Multiresponsive Nanoscale Self-Assembly of Azurin-Elastin-like Polypeptide Fusion Protein for Enhanced Prostate Cancer Therapy.

A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 µM concentration. The flow cytometric results of these micelles at 20 µM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.

Engineered Recombinant EGFP-Azurin Theranostic Nanosystem for Targeted Therapy of Prostate Cancer.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein his6EGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the in silico binding study, where the analogue of ephrinA, his6EGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The his6EGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.

A magnetic field augmented ultra-thin layer chromatography coupled surface enhanced Raman spectroscopy separation of hemozoin from bacterial mixture.

Malaria is considered as one the most widespread disease with highest possibility of co-infection at all levels of the disease prognosis. Rapid detection and discrimination of malaria from other co-infections remains a challenge. Hemozoin is a metabolic biproduct of malaraia possessing paramagnetic property due to presence of iron at its centre. Here, we report a label free, rapid and highly sensitive magnetic field based ultra-thin layer chromatography (UTLC) coupled with surface enhanced Raman spectroscopy (SERS) technique for detection and separation of hemozoin from a bacterial mixture. Highly optimized silver nanorods chip fabricated using glancing angle deposition (GLAD) is explored for the UTLC-SERS separation. These chips possessing channel like characteristic and high surface to the volume ratio serve as excellent UTLC plates. The magnetic nature of hemozoin has been exploited for its separation from the mixture of P. aeruginosa (Gram-negative) and S. aureus (Gram-positive) by allocating a 0.6 T magnet over the UTLC flow setup. The solvent front migrated approximately to a distance of 13 mm from the sample point due to the magnetic environment. Spatially resolved SERS data was collected along the mobile phase and separation of mixture was confirmed. Further, staining of hemozoin, P. aeruginosa and S. aureus was done using methylene blue, acridine orange and rhodamine 6 G respectively. The separation was confirmed for the stained analytes. The present developed method provides plate height as low as 18 µm and hemozoin detection limit as <10 parasites/mL. Therefore, we establish a highly specific and sensitive technique capable of separating small amounts of bioanalytes, aiding in the removal of co-infections from the disease at a very early stage of infection.

Successful management of cervical dysgenesis: Case report and review.

Cervical agenesis or dysgenesis is a rare Mullerian anomaly that is usually associated with vaginal aplasia. A literature review revealed reports of 83 cases including ours, of which 57 (68.6%) presented with obstruction of the external OS, 11 (13.2%) had the cervix replaced by a fibrous cord and 5 (6.02%) had a fragmented cervix. A total of 24 (28.9%) were managed by core and drilling technique (CDT), 16(19.2%) patients underwent uterovaginal anastomosis (UVA), 7(8.4%) underwent total abdominal hysterectomy preserving the ovaries and 5 (6.02%) were managed by cervical reconstruction. Unfortunately, 31 failed to return after their clinical and radiological diagnosis was confirmed. Early diagnosis and treatment are necessary to avoid long-term complications.

Histopathological Changes in Placenta of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) Infection and Maternal and Perinatal Outcome in COVID-19.

Background: Whether vertical transmission or placental pathology occurs after maternal infection during pregnancy remains unknown. There is a clear need for studies on the impact of COVID-19 on pregnancy outcome. A systemic inflammatory or hypercoagulable state may be the contributing factor for placental pathology. Methods: The pregnant women with COVID-19 who delivered between May 2020 and May 2021 were followed and data were collected about pregnancy course and placentas were examined for macro- and microscopical changes and were compared to controls with non-infected women. Results: Placenta of COVID-19-infected females had increased prevalence of decidual arteriopathy and placental injury reflecting hypoxia and uteroplacental insufficiency within the intervillous space. Features of maternal vascular malperfusion such as increased syncytial knots were present in 100% cases. Fibrinoid necrosis was seen in 100% cases and increased focal perivillous fibrin depositions were presented in 37.7% cases. About one fourth infected placentas had evidence of villitis. Even after matching for comorbidities like preeclampsia, these changes were present. Conclusion: The most common pathological findings of the placenta of COVID-19 infections are signs of maternal and fetal malperfusion. Future studies should target infections in different stage of gestation, including first and second trimesters.

An all-aqueous and phosphine-free integrated amine-assisted CO2 capture and catalytic conversion to formic acid.

A phosphine-free Ir(III)-NHC-based efficient catalytic system is developed for integrated CO2 capture with tetramethylguanidine as a capturing agent and conversion to formate with H2 gas, conducting both the steps in water, affording product yield up to 85% and TON up to 19 171 in just 12 h. In the segment of "integrated CO2-capture and conversion to formate", this system represents not only the first phosphine-free module, but also one of the few best known homogeneous catalysts.

MTHFR C677T predisposes to POAG but not to PACG in a North Indian population: a case control study.

Hyperhomocysteinemia induced by the C677T genetic variant in MTHFR (methylenetetrahydrofolate reductase) has been implicated in neuronal cell death of retinal ganglion cells (RGC), which is a characteristic feature of glaucoma. However, association of MTHFR C677T with glaucoma has been controversial because of inconsistent results across association studies. Association between MTHFR C677T and glaucoma has not been reported in Indian population. Therefore, with a focus on neurodegenerative death of RGC in glaucoma, the current study aimed to investigate association of MTHFR C677T with Primary Open Angle Glaucoma (POAG) and Primary Angle Closure Glaucoma (PACG) in a North Indian population. A total of 404 participants (231 patients and 173 controls) were included in this study. Genotyping was performed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. A few random samples were also tested by direct sequencing. Genotypic and allelic distributions of the POAG and PACG cohorts were compared to that of controls by chi-square test and odds ratios were reported with 95% confidence intervals. Genotypic and allelic distributions between POAG cases and controls were significantly different (p = 0.03 and p = 0.01 respectively). Unlike POAG, we did not find significant difference in the genotypic and allelic distributions of C677T between PACG cases and controls (p>0.05). We also observed a higher proportion of TT associated POAG in females than that in males. However, this is a preliminary indication of gender specific risk of C677T that needs to be replicated in a larger cohort of males and females. The present investigation on MTHFR C677T and glaucoma reveals that the TT genotype and T allele of this polymorphism are significant risk factors for POAG but not for PACG in North Indian population. Ours is the first report demonstrating association of MTHFR C677T with POAG but not PACG in individuals from North India.