Potential role of Candida albicans secreted aspartic protease 9 in serum induced-hyphal formation and interaction with oral epithelial cells.

INTRODUCTION: Candida albicans possesses the ability to switch rapidly between yeast to hyphal forms. Hyphal formation is a remarkable pathogenic characteristic, which allows C. albicans to invade into host cells. OBJECTIVES: This study was to investigate the role of the C. albicans SAP9 gene in hyphal formation and invasion ability. METHODS: The morphology of fungal cells in the hyphal-inducing liquid media (YPD+10% fetal bovine serum) was observed by the microscopy. And the morphology of the colony on solid agar plates of YPD+10% fetal bovine serum was photographed by the digital camera. The mRNA expressions of hypha-associated genes in serum medium were also analyzed by real time PCR. Then for the interaction between C. albicans and oral epithelial cells, endocytosis essay, invasion essay and damage assay were performed to compare the differences between the sap9Δ/Δ mutant strain and wild type strain. RESULTS: Compared with the wild type strain, the sap9Δ/Δ mutant strain exhibited a deficient yeast-to-hyphal morphological transition under serum hyphal-inducing conditions. Furthermore, the SAP9 knockout strain revealed a significant down-regulation of the expression of EFG1 (~40%), which is a transcription factor gene that mediates hyphae formation in C. albicans. Compared with the wild type strain, a 70% reduction in the endocytosis of the sap9Δ/Δ mutant strain by host cells was observed, as well as a 25% attenuation of active penetration and a 40% attenuation of host cell damage (P <0.05). CONCLUSIONS: Our data strongly suggests that C. albicans Sap9 is a potential hyphal-associated factor that responds to serum hyphal-inducing stimuli via a cAMP-protein kinase A pathway mediated by EFG1, and contributes to the process of invasion of Candida into the epithelial cells, leading to host cell damage.

Matrilin-1 is an inhibitor of neovascularization.

In the course of conducting a series of studies whose goal was to discover novel endogenous angiogenesis inhibitors, we have purified matrilin-1 (MATN-1) and have demonstrated, for the first time, that it inhibits neovascularization both in vitro and in vivo. Proteins were extracted from cartilage using a 2 m NaCl, 0.01 m HEPES buffer at 4 °C, followed by concentration of the extract. The concentrate was fractionated by size exclusion chromatography, and fractions were then screened for their ability to inhibit capillary endothelial cell (EC) proliferation in vitro. Fractions containing EC inhibitory activity were pooled and further purified by cation exchange chromatography. The resulting fractions from this step were then screened to isolate the antiangiogenic activity in vitro. This activity was identified by tandem mass spectrometry as being MATN-1. Human MATN-1 was cloned and expressed in Pichia pastoris and purified to homogeneity. Purified recombinant MATN-1, along with purified native protein, was shown to inhibit angiogenesis in vivo using the chick chorioallantoic membrane assay by the inhibition of capillary EC proliferation and migration. Finally, using a MATN-1-deficient mouse, we showed that angiogenesis during fracture healing was significantly higher in MATN-1(-/-) mice compared with the wild type mice as demonstrated by in vivo imaging and by elevated expression of angiogenesis markers including PECAM1, VEGFR, and VE-cadherin.

Using estradiol and progesterone concentrations to assess individual variability in the reproductive cyclicity of captive female little skates, Leucoraja erinacea, from the western Gulf of Maine.

In the current study, plasma steroid hormones were used to assess the individual variability of Leucoraja erinacea over the course of 12 months, in hopes of further defining its reproductive cycle. No statistical differences in hormone concentrations were observed between the isolated and non-isolated female skates. Monthly E2 concentrations ranged from 1,430 pg ml(-1) in August to 3,940 pg ml(-1) in March, indicating the presence of mature ovarian follicles and supporting the conclusions from previous studies that L. erinacea is capable of reproducing year-round. Concentrations of E2 were significantly elevated or depressed during some months (February, March, June, July, August, and September) of the year, suggesting that reproductive activity may vary over the annual cycle. Even though monthly P4 concentrations were highly variable, ranging from 82 pg ml(-1) in November to 816 pg ml(-1) in September, no significant reproductive peaks were observed. In addition, a persistently large variation in E2 and P4 concentrations, indicative of reproductive asynchrony within (mean CV 62% and CV 69%, respectively) and between (mean range CV 78 and 125%, respectively) individual skates, was observed throughout the study. Collectively, the continually high E2 concentrations and variability in both hormones observed in the current study are indicative of an oviparous species that reproduces actively throughout the year. However, the weekly sampling frequency revealed that plasma E2 concentrations, not P4, were more useful to assess reproductive status in asynchronous continuously breeding oviparous elasmobranchs.

A randomized trial of the effectiveness of an ultrasonic denture hygiene intervention program among community dwelling elders.

Purpose: This study aimed to assess the effectiveness of ultrasonic denture hygiene interventions in improving denture cleanliness among elderly individuals. Materials and methods: Sixty-six participants who had received upper metal framework removable partial dentures within the past 5 years were randomly allocated into three denture hygiene intervention groups: group 1 (mechanical cleaning with a toothbrush and ultrasonic cleaning with cetylpyridinium chloride), group 2 (mechanical cleaning with a toothbrush and ultrasonic cleaning with distilled water), and control (mechanical cleaning with a toothbrush only). Denture cleanliness was assessed at baseline and 1-month using: i) Denture Cleanliness Index (DCI) scores; ii) plaque coverage percentage; and (iii) microbiological samples for bacterial and yeast detection. Differences between groups were assessed with one-way analysis of variance and Chi-squared tests. Results: Mean DCI scores and mean percentages of plaque coverage area were significantly reduced in group 1 and group 2, compared to the control group for both cobalt chromium (CoCr) and acrylic fitting surfaces (p<0.001). No significant differences were found between groups 1 and 2 with regard to the prevalence and viable counts of yeasts or total microbial viable counts. No significant differences in the investigated clinical and microbiological parameters were observed between CoCr and acrylic surfaces following the intervention period. Conclusion: The ultrasonic cleaner was significantly more effective than mechanical cleaning in the reduction of biofilm coverage on metal framework removable partial dentures over a 1-month intervention period. Nevertheless, the adjunctive use of cetylpyridinium chloride with ultrasonic cleaning did not yield additional benefits.

A novel approach in predicting virtual garment fitting sizes with psychographic characteristics and 3D body measurements using artificial neural network and visualizing fitted bodies using generative adversarial network.

Advances in technology have brought accessibility to garment product fitting procedures with a virtual fitting environment and, in due course, improved the supply chain socially, economically, and environmentally. 3D body measurements, garment sizes, and ease allowance are the necessary factors to ensure end-user satisfaction in the apparel industry. However, designers find it challenging to recognize customers' motivations and emotions towards their preferred fit and define ease allowances in the virtual environment. This study investigates the variations of ease preferences for apparel sizes with body dimensions and psychological orientations by developing a virtual garment fitting prediction model. An artificial neural network (ANN) was employed to develop the model. The ANN model was proved to be effective in predicting ease preferences from two major components. A non-linear relationship was modeled among pattern parameters, body dimensions, and psychographic characteristics. Also, to visualize the fitted bodies, a generative adversarial network (GAN) was applied to generate 3D samples with the predicted pattern parameters from the ANN model. This project promotes mass customization using psychographic orientations and provides the perfect fit to the end users. New size-fitting data is generated for improved ease preference charts, and it enhances end-user satisfaction with garment fit.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 µg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 µg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

The corpus luteum (CL) is a transient ovarian endocrine gland that secretes progesterone, the hormone of pregnancy. Development of an optimally functioning CL requires the creation of a dense capillary bed through growth of new blood vessels, which is an intricate process called angiogenesis. A myriad of factors regulates angiogenesis, including the angiogenic inducer protein, cellular communication network factor 1 (CCN1). Although it is highly expressed in the early-cycle bovine CL, the mechanisms of CCN1 regulation have not been fully elucidated. In the present study, we showed that CCN1 expression in steroidogenic luteal cells from the early-cycle bovine CL was induced by Ras homolog family member A (RhoA) and by thrombin, but not by luteinizing hormone (LH). To the best of our knowledge, the involvement of thrombin and its signaling partner, RhoA, in regulating CCN1 in bovine steroidogenic luteal cells has not been previously reported. These findings will inform our future work to determine how RhoA activation by thrombin leads to increased expression of CCN1.

A role for cysteine-rich 61 in the angiogenic switch during the estrous cycle in cows: regulation by prostaglandin F2alpha.

The development and demise of the corpus luteum (CL) are accompanied by angiogenic and angioregressive processes; however, the mediators of these processes have not been fully identified and characterized. Transcriptional profiling studies revealed the upregulation of cysteine-rich 61 (CYR61) in the CL, about which nothing was previously known. In the present study, we found that over a 12-h period following a single injection of prostaglandin F(2alpha) (PGF(2alpha)), RT-PCR revealed the upregulation of CYR61 at 0.5 and 1 h, after which it declined. We also determined that luteal-derived endothelial cells as well as luteal steroidogenic cells are sources of CYR61. Treatment with PGF(2alpha) in vitro had no effect on CYR61 expression in luteal-derived endothelial cells, but it increased CYR61 expression in luteal steroidogenic cells. During the estrous cycle, CYR61/CYR61 (transcript/protein) was increased in the Day 4 but not in the Day 10 and Day 16 CL, suggesting that it may be associated with the switch to the angiogenic phenotype. In addition, the specific but transient upregulation of CYR61 by PGF(2alpha) in vivo, and in luteal steroidogenic cells but not endothelial cells in vitro, may be part of the mechanism underlying the previously reported transient increase in blood flow during the early onset of luteolysis. This is supported by our preliminary finding that CYR61 transiently inhibited endothelial cell expression of endothelin-converting enzyme 1 mRNA but not endothelin 1. Collectively, the increased expression of CYR61 in the Day 4 CL and its transient increase by PGF(2alpha) in Day 6, Day 10, and Day 16 CL indicate that CYR61 may play a role in regulating angiogenesis over the life span of the CL.

Differential phytate utilization in Candida species.

The present study was undertaken to evaluate and characterize the phytase activity in different Candida species. A total of 113 Candida isolates representing eight species were examined for phytase activity by an agar plate assay using the calcium salt of phytic acid as the sole phosphorus source. A phytase-positive phenotype was identified by the formation of a clear halo around a fungal colony. Cell-bound differential phytase activity was observed in Candida isolates at inter- and intra-species levels. Although phytase activity was not affected by the supplementation of external phosphate in C. albicans, C. dubliniensis, C. glabrata, and C. kefyr, elevated phytase activity was evident in C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis in phosphate-free medium. Further characterization showed that, in general, relatively higher phytase activity was observed at more acidic pHs, and the phytase activity increased with incubation temperature, reaching a maximum at 55 or 65°C. Taken together, the findings demonstrated, for the first time, differential phytase activities in different Candida species. Phytase activity may be a contributing factor to fungal survival and proliferation within the human gastrointestinal tract, where nutrients are usually scarce.

Isolation and characterization of Candida kefyr orotidine-5'-phosphate decarboxylase (URA3) gene.

Candida kefyr is a common yeast species that can be found in fermented milk and cheeses. As a first step to developing a gene transfer system for C. kefyr, the orotidine-5'-phosphate decarboxylase (URA3) gene was cloned, using degenerate PCR and genome walking. The uninterrupted open reading frame of the C. kefyr URA3 gene spans 801 bp, corresponding to 267 amino acid residues. The functionality of the gene was confirmed by complementation of ura3 auxotrophs of C. albicans and Saccharomyces cerevisiae. Phylogenetic analysis of the deduced amino acid sequence indicated that it shares a high degree of homology with other Candida URA3 homologues.

Novel antifungal activity of purpurin against Candida species in vitro.

The antifungal activity of purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated by a broth microdilution assay against a total of 24 Candida isolates representing six species. The minimum inhibitory concentration (MIC) range of purpurin was 1.28-5.12 µg/ml. Mechanistic studies using the rhodamine 6G extrusion assay indicated that purpurin inhibited the energy-dependent efflux pumps of the Candida isolates in a dose-dependent manner. Furthermore, purpurin demonstrated a dose-dependent depolarization of mitochondrial membrane potential, one of the biochemical checkpoints regulating cell death in eukaryotic cells, suggesting a possible linkage between purpurin antifungal mechanism and Candida apoptosis.

Photodynamic inactivation of Candida albicans by BAM-SiPc.

Photodynamic therapy is a treatment that combines the use of three non-toxic components, viz. photosensitiser, light and oxygen to cause localised oxidative photodamage. In the present study, the antifungal effect of the photosensitiser, BAM-SiPc, an unsymmetrical bisamino phthalocyanine, was investigated. BAM-SiPc was effective in photo-inactivating Candida albicans in a dose-dependent manner. The cell viability as determined by the clonogenic assay was reduced to c. 10% at 0.02 micromol l(-1) BAM-SiPc with a total fluence of 12 J cm(-2) at a cell density of 10(7) cells ml(-1). A short incubation time of 5-15 min was sufficient to allow the photosensitiser to exert its optimal antifungal activity. Microscopical analysis showed that BAM-SiPc was effectively internalised by the fungal cells. Photodynamic treatment led to an increase in the intracellular reactive oxygen species level and disturbed the membrane integrity of the fungal cells.

Antifungal activity of baicalein against Candida krusei does not involve apoptosis.

The present study was designed to evaluate the antifungal activity of baicalein against Candida krusei isolates. Using a broth microdilution assay, baicalein exhibited potent in vitro antifungal activity against C. krusei isolates with a minimum inhibitory concentration of 2.7 µg/ml. Flow cytometric study indicated that baicalein depolarized mitochondrial membrane potential in a concentration-dependent manner. However, mechanistic analyses showed that the intracellular reactive oxygen species (ROS) level was virtually unchanged, and massive DNA fragmentation was not observed in C. krusei isolates after baicalein treatment even at a concentration which was apoptotic in C. albicans. Taken together, we conclude that the antifungal activity of baicalein in C. krusei isolates occurs through perturbation in mitochondrial homeostasis without causing elevation of the intracellular ROS level and does not involve apoptosis.

Engineering and optimization of phosphate-responsive phytase expression in Pichia pastoris yeast for phytate hydrolysis.

Phytate is the major storage form of phosphorus in plants. It is present in cereals and raw materials of vegetable origin used in animal and human diets. However, non-ruminant animals have little phytase activity in their guts and, therefore, cannot digest phytate. As a result, almost all dietary phytate is discharged into the environment, causing phosphorus pollution. Phytate is also considered as an "antinutrient" for its ability to form insoluble and stable complexes with metal ions, thus reducing dietary absorption of essential minerals. It is a dire need to develop sustainable approaches for environmentally-friendly utilization for this valuable and abundant natural resource. To this end, we engineered Pichia pastoris to express and secrete phytase in a "made-to-order" fashion in response to external level of inorganic phosphate (Pi). Responsiveness to external Pi level was achieved by generating a Pi-responsive promoter library using directed evolution. The resultant yeast strains were proven to liberate Pi from wheat-based meal in a simulated in vitro digestion model. These yeast-based whole cell biocatalysts may serve as platform hosts with potential applications in food processing industry and animal waste treatment.

Candida albicans orf19.3727 encodes phytase activity and is essential for human tissue damage.

Candida albicans is a clinically important human fungal pathogen. We previously identified the presence of cell-associated phytase activity in C. albicans. Here, we reveal for the first time, that orf19.3727 contributes to phytase activity in C. albicans and ultimately to its virulence potency. Compared with its wild type counterpart, disruption of C. albicans orf19.3727 led to decreased phytase activity, reduced ability to form hyphae, attenuated in vitro adhesion, and reduced ability to penetrate human epithelium, which are the major virulence attributes of this yeast. Thus, orf19.3727 of C. albicans plays a key role in fungal pathogenesis. Further, our data uncover a putative novel strategy for anti-Candidal drug design through inhibition of phytase activity of this common pathogen.

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the alpha/beta-hydrolase fold superfamily.

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans alpha-amino acid ester hydrolase (AtAEH), members of the alpha/beta-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an alpha/beta-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the alpha/beta-hydrolase fold superfamily with a serine-histidine-aspartate catalytic triad.

Temporal and spatial expression of tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2) in the bovine corpus luteum.

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), may mediate the dramatic structural and functional changes in the corpus luteum (CL) over the course of its life span. In addition to regulating MMP activity, TIMPs are also involved in a variety of cellular processes, including cell proliferation and steroidogenesis. In a series of initial studies, we determined that matrix metalloproteinase inhibitory activity was present in protein extracts from early (4 days old, estrus = day 0), mid (10-12 days old) and late (16 days old) CL (n = 3 for each stage). Reverse zymography revealed four metalloproteinase inhibitory protein bands with relative molecular masses that are consistent with those reported for TIMP-1 to -4. In order to gain a better understanding of TIMPs and their role in luteal function, we further characterized this inhibitory activity with a particular focus on the temporal and spatial expression of TIMP-1 and TIMP-2 in the bovine CL. Northern blotting revealed that the TIMP-1 transcript (0.9 kb) was expressed at a higher (p < 0.05) level in early and mid cycle CL than in the late stage. In contrast, two TIMP-2 mRNA species, one major 1 kb species and one minor 3.5 kb species, were significantly (p < 0.05) increased in the mid and late cycle CL than in the early. Western blotting analyses demonstrated no differences in TIMP-1 (29 kDa) protein levels between early and mid stages, while its levels decreased (p < 0.05) from the mid to late stage CL. Conversely, TIMP-2 (22 kDa) protein was detected at a low level in the early CL, but significantly (p < 0.05) increased in the mid and late stages. Immunohistochemistry revealed that both TIMP-1 and -2 were localized to large luteal cells from all three ages of CL. TIMP-1 was also localized in capillary smooth muscle cells, while TIMP-2 was restricted to the endothelial cells in the capillary compartment. In conclusion, the different temporal expression patterns of TIMP-1 and TIMP-2 suggest that TIMP-1 may be important for luteal formation and development, while TIMP-2 may play significant roles during luteal development and maintenance. Furthermore, the distinct localization of these two inhibitors in the vascular compartment indicates that they may serve diverse physiological functions during different stages of luteal angiogenesis.

Charting epilepsy by searching for intelligence in network space with the help of evolving autonomous agents.

The problem of demarcating neural network space is formidable. A simple fully connected recurrent network of five units (binary activations, synaptic weight resolution of 10) has 3.2 *10(26) possible initial states. The problem increases drastically with scaling. Here we consider three complementary approaches to help direct the exploration to distinguish epileptic from healthy networks. [1] First, we perform a gross mapping of the space of five-unit continuous recurrent networks using randomized weights and initial activations. The majority of weight patterns (>70%) were found to result in neural assemblies exhibiting periodic limit-cycle oscillatory behavior. [2] Next we examine the activation space of non-periodic networks demonstrating that the emergence of paroxysmal activity does not require changes in connectivity. [3] The next challenge is to focus the search of network space to identify networks with more complex dynamics. Here we rely on a major available indicator critical to clinical assessment but largely ignored by epilepsy modelers, namely: behavioral states. To this end, we connected the above network layout to an external robot in which interactive states were evolved. The first random generation showed a distribution in line with approach [1]. That is, the predominate phenotypes were fixed-point or oscillatory with seizure-like motor output. As evolution progressed the profile changed markedly. Within 20 generations the entire population was able to navigate a simple environment with all individuals exhibiting multiply-stable behaviors with no cases of default locked limit-cycle oscillatory motor behavior. The resultant population may thus afford us a view of the architectural principles demarcating healthy biological networks from the pathological. The approach has an advantage over other epilepsy modeling techniques in providing a way to clarify whether observed dynamics or suggested therapies are pointing to computational viability or dead space.

Purpurin triggers caspase-independent apoptosis in Candida dubliniensis biofilms.

Candida dubliniensis is an important human fungal pathogen that causes oral infections in patients with AIDS and diabetes mellitus. However, C. Dubliniensis has been frequently reported in bloodstream infections in clinical settings. Like its phylogenetically related virulent species C. albicans, C. Dubliniensis is able to grow and switch between yeast form and filamentous form (hyphae) and develops biofilms on both abiotic and biotic surfaces. Biofilms are recalcitrant to antifungal therapies and C. Dubliniensis readily turns drug resistant upon repeated exposure. More than 80% of infections are associated with biofilms. Suppression of fungal biofilms may therefore represent a viable antifungal strategy with clinical relevance. Here, we report that C. dubliniensis biofilms were inhibited by purpurin, a natural anthraquinone pigment isolated from madder root. Purpurin inhibited C. dubliniensis biofilm formation in a concentration-dependent manner; while mature biofilms were less susceptible to purpurin. Scanning electron microscopy (SEM) analysis revealed scanty structure consisting of yeast cells in purpurin-treated C. dubliniensis biofilms. We sought to delineate the mechanisms of the anti-biofilm activity of purpurin on C. Dubliniensis. Intracellular ROS levels were significantly elevated in fungal biofilms and depolarization of MMP was evident upon purpurin treatment in a concentration-dependent manner. DNA degradation was evident. However, no activated metacaspase could be detected. Together, purpurin triggered metacaspase-independent apoptosis in C. dubliniensis biofilms.

Assessing reproductive status in elasmobranch fishes using steroid hormones extracted from skeletal muscle tissue.

Elasmobranch fishes (sharks, skates, and rays) are particularly susceptible to anthropogenic threats, making a thorough understanding of their life history characteristics essential for proper management. Historically, elasmobranch reproductive data have been collected by lethal sampling, an approach that is problematic for threatened and endangered species. However, recent studies have demonstrated that non-lethal approaches can be as effective as lethal ones for assessment of the reproductive status of an animal. For example, plasma has been used to examine concentrations of steroid hormones. Additionally, skeletal muscle tissue, which can be obtained non-lethally and with minimal stress, can also be used to quantify concentrations of steroid hormones. Skeletal muscle progesterone, testosterone, and estradiol concentrations were determined to be statistically significant indicators of reproductive status in the oviparous Leucoraja erinacea, the yolk-dependent viviparous Squalus acanthias, and the yolk-sac placental viviparous Rhizoprionodon terraenovae. The results of the present study demonstrate that steroid hormones present in non-lethally harvested skeletal muscle tissue can be used as reliable indicators of reproductive status in elasmobranchs.

Novel Mechanism of Glass Delamination in Type 1A Borosilicate Vials Containing Frozen Protein Formulations.

Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. A significant issue was observed when small, flake-like pieces of glass particles (lamellae) appeared in vials containing thawed protein product. The occurrence of glass particles during freeze-thaw results in product rejection and potentially, adverse events. In recent years, glass flakes due to chemical delamination have been observed in parenteral liquid formulations after long-term storage, resulting in a number of product recalls. In this study, for the first time, glass delamination is reported in pharmaceutical glass vials containing frozen protein formulation, caused by a novel mechanism involving thermally-induced mechanical stress. In this article, a monoclonal antibody drug product in glass vials and the corresponding placebo vials were studied to identify the contributing factors from the freeze-thaw process, such as freezing temperature, the presence or absence of protein, and other handling conditions. Freezing temperature was found to be the most critical factor. Glass lamellae were only observed when the products were frozen to -70 °C, while freezing only to -30 °C did not cause any lamellae formation even after multiple freeze-thaw cycles. Protein concentration and the handling of the vials were also identified as contributing factors. A concentration gradient which formed after freeze-thaw induced a higher rate of lamellae occurrence in a subsequent freeze-thaw cycle compared to vials without the concentration gradient. Analyses by Fourier transform infrared spectroscopy and scanning electron microscopy/energy dispersive spectroscopy confirmed that the flake-like lamellae were thin, flat glass particles. Defects corresponding to the glass flakes were observed by scanning electron microscopy on the inner surface of the vials that contained lamellae. In addition, inductively coupled plasma mass spectrometry testing did not show elevated levels of silicon in the drug product solution, suggesting that the glass lamellae formed in the frozen vials was a local, event-based phenomenon rather than silica dissolution from the product contact surface or glass degradation caused by corrosive attack. These findings can be explained by the same thermally-induced mechanical stress which caused vial breakage. Frozen protein formulations contracted below -30 °C, causing an inward glass deformation and a subsequent rapid movement of the glass when the frozen plug of drug product solution separated from the vial inner surface at approximately -50 to -60 °C. The mechanical stress released during this separation caused vial breakage. The incidence of vial breakage increased with more concentrated product and higher fill volume-to-vial volume ratios. The same mechanism applies to lamellae formation. As the rapid surface separation occurred, small, thin pieces of glass were pulled from the glass surface by the frozen plug, and, as a result, glass lamellae particles appeared in the drug product solution after thawing. LAY ABSTRACT: In recent years, glass flakes have been observed in parenteral liquid formulations due to chemical delamination during long-term storage, resulting in a number of product recalls. In our study, we discovered a novel mechanism of glass delamination in vials containing frozen protein formulations. This glass delamination mechanism has never been reported before, and we believe this work will benefit the pharmaceutical scientific community, especially the biotechnology and parenteral drug industries. Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. In this study, we observed that after thawing, small, flake-like pieces of glass particles (i.e., lamellae) appeared in vials containing frozen protein formulation. To investigate the root cause, we performed a series of freeze-thaw experiments and characterized the lamellae particles, the vial inner surface, and the elemental composition of the solution. The root cause was determined to be mechanical stress caused by thermal contraction of frozen protein formulations below -30 °C. This contraction caused an inward glass deformation on the vial sidewall and, subsequently, the glass vial surface abruptly separated from frozen protein formulation. Under this mechanical stress, small, thin glass pieces were peeled from the vial inner surface by the frozen formulation, causing lamellae formation. The experimental design and results leading to the discovery of the novel glass delamination mechanism are presented in detail in this article.