Design, synthesis, X-ray crystal structures, anticancer, DNA binding, and molecular modelling studies of pyrazole-pyrazoline hybrid derivatives.

We have designed and synthesized three pyrazole analogs (4, 5a, 5b), pyrazole-based chalcones (6a-6d) and (8a-8h), and N-formyl/acetyl 1,3,5-trisubstituted pyrazoline analogs (7a-7d), (9a-9d). FT-IR, 1H, 13C NMR, and mass spectrometry techniques were used to describe the structures of all the synthesized analogs. The single crystal X-ray method was used to identify the molecular structure of derivatives 4 and 5a. All synthesized analogs were screened by MTT assay on two cancer cell lines, the human lung cancer cell line (A549) and cervical cancer cell line (HeLa). Among all compounds, analog 9d demonstrates significant anticancer activity against HeLa (IC50 = 23.6 µM) and A549 (IC50 = 37.59 µM). The non-interactive interaction of active compound (9d) with Calf thymus DNA (Ct-DNA) has been investigated through various methods, such as UV-vis absorption, emission, cyclic voltammetry and circular dichroism. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical has been used to measure the antioxidant capacity of the pyrazoline derivative (9d). The outcomes showed that active analog has significant antioxidant activity. In addition, MD simulation of the EGFR tyrosine kinase protein-ligand complex was performed at a time scale of 100 ns. The MMGBSA data of ligand-protein complex are showed stable interactions up to 100 ns.

Access, timeliness and retention for HIV testing under early infant diagnosis (EID) program, India.

Early Infant Diagnosis of HIV infection services are crucial for managing the perinatally acquired HIV infection. Assessing the performance of the EID services and its underlying determinants is important for the National AIDS Control Program, India. The objectives of this study were to find out access to HIV testing, the timeliness of the testing cascade, and the proportion of HIV exposed infants who are followed up to 18 months for a definitive diagnosis of HIV. The study design was a mixed method. A total of 11 states accounting for 80% of HIV-positive pregnant women were selected. Program records from a total of 62 Integrated counselling and testing centres (ICTCs) served as the source of information. The qualitative component included interviews of program managers at the state and district level, service providers at the ICTC level, and caregivers of HIV exposed infants. In the sampled 62 ICTCs, 78% of the HIV exposed infants had at least one HIV test. Of the infants who had HIV tests, 50% had at first sample collected by 8 weeks of age. The median turnaround time from sample collection to DNA PCR testing was 36 (IQR 19-70) days and that to next sample collection in case of detection of virus in the first sample was 66 (IQR 55-116) days. At 18 months of age, 544 (62%) HIV exposed infants were retained in the EID testing cascade. A total of 30 infants were diagnosed with HIV at a median age of 421 (IQR 149-650) days. More than three fourth of the HIV exposed infants had access to early infant diagnosis (EID) services. Both demand and supply-side factors contribute to access, timeliness and retention and there is a need to address these factors.

COVID-19 patients' clinical profile and outcome with respect to their vaccination status: A prospective observational multicentre cohort study during third wave in Western India.

PURPOSE: To understand the benefits of COVID-19 vaccination (Covishield, Covaxin) on clinical features and outcome of COVID-19 during the third wave in India. MATERIALS AND METHODS: The primary study aim was to describe the clinical profile and outcome of COVID-19 regarding their vaccination and to identify risk factors for disease progression in vaccinated patients. This was a prospective observational multicentric study of COVID-19 attended by Infectious Disease physicians during January 15, 2022 to February 15, 2022. Adult patients with positive RT-PCR or rapid antigen test for COVID-19 were enrolled. Patient received treatment as per local institutional protocol. Chi square test for categorical and Mann Whitney test for continuous variables were applied for the analysis. Logistic regression was used to calculate adjusted odds ratios. RESULTS: A total of 788 patients were included in analysis out of 883 enrolled patients from 13 centers across Gujarat. By the end of two weeks' follow up, 22 patients (2.8%) had expired. The Median age of subjects was 54 years, with a (55.8%) male. 90% of the subjects were vaccinated, majority (77%) of them had received 2 doses of vaccine with Covishield (659, 93%). Mortality among the non-vaccinated was significantly (11.4%) higher than vaccinated (1.8%). Logistic regression analysis showed numbers of comorbidities (p â€‹= â€‹0.027), baseline higher WBC count (p â€‹= â€‹0.02), higher NLR (p â€‹= â€‹0.016), and Ct value (p â€‹= â€‹0.046) were associated with mortality while vaccination was associated with survival (p â€‹= â€‹0.001). The factors associated with mortality among vaccinated were age, comorbidities, baseline higher WBC, NLR, and CRP. CONCLUSIONS: Omicron variant was associated with mild symptoms. Clinical and laboratory risk factors for getting severe disease with Omicron variant were the same with previous SARS CoV-2 strain. Two doses of vaccine protect people against severe disease and death. Age, comorbidities, baseline leucocytosis, high NLR, elevated CRP are the risk factors for poor outcome in vaccinated patients.

Synthesis, spectral characterization of pyrazole derived Schiff base analogs: molecular dynamic simulation, antibacterial and DNA binding studies.

We have synthesized the pyrazole-bearing Schiff base derivatives (5a-5e) and (6a-6h) then the structural confirmation was supported by various spectral analyses. The antibacterial activity of all analogs was screened against bacterial strains Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonieae and Pseudomonas aeruginosa. In comparison to the reference drug ciprofloxacin, the lead analogs 5c and 6c showed potent activity, with MIC values of 64 µg/mL against E. coli and B. subtilis. Compound 5c showed a moderate effect with a MIC value of 128 µg/mL against B. subtilis, P. aeruginosa and K. pneumonieae, while compound 6c was against E. coli and P. aeruginosa. Furthermore, the compounds 5c and 6c displayed groove binding mode towards CT-DNA by absorption, emission, competitive fluorescence studies using EtBr, CD and time-resolved fluorescence studies. Thermodynamic parameters of analogs 5c and 6c with CT-DNA were also calculated at 298, 303 and 308K temperatures by UV-visible spectroscopy. The molecular docking studies give the docking score for all compounds with PDB codes: 1BNA and 2XCT. The MD simulation study of analogs 5c and 6c was also carried out. The pharmacokinetic and ADME properties were calculated for all of the synthesized analogs (5a-5e) and (6a-6h).Communicated by Ramaswamy H. Sarma.

Oxadiazole Schiff Base as Fe3+ Ion Chemosensor: "Turn-off" Fluorescent, Biological and Computational Studies.

Compound, (E)-5-(4-((thiophen-2-ylmethylene)amino)phenyl)-1,3,4-oxadiazole-2-thiol (3) was synthesized via condensation reaction of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol with thiophene-2-carbaldehyde in ethanol. For the synthesis and structural confirmation the FT-IR, 1H, 13C-NMR, UV-visible spectroscopy, and mass spectrometry were carried out. The long-term stability of the probe (3) was validated by the experimental as well as theoretical studies. The sensing behaviour of the compound 3 was monitored with various metal ions (Ca2+, Cr3+, Fe3+, Co2+, Mg2+, Na+, Ni2+, K+) using UV- Vis. and fluorescence spectroscopy techniques by various methods (effect of pH and density functional theory) which showing the most potent sensing behaviour with iron. Job's plot analysis confirmed the binding stoichiometry ratio 1:1 of Fe3+ ion and compound 3. The limit of detection (LOD), the limit of quantification (LOQ), and association constant (Ka) were calculated as 0.113 µM, 0.375 µM, and 5.226 × 105 respectively. The sensing behavior was further confirmed through spectroscopic techniques (FT-IR and 1H-NMR) and DFT calculations. The intercalative mode of binding of oxadiazole derivative 3 with Ct-DNA was supported through UV-Vis spectroscopy, fluorescence spectroscopy, viscosity, cyclic voltammetry, and circular dichroism measurements. The binding constant, Gibb's free energy, and stern-volmer constant were find out as 1.24 × 105, -29.057 kJ/mol, and 1.82 × 105 respectively. The cleavage activity of pBR322 plasmid DNA was also observed at 3 × 10-5 M concentration of compound 3. The computational binding score through molecular docking study was obtained as -7.4 kcal/mol. Additionally, the antifungal activity for compound 3 was also screened using broth dilution and disc diffusion method against C. albicans strain. The synthesized compound 3 showed good potential scavenging antioxidant activity against DPPH and H2O2 free radicals.

Resistance to unfolding by acidic pH and resistance to lysosomal degradation explains disease-association of HLA-B27 subtypes.

Several hypotheses have been proposed to explain the high rate of disease association of HLA-B27 with ankylosing spondylitis (AS), including formation of disulfide-bonded dimers and misfolding of the heavy chain (HC), involving formation of high molecular weight (HMW) multimers. Recently, we have shown that the HMW entities of non-disease associated (non-DA) subtypes cause activation of endosomal-lysosomal pathways, while disease-associated (DA) subtypes of HLA-B27 cause activation of autophagy and unfolded protein response (UPR) pathways. In this paper, we seek an explanation for the failure of these pathways to degrade the HMW entities of DA subtypes of HLA-B27, using a combination of in vitro assays, using extracellular domains of heavy chains (EDHC), as well as in vivo assays, using stable transfectants of the full lengths of heavy chains (FLHC) of DA and non-DA subtypes. Our data shows that both DA and non-DA subtypes form HMW entities. However, non-DA HMW entities display far greater levels of degradation than DA HMW species. Non-DA EDHC display greater loss of structure at lysosomal pH in vitro. This was confirmed by experiments showing that (i) DA FLHCs co-localize with LAMP1, and (ii) induction of autophagy by rapamycin causes significant decrease in levels of non-DA HMW entities, but not that of DA HMW entities. These results point towards lack of facile lysosomal clearance of FLHCs of DA subtypes, suggesting that disease association of HLA-B27 subtypes is correlated with higher persistence of HMW entities in the low pH of lysosomes, with higher potential to trigger immune response.

Correction to "Design and Synthesis of Carbothioamide/Carboxamide-Based Pyrazoline Analogs as Potential Anticancer Agents: Apoptosis, Molecular Docking, ADME Assay, and DNA Binding Studies".

[This corrects the article DOI: 10.1021/acsomega.2c02033.].

Design and Synthesis of Carbothioamide/Carboxamide-Based Pyrazoline Analogs as Potential Anticancer Agents: Apoptosis, Molecular Docking, ADME Assay, and DNA Binding Studies.

To discover anticancer drugs with novel structures and expand our research scope, pyrazoline derivatives (3a-3l) were designed and synthesized through cyclization of chalcones with thiosemicarbazide/semicarbazide in CH3COOH as a solvent. All newly synthesized pyrazoline derivatives were fully characterized using several spectroscopic experiments such as 1H, 13C NMR, FT-IR spectroscopy, and mass analysis. By HPLC, the purity of all analogs was found above 95% and both lead compounds (3a and 3h) were also validated by HRMS. Anticancer activity of synthesized pyrazoline derivatives (3a-3l) was investigated by the MTT assay against the human lung cancer cell (A549), human cervical cancer cell (HeLa), and human primary normal lung cells (HFL-1). Staurosporine (STS) was used as a standard drug. The anticancer results showed that two potent analogs 3a and 3h exhibit excellent activity against A549 (IC50 = 13.49 ± 0.17 and 22.54 ± 0.25 µM) and HeLa cells (IC50 = 17.52 ± 0.09 and 24.14 ± 0.86 µM) and low toxicity against the HFL-1 (IC50 = 114.50 ± 0.01 and 173.20 ± 10 µM). The flow cytometry was further used to confirm the anticancer activity of potent derivatives against the A549 cancer cell line. DNA binding interaction of anticancer agents 3a and 3h with Ct-DNA has been carried out by absorption, fluorescence, EtBr (dye displacement assay), circular dichroism, cyclic voltammetry and time-resolved fluorescence, which showed noncovalent binding mode of interaction. Anticancer activity of both lead compounds (3a and 3h) may be attributed to DNA binding. The evaluation of the antioxidant potential of pyrazoline analogs 3a and 3h by 2,2-diphenyl-1-picrylhydrazyl free radical showed promising antioxidant activity with IC50 values of 0.132 ± 0.012 and 0.215 ± 0.025 µg/mL, respectively. In silico molecular docking of pyrazoline derivatives was also performed using autodock vina software against the DNA hexamer with PDB ID: 1Z3F and ADMET properties to explore their best hits.

Binding and thermodynamic study of thalidomide with calf thymus DNA: Spectroscopic and computational approaches.

The thalidomide-DNA interactions have been investigated in detail by numerous biophysical techniques such as UV-vis, dye displacement assay, viscosity, cyclic voltammetry, circular dichroism, molecular docking, molecular dynamic simulation, FT-IR and 1H NMR spectroscopy. CD spectroscopy, thermal denaturation and viscosity measurement explained that thalidomide is groove binder. Molecular docking analysis highlighted that thalidomide binds trough minor groove of calf thymus DNA which also confirmed from dye displacement experiment. To our knowledge, this is the first instance thalidomide was shown to binds with calf thymus DNA. Molecular dynamic simulation indicated that the thalidomide-DNA system was stabilized by electrostatic attraction as the main interaction and mode of binding is minor groove. Our study provides a better understanding to the DNA-thalidomide binding affinity and it mechanism. Overall, all these in formations can be used for further understanding the pharmacological effects of thalidomide.

2-Amino-5-substituted-1,3,4-oxadiazole as chemosensor for Ni(II) ion detection: antifungal, antioxidant, DNA binding, and molecular docking studies.

An oxadiazole derivative 2 was prepared by condensation reaction through cyclization of semicarbazone in the presence of bromine; the structural confirmation was supported by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy, Fourier transform-infrared spectroscopy, and liquid chromatography-mass spectrometry. Its sensing ability towards Ni2+ ion was examined showing a binding constant of 1.04 × 105 compared with other suitable metal cations (Ca2+ , Co2+ , Cr3+ , Ag+ , Pb2+ , Fe3+ , Mg2+ , and K+ ) using ultraviolet-visible (UV-vis) and fluorescence spectroscopic studies. The minimum concentration of Ni2+ ions and limit of detection was found to be 9.4 µM. A job's plot gave the binding stoichiometry ratio of oxadiazole derivative 2 vs Ni2+ ions as 2:1. Furthermore, the intercalative binding mode of oxadiazole derivative 2 with calf thymus DNA was supported by ultraviolet-visible (UV-vis) and fluorescent light, viscosity, cyclic voltammetry, time-resolved fluorescence, and circular dichroism measurements. The molecular docking result gave the binding score for oxadiazole derivative 2 as -6.5 kcal/mol, which further confirmed the intercalative interaction. In addition, the antifungal activity of oxadiazole derivative 2 was also screened against several fungal strains (C. albicans, C. glabrata, and C. tropicalis) by broth dilution and disc diffusion methods. In antioxidant studies, the oxadiazole derivative 2 showed potential scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and H2 O2 free radicals.

Determinants of severity among hospitalised COVID-19 patients: Hospital-based case-control study, India, 2020.

BACKGROUND: Risk factors for the development of severe COVID-19 disease and death have been widely reported across several studies. Knowledge about the determinants of severe disease and mortality in the Indian context can guide early clinical management. METHODS: We conducted a hospital-based case control study across nine sites in India to identify the determinants of severe and critical COVID-19 disease. FINDINGS: We identified age above 60 years, duration before admission >5 days, chronic kidney disease, leucocytosis, prothrombin time > 14 sec, serum ferritin >250 ng/mL, d-dimer >0.5 ng/mL, pro-calcitonin >0.15 µg/L, fibrin degradation products >5 µg/mL, C-reactive protein >5 mg/L, lactate dehydrogenase >150 U/L, interleukin-6 >25 pg/mL, NLR ≥3, and deranged liver function, renal function and serum electrolytes as significant factors associated with severe COVID-19 disease. INTERPRETATION: We have identified a set of parameters that can help in characterising severe COVID-19 cases in India. These parameters are part of routinely available investigations within Indian hospital settings, both public and private. Study findings have the potential to inform clinical management protocols and identify patients at high risk of severe outcomes at an early stage.

Pyrazoline analogs as potential anticancer agents and their apoptosis, molecular docking, MD simulation, DNA binding and antioxidant studies.

N-formyl pyrazoline derivatives (3a-3l) were designed and synthesized via Michael addition reaction through cyclization of chalcones with hydrazine hydrate in presence of formic acid. The structural elucidation of N-formyl pyrazoline derivatives was carried out by various spectroscopic techniques such as 1H, 13C NMR, FT-IR, UV-visible spectroscopy, mass spectrometry and elemental analysis. Anticancer activity of the pyrazoline derivatives (3a-3l) was evaluated against human lung cancer (A549), fibrosarcoma cell lines (HT1080) and human primary normal lung cells (HFL-1) by MTT assay. The results of anticancer activity showed that potent analogs 3b and 3d exhibited promising activity against A549 (IC50 = 12.47 ± 1.08 and 14.46 ± 2.76 µM) and HT1080 (IC50 = 11.40 ± 0.66 and 23.74 ± 13.30 µM) but low toxic against the HFL-1 (IC50 = 116.47 ± 43.38 and 152.36 ± 22.18 µM). The anticancer activity of potent derivatives (3b and 3d) against A549 cancer cell line was further confirmed by flow cytometry based approach. DNA binding interactions of the pyrazoline derivatives 3b and 3d have been carried out with calf thymus DNA (Ct-DNA) using absorption, fluorescence and viscosity measurements, circular dichroism and cyclic voltammetry. Antioxidant potential of N-formyl pyrazoline derivatives (3a-3l) has been also estimated through DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical and H2O2. Results revealed that all the compounds exhibited significant antioxidant activity. In silico molecular modelling and ADMET properties of pyrazoline derivatives were also studied using PyRx software against topoisomerase II receptor with PDB ID: 1ZXM to explore their best hits. MD simulation of 3b and 3d was also carried out with topoisomerase II for structure-function correlation in a protein. HuTopoII inhibitory activity of the analogs (3a-3l) was examined by relaxation assay at varying concentrations 100-1000 µM.

Paraneoplastic Leukemoid Reaction in Gastroesophageal Junction Adenocarcinoma: A Case Report.

BACKGROUND The presence of leukocytosis associated with non-hematological malignancy after ruling out other causes is defined as paraneoplastic leukemoid reaction (PLR). PLR is a rare manifestation of various solid tumors. It is associated with poor prognosis unless receiving effective antineoplastic treatments. CASE REPORT A 72-year-old female was referred to a hematologist/oncologist for the evaluation of leukocytosis with neutrophilia. Initial workup was unremarkable; however, she had progressively worsening leukocytosis with neutrophilia, associated with severe anemia and dysphagia. Computed tomography (CT) scan revealed wall thickening at the gastroesophageal junction (GEJ) and multiple hypodensities of the liver. Esophagogastroduodenoscopy (EGD) confirmed the diagnosis of GEJ tumor and biopsy returned as adenocarcinoma with human epidermal growth factor receptor 2 (HER2) overexpression. Leukocytosis resolved after the first round of chemotherapy and the patient remains progression-free with the addition of trastuzumab to her chemotherapy regimen. CONCLUSIONS We report a rare case of PLR caused by GEJ adenocarcinoma. This is the first case of PLR in a patient with metastatic GEJ adenocarcinoma with HER2 overexpression in the Caucasian population. It is important to workup leukocytosis promptly, to keep malignancy in the differential diagnosis and to seek early hematology/oncology consultation.

Formin-dependent TGF-ß signaling for epithelial to mesenchymal transition.

The role of distinct actin filament architectures in epithelial plasticity remains incompletely understood. We therefore determined roles for formins and the Arp2/3 complex, which are actin nucleators generating unbranched and branched actin filaments, respectively, in the process of epithelial to mesenchymal transition (EMT). In clonal lung, mammary, and renal epithelial cells, the formin activity inhibitor SMIFH2 but not the Arp2/3 complex activity inhibitor CK666 blocked EMT induced by TGF-ß. SMIFH2 prevented the proximal signal of increased Smad2 phosphorylation and hence also blocked downstream EMT markers, including actin filament remodeling, decreased expression of the adherens junction protein E-cadherin, and increased expression of the matrix protein fibronectin and the transcription factor Snail. The short hairpin RNA silencing of formins DIAPH1 and DIAPH3 but not other formins phenocopied SMIFH2 effects and inhibited Smad2 phosphorylation and changes in Snail and cadherin expression. Formin activity was not necessary for the cell surface expression or dimerization of TGF-ß receptors, or for nuclear translocation of TAZ, a transcription cofactor in Hippo signaling also regulated by TGF-ß. Our findings reveal a previously unrecognized role for formin-dependent actin architectures in proximal TGF-ß signaling that is necessary for Smad2 phosphorylation but not for cross-talk to TAZ.

Differences in conformational stability of the two alpha domains of the disease-associated and non-disease-associated subtypes of HLA-B27.

The MHC Class I molecule, HLA-B27, is strongly linked with development of the inflammatory arthritic disease, ankylosing spondylitis (AS); whereas the B*2705 subtype shows strong association, B*2709 is not associated with disease, even though the two subtypes differ in only a single residue at position 116. Currently, attention is focused on the misfolding propensities of these two subtypes, including studies of disulfide-linked dimers and non-covalently formed high molecular weight (HMW) aggregates. Using mutants retaining only a single cysteine at positions C67 or C164, and using a cysteine-reactive, environment-sensitive, fluorescence probe (acrylodan), we find that within the same overall population of identical single-cysteine HLA-B27 molecules, there exist sub-populations which (a) possess free cysteines which react with acrylodan, (b) form disulfide-linked dimers, and (c) form HMW aggregates. Further, using acrylodan fluorescence, we find (d) that the α1 and α2 domains unfold independently of each other in HMW aggregates, (e) that these two domains of B*2709 are less stable to chemical and thermal denaturation than the corresponding domains of B*2705, suggesting easier clearance of misfolded molecules in the former, and (f) C67 is much more exposed in B*2705 than in B*2709, which could potentially explain how B*2705 more easily forms C67-mediated disulfide-bonded dimers.

Multi-modal Binding of a 'Self' Peptide by HLA-B*27:04 and B*27:05 Allelic Variants, but not B*27:09 or B*27:06 Variants: Fresh Support for Some Theories Explaining Differential Disease Association.

A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169-181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (ß2 microglobulin), and in (b) peptide binding to ß2 microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27's disease association.

A study on metabolic prowess of Pseudomonas sp. RPT 52 to degrade imidacloprid, endosulfan and coragen.

A bacterial strain identified as Pseudomonas sp. RPT 52, was isolated from an agricultural field by soil enrichment technique. The bacterial strain was able to metabolize three different chlorinated pesticides; imidacloprid, endosulfan and coragen (belonging to neonicotinoid, organochlorine and anthranillic diamide categories, respectively). RPT 52 was able to degrade 46.5%, 96.6%, 92.7% and 80.16% of 0.5 mM of imidacloprid, endosulfan α, endosulfan ß and coragen, respectively, in minimal medium over a period of 40 h, when provided as sole source of carbon and energy. Degradation kinetics showed that imidacloprid, endosulfan α and endosulfan ß followed first order kinetics whereas coragen followed zero order kinetics. Toxicity studies show reduction in toxicity of the parent compound when degraded by RPT 52. Laboratory scale, soil microcosm studies showed that strain RPT 52 is a suitable candidate for bioremediation of endosulfan and coragen contaminated sites. Thus, RPT 52 holds potential for toxicity reduction in the affected environment.

Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

Increased production and assembly of extracellular matrix proteins during transdifferentiation of epithelial cells to a mesenchymal phenotype contributes to diseases such as renal and pulmonary fibrosis. TGF-ß and hypoxia, two cues that initiate injury-induced fibrosis, caused human kidney cells to develop a mesenchymal phenotype, including increased fibronectin expression and secretion. However, upon hypoxia, assembled extracellular fibronectin fibrils were mostly absent, whereas treatment with TGF-ß led to abundant fibrils. Fibrillogenesis required cell-generated force and tension. TGF-ß, but not hypoxia, increased cell contractility, as determined by phosphorylation of myosin light chain and quantifying force and tension generated by cells plated on engineered elastomeric microposts. Additionally, TGF-ß, but not hypoxia, increased the activation of integrins. However, experimentally activating integrins markedly increased the levels of phosphorylated myosin light chain and fibronectin fibril assembly upon hypoxia. Our findings show that deficient integrin activation and subsequent lack of cell contractility are mechanisms that mediate a lack of fibrillogenesis upon hypoxia and they challenge current views on oxygen deprivation being sufficient for fibrosis.

The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit.

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics.