Protein A-Nanoluciferase fusion protein for generalized, sensitive detection of immunoglobulin G.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Conservative Management of Major Anastomotic Leaks Occurring after Primary Repair in Esophageal Atresia with Fistula: Role of Extrapleural Approach.

AIMS: We are reporting single-institution's experience regarding the role of conservative management in 38 cases of minor and major anastomotic leaks [AL] occurring after primary surgery of esophageal atresia [EA] with tracheo-esophageal fistula [TEF] during last 17 years between 2000 and 2017. In this retrospective review, we are sharing our experience and protocol of management of AL with more emphasis to evaluate: (a) role of conservative treatment in major AL (b) role of extra-pleural approach in enhancing the success rate in conservative treatment in major AL (c) to define the criteria for major & minor leaks and (d) to evaluate the role of ventilation in primary EA surgery to control AL. METHODS: All these cases were operated through extra-pleural approach and out of total 203 cases, 38[18.7%] developed anastomotic leaks. In 29 of the 38 cases [14.3%], leak was minor and in 9 cases [4.4%] the leak was a major one. All these cases of leaks were managed conservatively. RESULTS: All cases of major and minor leaks showed spontaneous healing except one case of minor leak that died before healing due to major cardiac anomaly. For minor leaks, average healing time was 9.5 days while for major leaks it was 17.4 days. Overall mortality was 14.8% and there was no mortality directly attributable to major or minor leak. During follow up, the incidence of stricture was 40% in cases having anastomotic leaks, while in cases without a leak, the incidence of stricture was 23.3%. These all cases of stricture responded to regular dilatations. CONCLUSION: We believe in cases of major AL, where primary repair is done by EP approach, a conservative treatment should be the treatment of choice. With this conservative approach of management of major AL, we not only save the native esophagus, the best conduit, but there is also less morbidity and mortality.

Endorepellin-evoked Autophagy Contributes to Angiostasis.

Endorepellin, the C-terminal domain of perlecan, is an angiostatic molecule that acts as a potent inducer of autophagy via its interaction with VEGFR2. In this study, we examined the effect of endorepellin on endothelial cells using atomic force microscopy. Soluble endorepellin caused morphological and biophysical changes such as an increase in cell surface roughness and cell height. Surprisingly, these changes were not accompanied by alterations in the endothelial cell elastic modulus. We discovered that endorepellin-induced autophagic flux led to co-localization of mammalian target of rapamycin with LC3-positive autophagosomes. Endorepellin functioned upstream of AMP-activated kinase α, as compound C, an inhibitor of AMP-activated kinase α, abrogated endorepellin-mediated activation and co-localization of Beclin 1 and LC3, thereby reducing autophagic progression. Functionally, we discovered that both endorepellin and Torin 1, a canonical autophagic inducer, blunted ex vivo angiogenesis. We conclude that autophagy is a novel mechanism by which endorepellin promotes angiostasis independent of nutrient deprivation.

Decorin causes autophagy in endothelial cells via Peg3.

Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells.

Endorepellin evokes autophagy in endothelial cells.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity via dual receptor antagonism, through concurrent binding to the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2). Here, we discovered that soluble endorepellin induced autophagy in endothelial cells by modulating the expression of Beclin 1, LC3, and p62, three established autophagic markers. Moreover, endorepellin evoked expression of the imprinted tumor suppressor gene Peg3 and its co-localization with Beclin 1 and LC3 in autophagosomes, suggesting a major role for this gene in endothelial cell autophagy. Mechanistically, endorepellin induced autophagy by down-regulating VEGFR2 via the two LG1/2 domains, whereas the C-terminal LG3 domain, the portion responsible for binding the α2ß1 integrin, was ineffective. Endorepellin also induced transcriptional activity of the BECN1 promoter in endothelial cells, and the VEGFR2-specific tyrosine kinase inhibitor, SU5416, blocked this effect. Finally, we found a correlation between endorepellin-evoked inhibition of capillary morphogenesis and enhanced autophagy. Thus, we have identified a new role for this endogenous angiostatic fragment in inducing autophagy through a VEGFR2-dependent but α2ß1 integrin-independent pathway. This novel mechanism specifically targets endothelial cells and could represent a promising new strategy to potentiate the angiostatic effect of endorepellin and perhaps other angiostatic matrix proteins.

The role of perlecan and endorepellin in the control of tumor angiogenesis and endothelial cell autophagy.

During tumor growth and angiogenesis there is a dynamic remodeling of tissue architecture often accompanied by the release of extracellular matrix constituents full of biological activity. One of the key constituents of the tumor microenvironment is the large heparan sulfate proteoglycan perlecan. This proteoglycan, strategically located at cell surfaces and within basement membranes, is a well-defined pro-angiogenic molecule when intact. However, when partially processed by proteases released during cancer remodeling and invasion, the C-terminal fragment of perlecan, known as endorepellin, has opposite effects than its parent molecule. Endorepellin is a potent inhibitor of angiogenesis by exerting a dual receptor antagonism by simultaneously engaging VEGFR2 and α2ß1 integrin. Signaling through the α2ß1 integrin leads to actin disassembly and block of endothelial cell migration, necessary for capillary morphogenesis. Signaling through the VEGFR2 induces dephosphorylation of the receptor via activation of SHP-1 and suppression of downstream proangiogenic effectors, especially attenuating VEGFA expression. A novel and emerging role of endorepellin is its ability to evoke autophagy by activating Peg3 and various canonical autophagic markers. This effect is specific for endothelial cells as these are the primary cells expressing both VEGFR2 and α2ß1 integrin. Thus, an endogenous fragment of a ubiquitous proteoglycan can regulate both angiogenesis and autophagy through a dual receptor antagonism. The biological properties of this natural endogenous protein place endorepellin as a potential therapeutic agent against cancer or diseases where angiogenesis is prominent.

Assessment of Shame and Stigma in Head and Neck Cancer: A Meta-Analysis.

Background: Head and neck cancer is the most common cancer around the globe, following lung cancer and breast cancer. Treatment at advanced stages of head and neck cancer is usually followed intense surgical procedures, which leads to mutilation among patients. Mutilation imparts a sense of disgrace and causes a feeling of shame and stigma in the patient. The feeling of shame and stigma persists over time and affects the overall long-term survival of patients by deteriorating their quality of life. Objectives: Since shame and stigma is an important psychological domain of head and neck cancer, the present article aims toward evaluating the studies published so far for the assessment of shame and stigma in head and neck cancer and highlighting the lacunae in the existing research designs. The present study also aims to design a checklist that could be followed while developing, translating, or validating a psychometric instrument that aims to measure shame and stigma in head and neck cancer. Methods: In the present metanalysis, all articles published in the past years on shame and stigma in head and neck cancer was compiled using a predefined data extraction matrix. The available literature was compiled for major objectives of the study, the sample size used, major findings, and critical lacunae that need to be addressed. Results: Shame and stigma is a very important domain of psychological well-being in head and neck cancer patients, which yet not appropriately addressed and further need to be researched. Conclusion: Future studies could be based on the lacunae highlighted in the existing literature, and the prescribed methodology checklist could be taken into consideration while conducting further studies involving developing, translating, or validating a psychometric instrument related to shame and stigma in the head and neck cancer.

Endorepellin affects angiogenesis by antagonizing diverse vascular endothelial growth factor receptor 2 (VEGFR2)-evoked signaling pathways: transcriptional repression of hypoxia-inducible factor 1α and VEGFA and concurrent inhibition of nuclear factor of activated T cell 1 (NFAT1) activation.

Endorepellin, the angiostatic C-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits angiogenesis by simultaneously binding to the α2ß1 integrin and the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) on endothelial cells. This interaction triggers the down-regulation of both receptors and the concurrent activation of the tyrosine phosphatase SHP-1, which leads to a signaling cascade resulting in angiostasis. Here, we provide evidence that endorepellin is capable of attenuating both the PI3K/PDK1/Akt/mTOR and the PKC/JNK/AP1 pathways. We show that hypoxia-inducible factor 1α (HIF-1α) transcriptional activity induced by VEGFA was inhibited by endorepellin independent of oxygen concentration and that only a combination of both PI3K and calcineurin inhibitors completely blocked the suppressive activity evoked by endorepellin on HIF1A and VEGFA promoter activity. Moreover, endorepellin inhibited the PKC/JNK/AP1 axis induced by the recruitment of phospholipase γ and attenuated the VEGFA-induced activation of NFAT1, a process dependent on calcineurin activity. Finally, endorepellin inhibited VEGFA-evoked nuclear translocation of NFAT1 and promoted NFAT1 stability. Thus, we provide evidence for a novel downstream signaling axis for an angiostatic fragment and for the key components involved in the dual antagonistic activity of endorepellin, highlighting its potential use as a therapeutic agent.

Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages--the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses.

Accuracy of a self-reported Measure in Psychological Assessment when the Instrument is self-administered by the Patient or when Administrated by the Clinician.

Background: Self-reported measures are the questionnaire-based instrument that are routinely used in the clinical scenario to assess psychological health. Technically, the self-reported measure should be administrated by the patients themselves but due to the complexity of tools and illiteracy among patients, clinicians often tend to interview the patients. Objective: Present article aims to compare the accuracy of a self-reported measure in the assessment of the psychological health of a patient when the instrument is self-administrated by the patient and when administrated by the clinician or researcher. Methods: We have recruited 43 patients of oral cancer in the study who have a tumor in the buccal mucosa region. The Hindi version of the shame and stigma scale was used to analyse the shame and stigma in patients. The questionnaire was first provided to the patient for the self-administration and after that clinician administrated the questionnaire to the patient by keeping the clinician blinded to the patient self-administrated responses. Results: There was no significant difference in the global mean score and mean score of various subdomains of shame and stigma scale in the self-administered and clinician-administered mode of interview. However, the clinician-administered mode could provide more accurate measures as it helps the patient towards a better understanding of questions. Conclusion: It is recommended that the newly developed or translated self-reported measure should be tested for both patient administrated and clinician administrated compatibility. Questionnaires could be administrated by the clinician in the case when the patient is illiterate or in the case when the patient does not understand the language of the instrument.

Endorepellin, the angiostatic module of perlecan, interacts with both the α2ß1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2): a dual receptor antagonism.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2ß1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2ß1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2ß1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2ß1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.

Quality of Life in Oral Cancer Patients Following Surgical Excision and Flap Reconstruction.

Background: Incidence of oral cancer have been increased aggressively in recent years, and many advanced surgical techniques have developed to facilitate the treatment of disease while preserving the patient's quality of life (QoL). Subjects and Methods: The present study aims toward the evaluation of patient's satisfaction toward the current treatment procedure for oral cancer by assessing the QoL preoperatively and postoperatively. In the study period of four years, we had operated 150 patients with oral cancer (Stage III and IVa) by surgical excision and flap reconstruction, and QoL was assessed using the WHO-QoL BREF questionnaire. Results: We found significant improvement after surgery in various aspects of QoL including the physical health, social health, psychological health and environmental health. Conclusions: The present study is evidence of patient satisfaction toward the current treatment protocols  of oral cancer. However, advanced surgical techniques that can enhance the rate of functional rehabilitation can have paramount importance in improving the patient's QoL.

Stigmatic Impact of COVID-19 Pandemic on Head & Neck Cancers Survivors.

Social stigma is the spoilation of the social image of an individual, which leads to the social disapproval of the individual by the community. Patients of many diseases like HIV, deafness, and reproductive disorders often face social disproval. Head & neck cancer survivors perceived stigma due to the mutilation that occurred after surgical treatment procedures. The novel coronavirus is a recently emerged zoonotic viral agent that affects the respiratory system of humans. Following its outbreak from the Wuhan city of China, the COVID-19 spreaded fiercely around the globe, forming a pandemic. Since COVID-19 is a contagious disease with no available treatment, social distancing is considered as the best strategy to prevent the geometric spread of infection. With the social distancing model, the head & neck cancer survivors along with the various other stakeholders perceived stigma being a high-risk group for COVID-19 infection. As the pool of caregivers is diminished due to pandemic, head & neck cancer survivors face increased social isolation and perceived stigma in asking for help from relatives. At the time of the pandemic, social support is critical to fighting against the disease. Social distancing should be maintained along with communication with the patients through calls, text, and online social platforms. It is not wise to stigmatize disease as, in that case, patients who are infected with the disease will try to hide it and avoid seeking medical care. With the promotion of social distancing, it is crucial to convey awareness regarding not to stigmatize the disease.

Media Commercials Conveying Awareness Regarding Prevention of Head and Neck Cancer by Focusing on Stigmatized Perspective of Disease: Right or Wrong?

Cancer in the head and neck region is among the most common cancer around the world, the incidence of which keep increasing in past years. Treatment of disease is usually done by the surgical excision which often leads to some degree of facial disfigurements which cause mutilation in patients. Mutilation imparts the feeling of stigma in patients, and patients usually tend to hide facial disfigurements using additional clothing. As a prevention strategy, awareness regarding the disease conveys to the mass population via media commercials. Media commercials which highlight the adverse outcomes of cancer are found to target the stigmatized perspective of disease. On the brighter side, more stigmatized is the patient image in the commercials, more motivation it will create in masses to avoid risk factors like tobacco, smoking and alcohol. But on the darker side, stigmatized commercials create a social environment in which people tend to maintain social distance to cancer patients, and patients have to bear social disapproval by society for their whole life. It reduces the self-esteem and quality of life of patients which affects their overall survival. In the present article, we review the status of stigma in head and neck cancer patients, tools that are available for assessment of stigma, and effects of the stigmatized media commercials on the patient's self-esteem. The present article represents the accurate picture of the problem and highlights the policies which could be employed to balance the paradox of stigmatized media commercials and a healthy social environment for cancer patients.

Yoga as a Preventive Intervention for Cardiovascular Diseases and Associated Comorbidities: Open-Label Single Arm Study.

Aim: Common Yoga Protocol (CYP) is a standardized yoga protocol authored by experts from all over the world under the aegis of the Ministry of AYUSH, Ayurveda, Yoga and Naturopathy, Unani, Siddha, Sowa Rigpa and Homeopathy (AYUSH). The potential of CYP can be determined as a cost-effective lifestyle modification to prevent the risk of developing cardiovascular diseases (CVD). Methods: In this prospective trial, we compared the effect of CYP at baseline and after 1 month. A total of 374 yoga-naïve participants performed CYP under the supervision of experienced trainers. Physiological [body mass index (BMI), blood pressure, percent oxygen saturation], biochemical (fasting blood glucose and lipid profile), and neurocognitive parameters were measured before and after the intervention. Results: At day 30 of yoga practice, serum levels of low-density lipoprotein (LDL), total cholesterol (TC), and high-density lipoprotein (HDL) were found significantly improved as compared to the baseline levels observed at the time of enrollment. Similarly, the lipid profile was also obtained from experienced trainers and found to be significantly different from those of yoga-naïve volunteers. When the intervention was compared between the healthy yoga-naïve participants with yoga-naïve participants suffering from medical issues, it was found that cholesterol profile improved significantly in the healthy-naive group as compared to the diseased group (hypertension, diabetes, underwent surgery, and CVD). Conclusion: These results highlight the need for further research to better understand the effects of yoga on the primary prevention of CVD.

Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407). METHODS: INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgG(AD))/d-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA. RESULTS: The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgG(AD)/d-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490). CONCLUSIONS: We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2. GENERAL SIGNIFICANCE: Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection.

Antibody mix-and-read assays based on fluorescence intensity probes.

Antibodies and Fc fusion proteins are a rapidly growing class of pharmaceuticals. Cell culture and purification process development and operation require frequent measurement of product concentrations, commonly by complex enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. Here we report a fast (<30 s), and simple antibody Fc assay based on mix-and-read reporting by fluorescence emission. A soluble fluorescein-labeled Fc-affinity reporter produced by standard peptide synthesis is mixed with an Fc-containing sample to produce an immediate shift in both fluorescence polarization and intensity, compatible with on- and at-line measurements and microbioreactor monitoring. We observed significant shifts in fluorescence intensity in Chinese hamster ovary cell culture fluid spiked with IgG and detected an adalimumab biosimilar down to 100 ng/mL (10-4 g/L), despite the interferents in the complex sample matrix. Neither the fluorescence polarization nor the fluorescence intensity assay is significantly affected by the addition of clarified lysate of 2 million CHO-k1 cells/mL, suggesting applicability even to cultures of low viability. Biochemical and molecular docking approaches suggest that the fluorescence intensity enhancement is caused by changes in the fluorophore's local microenvironment upon binding to IgG Fc, especially by interactions with Fc His433.Abbreviations: CCF: Cell Culture Fluid; CHO: Chinese Hamster Ovary cells; ELISA: Enzyme Linked Immunosorbent Assay; Fc: Fragment Crystallizable of antibody; HPLC: High-Performance Liquid Chromatography; HPßCD: hydroxypropyl-ß-cyclodextrin; IgG: ImmunoglobulinG; mAb: Monoclonal Antibody; PBS: Phosphate-Buffered Saline; PDB: Protein Data Bank; SpA: Staphylococcal protein A; SpG: Staphylococcal protein G.

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in INT-407 cells.

Enteroaggregative Escherichia coli (EAEC) is emerging as a cause of acute and persistent diarrhea in developing countries. An important feature of EAEC pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. In this article, we have shown that EAEC-induced activation of mitogen-activated protein kinases (MAPK) (ERK-1/2, JNK and p38MAPK) in cultured human intestinal epithelial cells (INT-407) leads to the induction of DNA-binding activity of NF-kappaB and AP-1, resulting in IL-8 production. Plasmid-cured EAEC could also activate the MAPK and the transcription factors leading to IL-8 secretion, but to a lesser extent than that of wild-type EAEC. Further, pretreatment of these cells with the highly specific MEK inhibitor (PD 098059), the JNK inhibitor (SP 600125), and the p38MAPK inhibitor (SB 203580) resulted in inhibition of the IL-8 secretion by EAEC (wild type as well as plasmid cured)-infected INT-407 cells. These findings demonstrate that the inflammatory response induced by EAEC may be due to the specific stimulation of MAPK signaling pathways leading to nuclear responses. To our knowledge, this is the first article regarding the detailed mechanism of IL-8 secretion from the EAEC-infected human intestinal epithelial cell line.