Secretory Products Research Articles

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells.

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.

The involvement of gamma-aminobutyric acid shunt in the endoplasmic reticulum stress response of Arabidopsis thaliana

The endoplasmic reticulum (ER) is the main site of secretory protein production and folding and its homeostasis under environmental stress is vital for the maintenance of the protein secretory pathway. The loss of homeostasis and accumulation of unfolded proteins in the ER is referred to as ER stress. Although, γ-aminobutyric acid (GABA) is an important regulator of stress response in plants, its roles during ER stress remains unclear. This study investigated the involvement of GABA in the ER stress response of plants. For this, changes in GABA metabolism under ER stress was analysed in Arabidopsis thaliana, then to study the response of the ER-folding machinery, plants were treated with exogenous GABA under ER stress. The antibiotic tunicamycin, which inhibits N-glycosylation was used to specifically induce ER stress. This stress up-regulated the expression of five glutamate decarboxylase (GAD) genes except GAD2 and GABA content of A. thaliana plants increased with an increasing concentration of tunicamycin (0.1 μg ml−1 and 0.25 μg ml−1). Moreover, expressions of genes involved in the conversion of GABA to succinate was also induced, while genes involved in transport across plasma and mitochondrial membrane showed no response to ER stress. The exogenous treatment of plants with 1-and 5-mM GABA increased plant performance under ER stress but 0.1 mM proved ineffective. Plants treated with GABA under ER stress had decreased expression of ER stress marker genes such as BIP1, BIP3 or CNX, but the expression of genes related to ER stress perception or ER-associated protein degradation showed no changes with respect to GABA treatments.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli.

Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

Low concentration IL-1β promotes islet amyloid formation by increasing hIAPP release from humanised mouse islets in vitro.

Aggregation of the beta cell secretory product human islet amyloid polypeptide (hIAPP) results in islet amyloid deposition, a pathological feature of type 2 diabetes. Amyloid formation is associated with increased levels of islet IL-1β as well as beta cell dysfunction and death, but the mechanisms that promote amyloid deposition in situ remain unclear. We hypothesised that physiologically relevant concentrations of IL-1β stimulate beta cell islet amyloid polypeptide (IAPP) release and promote amyloid formation. We used a humanised mouse model of endogenous beta cell hIAPP expression to examine whether low (pg/ml) concentrations of IL-1β promote islet amyloid formation in vitro. Amyloid-forming islets were cultured for 48h in the presence or absence of IL-1β with or without an IL-1β neutralising antibody. Islet morphology was assessed by immunohistochemistry and islet mRNA expression, hormone content and release were also quantified. Cell-free thioflavin T assays were used to monitor hIAPP aggregation kinetics in the presence and absence of IL-1β. Treatment with a low concentration of IL-1β (4pg/ml) for 48h increased islet amyloid prevalence (93.52 ± 3.89% vs 43.83 ± 9.67% amyloid-containing islets) and amyloid severity (4.45 ± 0.82% vs 2.16 ± 0.50% amyloid area/islet area) in hIAPP-expressing mouse islets in vitro. This effect of IL-1β was reduced when hIAPP-expressing islets were co-treated with an IL-1β neutralising antibody. Cell-free hIAPP aggregation assays showed no effect of IL-1β on hIAPP aggregation in vitro. Low concentration IL-1β did not increase markers of the unfolded protein response (Atf4, Ddit3) or alter proIAPP processing enzyme gene expression (Pcsk1, Pcsk2, Cpe) in hIAPP-expressing islets. However, release of IAPP and insulin were increased over 48h in IL-1β-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3pmol/5 islets), and this effect was blocked by co-treatment with IL-1β neutralising antibody. Under amyloidogenic conditions, physiologically relevant levels of IL-1β promote islet amyloid formation by increasing beta cell release of IAPP. Neutralisation of this effect of IL-1β may decrease the deleterious effects of islet amyloid formation on beta cell function and survival.

Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators.

Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire "expression cassette" exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3' end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In Pichia pastoris (Komagataella phaffi), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of Candida antarctica lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of P. pastoris; (2) terminator regions of well-expressed and housekeeping genes of P. pastoris; and (3) terminators of other yeast genes like Saccharomyces cerevisiae. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The Calb transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.Key points• Influence of transcription terminators on Calb gene expression• Modulation of gene expression by enhancing transcript stability.

Influence of pulmonary neuroendocrine cells on lung homeostasis

Pulmonary neuroendocrine cells (PNECs) – a unique cell population identified at all levels in the epithelium of the respiratory tract, histophysiology of which is still poorly understood. Given its important role as one of the main regulators of the respiration processes and homeostasis, its studying is one of the urgent tasks of medicine.According to the International Terms for Human Cytology and Histology published by the Federative International Committee on Anatomical Terminology (FICAT) under the writing of Wolters Kluwer and Lippincott Williams & Wilkins (2008), these cells are called respiratory neuroendocrine cells (in the trachea) or respiratory endocrine cells (in the bronchial tree). However, these cells have documented in modern international scientific literature as pulmonary neuroendocrine cells.The aim of this work is to analyze the modern scientific literature data on the effect of PNECs on the lung homeostasis in normal and pathological conditions.PNECs and their clusters – neuroepithelial bodies act as factors that regulate lung growth and maturation in embryogenesis via secretion of serotonin and gastrin-releasing hormone. In postnatal ontogenesis, PNEC secretion products – amines and neuropeptides, are characterized by participation in various physiological and pathological processes in the lung. PNECs normally maintain neurohumoral control over vascular and airway smooth muscle tone, act as peripheral chemoreceptors, and also are responsible for regulation of cell proliferation, differentiation, and mucus production from the respiratory epithelium. In case of respiratory tract damage, PNECs are capable of transdifferentiation by activating the Notch signaling pathway and renewal of other cellular types of respiratory epithelium. PNECs have a neuroimmunomodulating effect by means of neuropeptides and neurotransmitters secretion, which maintain and enhance the airways inflammatory response to an allergen. After the allergen exposition, PNECs activate type 2 innate lymphoid cells (ILC2) which being modulated by the neuropeptide CGRP produce type 2 cytokines IL-5 and IL-13, thereby contributing to an allergic response in the airways. At the same time, secreted by the PNECs neurotransmitter γ-aminobutyric acid (GABA) interacts with IL-13 to activate goblet cell mucus secretion. ILC2 induce eosinophilic inflammation and airways hypersensitivity. Recent studies have shown that ILC2 cells also stimulate Th2-associated immune response. Thus, CGRP and GABA are the key products of PNEC, which stimulate the Th2-associated immune response in the lung.Conclusions. Pulmonary neuroendocrine cells together with immune cells form a neuroimmunological module for the reception and response to environmental chemoattractants. The data on the role of pulmonary neuroendocrine cells in the airways allergic inflammation are still controversial in the literature, which necessitates further study of this issue.

Effect of heat stress during the early and late dry period on mammary gland development of Holstein dairy cattle

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ∼45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, CSN2, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Testicular immune cells and vasculature in Klinefelter syndrome from childhood up to adulthood.

Is the distribution of immune cells and the testicular vasculature altered in testicular biopsies from patients with Klinefelter syndrome (KS)? Increased numbers of macrophages and mast cells, an increased expression of decorin and an increased blood vessel density were found in KS samples compared to controls. Most KS patients are infertile due to an early germ cell loss. From puberty onwards, testicular fibrosis can be detected. How this fibrotic process is initiated remains unknown. In this study, the number of macrophages, mast cells and their secretory products were evaluated in KS, Sertoli cell only (SCO) and control patient samples. The association between immune cell numbers and level of fibrosis in KS tissue was examined. In addition, the vascularization within these testicular tissue biopsies was studied. For immunohistochemical evaluation, KS patients at different stages of testicular development were included: prepubertal (aged 4-7 years; n = 4), peripubertal (aged 11-17 years; n = 21) and adult (aged >18 years; n = 37) patients. In addition, testicular tissue biopsies of adult SCO (n = 33) and control samples for the three KS age groups (prepubertal n = 9; peripubertal n = 5; adult n = 25) were analysed. Gene expression analysis was performed on adult testicular tissue from KS (n = 5), SCO (n = 5) and control (n = 5) patients. Adult (>18 years) KS, SCO and control testicular tissue biopsies were obtained during a testicular sperm extraction procedure. KS peripubertal (11-18 years), prepubertal (<11 years) and age-matched control biopsies were obtained from the biobank of the university hospital. Immunohistochemistry was used to determine the tubular structure (H/PAS), the number of spermatogonia (MAGE-A4), macrophages (CD68) and mast cells (tryptase) and the blood vessel density (Von Willebrand factor). In addition, quantitative real-time polymerase chain reaction was used to determine the expression of secretory products of macrophages and mast cells (tryptase, tumour necrosis factor alpha and decorin). A significant increase in the number of macrophages (P < 0.0001) and mast cells (P = 0.0008) was found in the peritubular compartment of testes of adult KS patients compared to control samples. However, no association between the number of immune cells and the degree of fibrosis was observed. In adult SCO samples, a significant increase was seen for peritubular macrophage (P < 0.0001) and mast cell (P < 0.0001) numbers compared to control samples. In the interstitial compartment, a significant increase in mast cell number was found in adult SCO samples compared to KS (P < 0.0001) and control (P < 0.0001) tissue. A significant difference (P = 0.0431) in decorin expression could be detected in adult KS compared to control patients. Decorin expression was mostly seen in the walls of the seminiferous tubules. When comparing the vascularization between KS patients and age-matched controls, a significant increase (P = 0.0081) in blood vessel density could be observed only in prepubertal KS testicular tissue. N/A. As controls for this study, testicular tissue biopsies of men who underwent a vasectomy reversal or orchiectomy were used, but these men may not represent fertile controls. In addition, a high variability in immune cell numbers, secretory products expression and number of blood vessels could be observed amongst all patient samples. Increased numbers of macrophages and mast cells have previously been described in non-KS infertile men. Our results show that these increased numbers can also be detected in KS testicular tissue. However, no association between the number of macrophages or mast cells and the degree of fibrosis in KS samples could be detected. Decorin has previously been described in relation to fibrosis, but it has not yet been associated with testicular fibrosis in KS. Our results suggest a role for this proteoglycan in the fibrotic process since an increased expression was observed in adult KS tissue compared to controls. Impaired vascularization in KS men was suggested to be responsible for the KS-related disturbed hormone levels. Our results show a significant difference in blood vessel density, especially for the smallest blood vessels, between prepubertal KS samples and age-matched controls. This is the first study to report differences between KS and control testicular tissue at prepubertal age. The project was funded by grants from the Vrije Universiteit Brussel (E.G.) and the scientific Fund Willy Gepts from the UZ Brussel (D.V.S.). D.V.S. is a post-doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO; 12M2819N). No conflict of interest is declared for this research project.

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

A new model for the HPA axis explains dysregulation of stress hormones on the timescale of weeks.

Stress activates a complex network of hormones known as the hypothalamic–pituitary–adrenal (HPA) axis. The HPA axis is dysregulated in chronic stress and psychiatric disorders, but the origin of this dysregulation is unclear and cannot be explained by current HPA models. To address this, we developed a mathematical model for the HPA axis that incorporates changes in the total functional mass of the HPA hormone‐secreting glands. The mass changes are caused by HPA hormones which act as growth factors for the glands in the axis. We find that the HPA axis shows the property of dynamical compensation, where gland masses adjust over weeks to buffer variation in physiological parameters. These mass changes explain the experimental findings on dysregulation of cortisol and ACTH dynamics in alcoholism, anorexia, and postpartum. Dysregulation occurs for a wide range of parameters and is exacerbated by impaired glucocorticoid receptor (GR) feedback, providing an explanation for the implication of GR in mood disorders. These findings suggest that gland‐mass dynamics may play an important role in the pathophysiology of stress‐related disorders.

MicroRNA-146a Signaling in Alzheimer's Disease (AD) and Prion Disease (PrD).

The mouse- and human-brain-resident, nuclear factor kappa B (NF-κB)-regulated, micro RNA-146a-5p (miRNA-146a-5p) is an inducible, 22-nucleotide, single-stranded non-coding RNA (sncRNA) easily detected in several brain and immunological cell types, and an important epigenetic modulator of inflammatory signaling and the innate-immune response in several neurological disorders. Among all studied microRNAs, miRNA-146a-5p (typically referred to as just miRNA-146a) has been well characterized and its pathological function in progressive, age-related, and lethal human inflammatory neurodegenerative disease states is well documented. This communication will review our current understanding of miRNA-146a, its induction by the NF-kB-stimulating actions of inflammatory mediators, including the secretory products of certain microbial species such as viral vectors, and Gram-negative bacteria (such as Bacteroides fragilis) that are normal residents of the human gastrointestinal (GI) tract microbiome, and how miRNA-146a appears to contribute to neuro-pathological, neuro-inflammatory, and altered neuro-immunological aspects of both Alzheimer's disease (AD) and prion disease (PrD).

Flavonoids from Scutellaria baicalensis inhibit senescence-associated secretory phenotype production by interrupting IκBζ/C/EBPβ pathway: Inhibition of age-related inflammation

BackgroundProlonged exposure to the senescence-associated secretory phenotype (SASP) with age leads to chronic low-grade inflammation in neighboring cells and tissues, causing many chronic degenerative diseases. PurposeThe effects on SASP production of the ethanol extract from Scutellaria radix and 17 isolated flavonoid constituents were examined in vitro and in vivo. MethodsCellular senescence was induced by bleomycin. Expression of the SASP and cell signaling molecules was detected using ELISA, RT-qPCR, Western blotting, and immunofluorescence staining. To investigate the in vivo effects, 21-month-old aged rats were used. ResultsThe ethanol extract and 5 compounds including 1 (Oroxylin A; 5,7-dihydroxy-6-methoxyflavone), 5 (2′,6′,5,7-tetrahydroxy-8-methoxyflavone), 8 (2′,5,7-trihydroxyflavone), 10 (2′,5,7-trihydroxy-8-methoxyflavone) and 11 (2′,5,7-trihydroxy-6-methoxyflavone) potently reduced IL-6 and IL-8 production and gene expression of the SASP, including IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2, and MMP-3. This finding indicates the important role of the B-ring 2′‑hydroxyl group in flavonoid molecules. Furthermore, compounds 8 and 11, the strongest SASP inhibitors, decreased the expression of IκBζ and C/EBPβ protein without affecting either BrdU uptake or the expression of senescence markers, such as pRb and p21. Finally, the oral administration of compound 8 to aged rats at 2 and 4 mg/kg/day for 10 days significantly inhibited the gene expression of SASP and IκBζ in kidneys. This is the first report of the strong SASP inhibitory action of flavonoids from Scutellaria radix on in vitro and in vivo senescence models. The inhibitory action was shown to be mediated mainly by interfering with the IκBζ/C/EBPβ signaling pathway. ConclusionTargeting production of the SASP using flavonoids from Scutellaria radix or its extract might help reduce low-grade sterile inflammation and control age-related diseases.

Abstract B134: Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines

Abstract Colorectal cancer (CRC) is the third most common cancer among African Americans (AAs) in the US. When compared to Caucasian Americans (CAs), AAs present with higher incidence and death rates as well as worse prognosis after treatment with 5-Fluorouacil (5-FU). Previous studies have shown a correlation between microsatellite instability (MSI) and response to 5-FU and our recent findings suggest that differences in the tumor immunology and MSI status of AA and CA CRC patients are associated with the observed disparities between these populations. Therefore, we examined if cytokines secretion, protein production and cellular response to 5-FU treatment, differs between colon cancer cell lines from AAs generated in Dr. Williams’ laboratory (SB521, SB501 and CHTN06) and colon cancer cell lines from CA patients (HT29 and HCT116). Methods: We performed whole transcriptome sequencing of colon cancer cell lines (RNAseq), utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina) and Partek Flow data analysis to correlate gene expression to immune-oncology pathways. ELISA assays (RayBiotech) were used to examine the secretion of cytokines in supernatants from cells treated with interleukin 1β and tumor necrosis factor alpha. We used western blotting for protein detection of the phosphorylated form of c-Jun N-terminal kinases (JNK) as well as cell viability assays for establishing the IC50 of the cell lines to 5-FU. Results: The gene expression results indicated that the immune profiles of AA cell lines differ from CA in genes and cytokines related to cellular anti-tumor activity, including CD8B, IL-1R, IL-1R2, Granzyme B and NFKB. ELISAs of supernatants from CA and AA CRC cell lines revealed a differential cytokines secretion between the two races, namely IL-8. Lastly, the MSI AA cell line showed sensitivity to 5-FU (tenfold less) when compared to the CA cell lines and a distinct protein production pattern. Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance and cancer progression in the CRC cell lines between the two races. These results were in accordance with the cytokines’ and protein’s expression patterns observed in the two cohorts of cell lines. Importantly, our data indicates that 5-FU is more efficient in reducing cell viability in the MSI AA cell line than in the two CA cell lines regardless of their MSI status. Altogether, our results illustrate the value of these in vitro models to study 5-FU treatment in AA patients with MSI and MMR mutations and to elucidate the differences in chemotherapy treatment responses between AAs and CAs. In conclusion, we demonstrated distinct immunological profiles and 5-FU sensitivity of AA and CA CRC cell lines. As such, these differences observed could be used to guide new therapeutic strategies. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Ping Ji, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B134.

Thyroidectomy induces thyroglobulin formation by parotid salivary glands in rats

Structural changes in parotid gland (PG) were previously reported following ablation of thyroid gland. However, the functional alterations (especially for proteins) have not been elucidated yet. Herein, we investigated the effect of rat thyroidectomy on PG structure and protein content and studied the ability of thyroxin-supplementation to alleviate the associated structural and functional changes. Male young adult 4-month old albino rats (n = 48) were allocated equally into 4 groups (control, sham-operated, thyroidectomized, and thyroxin-supplemented). PGs were examined histologically, and their proteins expression and localization were analyzed using western blot (WB) and immunohistochemistry (IHC), respectively at 3 w and 5 w post-surgery. Functionally, PGs of thyroidectomized rats formed a newly expressed 300 KDa protein, which was confirmed to be thyroglobulin (TG) by WB and IHC, with higher expression at 5 w. TG was localized in the interstitium, within capillaries, in the cytoplasm of the intralobular ductal cells, in the secretory products within the ductal lumen, and in the cytoplasm of individual small cells at the periphery of the acini. This functional change accompanied by structural changes in PGs (presence of dark and light acinar cells, TG-like colloid material, and high periductal vasculature). Noteworthy, PG of the thyroxin-supplemented depicted vanishment of TG. From these data, it could be concluded that thyroidectomy could alter the morphology and function of the parotid that induce a thyroid-like reprogramming of the parotid to secrete TG and thyroxin supplementation could alleviate this effect.

Experimental Analysis of E2BB (LTIIb) Signal Peptide in Secretory Production of Reteplase in Escherichia coli

Recombinant reteplase is the truncated form of tissue plasminogen activator. Signal peptides play a pivotal role in the secretion of recombinant proteins. This study aimed to evaluate the effect of LTIIb signal peptide on the recombinant reteplase secretion in Escherichia coli. In the current study, cloning and expression of reteplase coding sequence with and without sequence corresponds to the peptide signal using pET 28a system was performed and its recombinant protein expression in E. coli BL21 (DE3) was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to the final concentrations 0.1 mM. The recombinant reteplase expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assay. The activity of reteplase in periplasmic fraction and supernatant was measured by colorimetric assay. Our results confirm that high levels of reteplase protein are expressed as inclusion bodies. In the presence of peptide signal LTIIb, in periplasmic fraction, reteplase activity was negligible compared to the reteplase standard. No activity observed for reteplase in the periplasmic and extracellular fraction. The comparison between results obtained for LTIIb-reteplase with those to reteplase revealed that LTIIb signal peptide is not the proper candidate for the secretory production of reteplase.

The role of meibography in ocular surface diagnostics: A review

The meibomian glands are lipid-secreting glands located in the tarsal plates, whose secretory products cover the tear film, thereby reducing evaporation as well as ensuring lubrication of the ocular surface. The meibomian glands can be visualized at different levels of magnification by infrared meibography, laser confocal microscopy, and optical coherence tomography. These imaging modalities have been subject to much research and progress in clinical practice and have shaped our current understanding of meibomian glands in health and disease. In this review, we explore the evolution of meibography over the past decades, the major contributions of various meibographic modalities, and discuss their clinical significance.

Islet pericytes convert into profibrotic myofibroblasts in a mouse model of islet vascular fibrosis.

Islet vascular fibrosis may play an important role in the progression of type 2 diabetes, but there are no mouse models allowing detailed mechanistic studies to understand how a dysfunctional islet microvasculature contributes to diabetes pathogenesis. Here we report that the transgenic AktTg mouse, unlike other mouse strains, shows an increased deposition of extracellular matrix (ECM) proteins in perivascular regions, allowing us to study the cellular mechanisms that lead to islet vascular fibrosis. Using immunohistochemistry, we labelled the islet microvasculature and ECM in pancreas sections of AktTg mice and human donors and performed lineage tracing to follow the fate of islet pericytes. We compared islet microvascular responses in living pancreas slices from wild-type and AktTg mice. We found that vascular pericytes proliferate extensively, convert into profibrotic myofibroblasts and substantially contribute to vascular fibrosis in the AktTg mouse model. The increased deposition of collagen I, fibronectin and periostin within the islet is associated with diminished islet perfusion as well as impaired capillary responses to noradrenaline (norepinephrine) and to high glucose in living pancreas slices. Our study thus illustrates how the AktTg mouse serves to elucidate a cellular mechanism in the development of islet vascular fibrosis, namely a change in pericyte phenotype that leads to vascular dysfunction. Because beta cells in the AktTg mouse are more numerous and larger, and secrete more insulin, in future studies we will test the role beta cell secretory products play in determining the phenotype of pericytes and other cells residing in the islet microenvironment under physiological and pathophysiological conditions. Graphical abstract.

Free ammonia is the primary stress factor rather than total ammonium to Chlorella sorokiniana in simulated sludge fermentation liquor

Microalgal growth was inhibited in high-strength ammonium wastewater, but the actual stress factor and their physiological responses remain to be elucidated. In this study, compared to either the total ammonium or pH alone, free ammonia (FA) concentration clearly exhibited negative correlation with the growth of Chlorella sorokiniana cultivated in the simulated sludge fermentation liquor (SFL), which could be well described by an exponential decay model, indicating of the primary stress factor. Subsequently, six concentrations of FA were selected for further investigation and samples were monitored for: 1) growth and ammonium removal; 2) changes in photosynthetic system; 3) changes in reactive oxygen species (ROS) and soluble extracellular polymeric substances (S-EPS); as well as 4) biomass biochemical composition. Both the growth performance and photosynthetic parameters Fv/Fm and YII negatively correlated with FA concentrations. The highest biomass production was observed at the lowest initial FA concentration (2.06 mg/L) and almost no growth was observed in the highest FA concentration (110.97 mg/L).Ammonium nitrogen was removed by algae uptake and evaporation. Cell stress was indicated by significantly increased production of ROS (2.53–12.92 FI/1010cells) and secretion of S-EPS (144.13–160.86 mg/g DW). High FA concentrations induced the accumulation of cellular carbohydrates and palmitic acid. The stress responses to FA observed provide a guide in strategies for microalgal adaptation and growth in high-strength ammonium wastewater.

The lack of terminal tubule cells in the submandibular gland of mice deficient in submandibular gland protein C.

The submandibular gland (SMG) of newborn mice has no mature acini but has the rudiments of acini called terminal tubules (TT). The TT are composed of TT cells with dark secretory granules and proacinar cells with lighter secretory granules, the latter being considered the immediate precursor of mature acinar cells. TT cells contain a specific secretory protein, submandibular gland protein C (SMGC) and they decrease in number postnatally at a higher rate in males than in females. In the present study, in order to clarify the biological roles of TT cells and their secretory product SMGC, we generated a knockout (KO) mouse strain deficient in SMGC. The KO mice of both sexes grew normally, had normal reproductive capacity and had normal acinar and duct systems in the SMG in adult ages. However, through the neonatal and early postnatal stages, the KO mice were deficient not only in the production of SMGC but also in TT cells. With electron microscopy of the SMG of newborn KO mice, TT cells with characteristic granules were absent and replaced by undifferentiated ductal cells, whereas proacinar cells were normal. These results suggested that the absence of SMGC inhibits the development of TT cells and that the absence of SMGC and TT cells has no notable influence on the postnatal development of the acinar and duct systems in the SMG.

Streptomyces lydicus M01 Regulates Soil Microbial Community and Alleviates Foliar Disease Caused by Alternaria alternata on Cucumbers.

Due to the adverse effect on the environment caused by excessive use of chemical fertilizers, the development of sustainable agriculture attracts a growing demand of biological based fertilizers composed of living microorganisms. In this study, an Actinobacteria Streptomyces lydicus M01 was isolated from the rhizosphere soil of Pyrus calleryana. This strain effectively promoted the plant growth and suppressed a foliar disease caused by Alternaria alternata on cucumbers. S. lydicus M01 exhibited growth promoting characteristics such as phosphate solubilization, IAA secretion, siderophore and ACC deaminase production. Through Illumina sequencing of the 16S rRNA gene and ITS gene of the soil microbes, we found that the application of S. lydicus M01 altered the composition of the microbial community by promoting beneficial groups, including bacteria genera Pseudarthrobacter, Sphingomonas, Rhodanobacter, and Pseudomonas, fungi genera Fusicolla, Humicola, Solicoccozyma, and Paraphaeosphaeria. Most of these bacteria and eukaryotes exhibit positive effects on growth promotion, such as nutrient accumulation, auxin secretion, abiotic stress alleviation, biological control, or bioremediation. Furthermore, studies on the reactive oxygen species (ROS) level and antioxidants of cucumber leaves revealed that S. lydicus M01 treatment reduced the ROS accumulation and increased the activities of antioxidases related with ROS scavenging, which indicated an enhanced disease resistance of cucumbers under biotic stress. Thus, our results suggest that the application of S. lydicus M01 can systemically affect plant microbiome interactions and represent a promising sustainable solution to improve agricultural production instead of chemical fertilizers.