisms and to test the efficacy of treatment strategies aimed at ameliorating SCD associated cardiorespiratory disease.Different transgenic mouse models for SCD have been generated to study certain aspects of the pathophysiology of SCD and evaluate the effectiveness of therapeutic agents (previously reviewed)31, we first investigated when lethality occurred at perinatal ages. At postnatal day (P) 3, 35% (88 SAD vs 162 WT) of mice genotyped were SAD positive. Prevalence of the SAD genotype decreased to 24% at P21 (77 SAD vs 250 WT). Given that the theoretically predicted rate of the SAD genotype is 50%, 46% of SAD mice died prior to P3 and 24% died between P3 and P21.Based on the lower frequency of adult SAD mice than expected>20). The remaining SAD had a body weight < 20% below WT (SAD<20); 20% weight reduction is related to severity of phenotype. Compared with WT, SAD>20, but not SAD<20, had decreased fR, and increased total apnea duration. Ten of 38 (26%) SAD>20 died between P3-P6. At P7-P8, SAD>20 (n = 8), but not SAD<20 (n = 8), had a decreased body weight  and increased total apnea duration (1.9 ± 0.2 s/min) compared to WT  at 7–8 months old . SADT had an increased fR , decreased VT , and increased VE  relative to WT (n = 39). Further, breathing was more regular relative to WT with decreased total apnea duration  and coefficients of variability (SD/mean) in fR  and VT . In contrast, the SADNT had indistinguishable breathing parameters from WT.We followed a larger cohort (n = 77) of SAD mice beyond the age of 6 months, all of which displayed similar body weights to WT. A major subset of SAD mice 48%, 37 of 77) with a unique respiratory phenotype was identified by tachypnea . By contrast, SAD mice had an initial increase in VE followed by period of marked apneas and behavioral seizures. Total apnea duration increased in SAD<20  and SAD>20  after 5 min hypoxia compared with WT after hypoxia (1.3 ± 0.2 s/min) and mutant in room air. Hypoxia-induced apnea and seizures were more severe in SAD>20 than SAD<20 mice  had sustained increase in VE (233.4 ± 11.8% relative to room air) and regular occurrence of sighs  without marked behavioral seizures. Compared with WT mice, hypoxia caused a blunted VE increase , accompanied by increased apnea duration  in SADT (n = 14), but not SADNT mice (n = 13). Hypoxia induced observed behavioral seizures occurred in most mutants and were more severe in SADT than SADNT mice. Further, 1 of 8 SADT died, but all 8 SADNT and 10 WT mice survived during 1 hour of 8% O2 challenge.The response of SAD mice to 8% Oths Fig. , but notNT mice had a decreased exercise capacity . The decrease in exercise capacity was more severe in SADT mice .The exercise capacity testing Fig.  and tranT mice as indicated by decreased PAAT (15.4 ± 0.9 ms) compared with WT  or SADNT  mice  assumed a spherical shape with increased diastolic RV internal diameter and cross-sectional area . The left ventricle had an increased mass  consistent with the hypertrophy detected in another SCD mouse model32.Pulmonary arterial hypertension was observed in 5 of 8 SADice Fig. . In 3 of2O/ml, consistent with previous reports on mouse strains34. Compared with WT, SADT (n = 7), but not SADNT (n = 7) mice, had a lowered lung capacity  and increased elastance .At 7–8 months of age Fig. , WT  and was further emphasized under hypoxic condition .In room air, a small percentage of erythrocytes were elongated, consistent with irreversible sickle cells in blood of SAD as well as of SCD patients>20 at P7-P8 were red rather than the normal pink-white color seen in WT and SAD<20 mice. Compared with normal lung histology in WT  showed mild or moderate microvascular occlusions and congestion, as evident by dilated capillaries and venules filled with densely packed and deformed erythrocytes. SAD<20 mice had normal histology or mild histological change. Infarcts of the lung, thrombosis or small pulmonary hemorrhages, pulmonary septal and alveolar thickening were observed in some SAD>20, but not in SAD<20 mice. More severe features of microvascular occlusions and congestion of pulmonary capillaries and venules were observed in SAD mice after 30 min of hypoxic challenge, with more severe pulmonary hemorrhages in SAD>20 than SAD<20. Examination of SAD mice at 7–8 months revealed normal lung histology in SADNT or subtle dilated pulmonary vessels and patchy vascular congestion, engorged alveolar capillaries in SADT  or in WT mice under hypoxia with no thrombi  demonstrated clearly abnormal respiratory patterns as well as pulmonary vascular hemorrhage, infarct, and signs of interstitial lung disease resulting in increased vulnerability. In addition, prolonged apnea duration in these smallest SAD mice in early life is likely to increase the occurrence of hypoxia and hypercapnia leading to greater erythrocyte sickling and further vaso-occlusion in the lung parenchyma and pulmonary vascular system. Further, 26% of P3 mice died before P7–8 and none survived beyond weaning. This overall pattern of increased vulnerability and abnormal respiratory pattern likely contributes to episodes of acute lung injury, chronic lung disease, and increased risk of mortality21. Notably, the remaining SAD mouse cohort (57%) with moderately decrease body weight displayed no distinguishable baseline respiratory pattern despite differences in response to hypoxia. Accordingly, lung histology in this SAD cohort appears relatively normal and mice survived into adulthood. Hence, mortality in young SAD mice is bimodal with a high proportion that die prior to P3, likely secondary to lung injury and altered respiratory rhythmogenesis35, and a significant, but lower proportion, dying before weaning from pathology that likely parrallels that of ACS. The difference in physiologic response and survival of the two SAD cohorts is independent of gene modifiers or genotype since all SAD mice are of a homogenous genetic background. In human SCD, infection, fat emboli and pulmonary infarction are identified as contributors to ACS2; mortality in the current SAD mouse cohorts, however, cannot be attributed to infections because of mice specific pathogen-free housing conditions. Collectively, this SAD mouse study highlights that microvascular occlusions and infarctions are critical to the pathogenic mechanism of acute lung injury in mice and potentially play a more important role in human ACS and childhood SCD mortality than previoulsy appreciated.Our study of SAD mice at neonatal and pre-weaning stages revealed several characteristics of acute and early respiratory disease. Although all SAD mice were anemic at P3The two pre-weaning SAD mouse cohorts could also be distinguished based on response to hypoxia challenge at P7–8, providing further validation for distinct SAD cohorts. The SAD mouse cohort of smallest size with abnormal baseline respiratory pattern also demonstrated longer periods of apnea, and higher frequency of seizures and death during prolonged hypoxia exposure. These physiological responses were consistent with histological findings of severe pulmonary intravascular occlusion, congestion, and hemorrhage. The second SAD mouse cohort with moderate reduction in size and normal baseline respiratory patterns exhibited an increase in apnea duration and seizures under hypoxia, compared to WT mice, and developed pulmonary intravascular occlusion and congestion which was milder then the SAD cohort of smallest size. These results provide further evidence of two clearly identifiable SAD subgroups; one with a lethal pre-weaning respiratory phenotype evident in normoxia and one with an intermediate phenotype, revealed during hypoxia. In addition, these results suggest that ventilatory response to hypoxia may be impaired in SCD; this would lead to greater risk for vasooclusive crises, decreased exercise tolerance, as well as greater risk for pulmonary hypertension and respiratory sleep disorders including obstructive sleep apnea.37, and adopt a tightly constrained breathing pattern, probably as a strategy for reducing dyspnea38. A recent study confirmed coupling of dyspnea perception and tachypneic breathing during hypercapnia39. Importantly, our results indicate that the absence of major or recurrent ACS does not protect against the progression of restrictive lung disease suggesting that ACS and restrictive lung disease, while clearly associated in human SCD, have different underlying mechanisms.The second critical respiratory phase in SAD mice was demonstrated by development of a restrictive lung disease in adulthood. Approximately half of adult SAD mice showed a tachypneic pattern of respiration, fast rate and decreased variability of breathing. The other half of adult SAD mice did not develop tachypnea and had normal lung function and morphology. The decreased total lung compliance and increased lung elasticity in the cohort of SAD mice with tachypnea depict a chronic restrictive pulmonary pattern. The lung dysfunction correlates with the morphological abnormalites including microvacular occlusion and fibrosis. Patients with restrictive lung defects are typically dyspneic, have an increase in overall respiratory centre drive, as a result of increased lung elasticity40, the current results on physical exercise capacity of older SAD mice reproduced another facet of SCD pathophysiology. The severe deficit in exercise tolerance for tachypneic SAD mice and moderate in non-tachypneic SAD mice correlated with the observations of dyspnea, restrictive lung defect and exercise limitations in adult SCD41. SAD mice showed typical SCD cardiopathologies with right and left ventricular hypertrophy42.When challenged by hypoxia and exercise tolerance tests, adult SAD mice systematically demonstrated altered respiratory pathophysiology. Under hypoxia, the periods of apnea and onset of seizures in adult SAD were similar to those in pre-weaning SAD mice with hypersensitivity to hypoxia, albeit, with fewer deaths and less severe histopathology. While there is evidence that long-term voluntary physical exercise of young SAD mice is likely to improve the oxidative stress status in SCD44 though the precise cause of death has not been characterized. In contrast to our finding of mild histological changes at P7–8, longitudinal study of other models has shown no tissue damage until 3–6 months of age45. In adult mice, hemorrhage, progressive iron deposition, capillary congestion, increased wall thickness of pulmonary arteries, RV enlargement, and pulmonary hypertension have been described in other models46. Interestingly, unlike the SAD mice, the lesions that characterize advanced pulmonary hypertension in humans as well as the interstitial fibrosis seen in adults with SCD are not seen in some models42. Prior study of oxygen response and hypoxia/reoxygenation using longer hypoxia exposure (>1 hour of exposure) then in our protocol have demonstrated impairments in microvascular function and exaggerated inflammatory response, both of which likely contribute to vasocclusive events including ACS49; our finding complement these by demonstrating the consequences of short hypoxia exposure. While all models demonstrate important components of cardiorespiratory pathophysiology, the SAD model appears to provide greater consistency with human SCD.Our results highlight some similarities as well as important differences with respect to cardiorespiratory pathophysiology in different mouse models of SCD. Similar to the SAD mice, there is a high rate of early mortality other SCD mouse models4. Early signs of pulmonary hypertension, including increased tricuspic rejurgitation jet velocity (TRV) has been described in SCD children < 10 years of age50 as well as impaired exercise capacity relative to non-SCD peers52. Our study further suggests that these complications may be present much earlier in the course of SCD and of wider scope than is evident from clinical studies. The longer duration of apnea in SAD young mice is consistent with the higher risk for nocturnal oxygen desaturation and obstructive sleep apnea in childhood SCD53. Moreover, the high prevalence of tachypnea at rest in adult SAD and a restrictive pattern of lung function strongly warrant systematic monitoring of adult SCD patients to identify those with disease progression.Overall, our longitudinal analysis revealed that SAD mice from the neonatal stage to adulthood display a spectrum of severe cardiorespiratory phenotypes mimicking the known cardiorespiratory impairments of human SCD. Cardiorespiratory disease in SCD children and adolescents is documented with a variable degree of decline in lung functionOur present results demonstrate distinct subgroups of SAD mice which can be used as a model to determine the molecular and cellular pathophysiologic mechanisms leading to each type- and phase-specific cardiorespiratory disease in SCD. There is clear evidence that cardiorespiratory disease is present earlier than is appreciated in clinical studies. Translating the findings from this study into therapy targeting the mechanisms responsible for cardiorespiratory disease is vital to improving childhood mortality and long-term outcomes in human SCD.54. Male SAD-I (Hbbs/s) transgenic mice were mated to female WT  mice. Red blood cells in the SAD mutant contained 19% human Hb SAD (α2human ß2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Pups of both sexes and adult males were examined; adult females were not tested to reduce the potential variability causes by hormonal fluctuations associated with the female reproductive cycle. For measurement of hematocrits and sickle cell fraction, tail blood samples were collected into 5% phosphate-buffered formaldehyde before and after hypoxia, as previously described55. Cells were placed in a grid rule slide, and 500–1000 erythrocytes were counted using light microscopy ; irreversibly sickled cells correspond to elongated cells with length-to-width ratio of 2 of more.Experimental procedures were approved by the University of Alberta Animal Welfare Committee. All experiments were performed in accordance with the standards of the Canadian Council on Animal Care. Testing and analyses were performed by individuals unaware of the animal genotype. A colony of SAD mice was established from breeding pairs provided by Dr. Marie Trudel. SAD mice were backcrossed on C57BL/6 J for >20 generations and are considered on a pure homogenous genetic background. SAD and control wild type (WT) mice within the same litter were processed together for comparison. Genotypes were determined by PCR analysis of DNA samples obtained by ear-punch or tail-clip biopsies, as described previously56. Custom made plexi-glass chambers with volumes of 10, 25, 50, 80, 260 ml were used to accommodate mice with body weights of 1–2, 3–4, 5–7, 8–14, and 15–40 g, respectively. Recordings were made 30 minutes after placing the mice in the suitable size chamber to optimize the signal/noise ratio while allowing the mice to move freely. Pressure changes were detected with a pressure transducer , signal conditioner , an analogue-digital board (Digidata 1322 A), data acquisition (Axoscope) and analysis (Clampfit) software . The chemoreceptive responses to altered O2 (5–60 minute after 1 min of gas exchange equilibration) were assessed by switching from room air to hypoxia (8% O2). It took approximately 30–60 seconds for the new gas to equilibrate within the chamber, as confirmed by measurements of oxygen content with Oxychek instrumentation . It should be noted that our plethysmographic recording setup is effective for studying respiratory frequency (fR) and detection of apneas (defined as the absence of a minimum of 2 respiratory cycles). However, it is not designed for precise quantification of tidal volume . The physical principle underlying whole-body plethysmography is the detection of pressure changes in the chamber resulting from the heating and humidification of inspired gas. However, tidal volume measurements may also be influenced by gas compression effects related to airway resistance. Because of these limitations, semi-quantitative measurement of VT was recorded and used to calculate minute ventilation (VE = fR × VT). For pups, the plethysmograph was contained within an infant incubator  to maintain the ambient temperature at the approximate nest temperature of 32 °C.Measurements in unrestrained mice were performed using whole-body plethysmographs that had inflow and outflow ports for the continuous delivery of a steady flow (twice the chamber volume per min) of fresh air and removal of expired carbon dioxide, as previously described57: 0, no response; I, wet-dog shake (WDS); II, WDS, pawing, and clonic jerks; III, WDS, pawing, clonic jerks, rearing, and falling; and IV, continuous grade III seizures for ≥ 1 min. I-III: ≥ 10 s; < 10 s, counted as no response. Seizure severity grade was recorded by two observers blinded to the study group.A seizure severity grade was assigned based on maximal response achieved on a scale from 0 to IV, modified from that described previouslyMice were acclimated and trained on a 0° uphill Exer 3/6 open treadmill (Columbus Instruments) for 2 days. On the test day, mice started running at the speed of 10 m/min with the treadmill speed increased at a rate of 2 m/min every 5 min for a total of 25 min to a maximum rate of 20 m/min, until exhaustion as defined as the point at which mice spent more than 5 s on the electric shocker without attempting to resume running. Total running time and distance were recorded for each mouse.58. After shaving the chest, a depilatory cream was applied. The ultrasound transmission gel was kept in the thermasonic gel warmer  at 37 C. Images were obtained with a broadband scan head centered at 30-MHz. M-mode and 2-D modalities were applied to measure right ventricle (RV) free wall thickness during end diastole using the right parasternal long-axis view. These images were obtained from the right side of the mouse, with the ultrasonic beam positioned perpendicularly to the wall of the mid-third of the RV. 2-D images of the pulmonary infundibulum were obtained from the parasternal long-axis view at the level of the aortic valve and pulsed-wave Doppler recording of the pulmonary blood flow was obtained. The pulsed-wave Doppler sample was positioned at the tip of the pulmonary valve leaflets and aligned to maximize laminar flow. M-mode measurements were performed from “leading edge to leading edge”  as recommended by the American Society of Echocardiography. Pulsed-wave Doppler was used to measure pulmonary artery flow . The pulmonary valve tends to close prematurely, and peak flow velocity is reached earlier in systole. Therefore, PAAT decreases as pulmonary pressure increases. In mice, right heart catheterization, a terminal procedure, is the only available technique to measure right ventricular systolic pressure (RVSP) and, therefore, serial measurements of RVSP are not possible. PAAT, measured using high frequency echocardiography, correlates closely with invasively measured RVSP with high sensitivity and specificity58. Enlarged right ventricle was noted in short axis view. Measurements were performed offline (Vevo 77 workstation) by two readers blinded to the condition or genotype of the mice. All measurements were averaged over 5 cardiac cycles.Transthoracic echocardiography was performed at 7–8 months using a Vevo 770 high-resolution imaging system equipped with a 30-MHz transducer  with mild anesthesia (isoflurane 1.2%) and a protocol similar to prior studies which characterized pulmonary arterial hypertension in rodents2O. Once ventilated, mice were paralyzed with 1 mg/kg pancuronium bromide ip . Pressure–volume curves were generated by a sequential and increasing delivery of air into the lungs from resting pressure (zero volume) to a standard pressure of +30 cm H2O to measure total lung capacity (TLC) followed by sequential expiratory steps during which air was incrementally released. The plateau pressure was recorded when airflow returned to zero at each step59. To determine compliance of the lung, the Salazar–Knowles equation was applied to the pressure measurements obtained between total lung capacity and functional residual capacity during the expiratory phase of the pressure–volume loop59.Mice were anesthetized with a mixture of 75 mg/kg ketamine sodium and 50 mg/kg xylazine , tracheotomized, and mechanically ventilated at a rate of 150 breaths per minute, tidal volume of 6 ml/kg, and positive end-expiratory pressure of 3 cm H55. Once euthanized, the mice were immediately dissected and major organs were weighed. Lung tissue was fixed with formaldehyde (10%), embedded in paraffin, sectioned and stained with Masson’s trichrome methods by technicians of histology core in the University of Alberta. Each slide was then evaluated by two separate investigators with strong background of pathology in a blinded manner using histology scores (0–5) modified from that previously described28. Specifically, microvascular occlusions and congestion, characterized by dilated capillaries and venules filled with densely packed and deformed erythrocytes was assessed quantitatively , the features of infarcts of the lung, thrombosis, and small pulmonary hemorrhages were counted as 4, marked pulmonary hemorrhages counted as 5. We used a simple method of estimating severity of pulmonary fibrosis on a numerical scale described previously60.Euthanasia was performed by cervical dislocation. This technique reduces the effect of hypoventilation secondary to sedationSigmaplot 11  was used to conduct statistical analyses. Data are expressed as mean ± SEM. We have reported mutant results referenced to WT where WT values are expressed as 100%. One-way ANOVA or two-way repeated measures (RM) ANOVA followed by Holm-Sidak method were used as appropriate for comparison of those data that passed the normality test (Shapiro-Wilk) and equal variance test. Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method were used for comparison for those data that failed the normality test. A p-value < 0.05 or critical value indicated statistically significant group differences.