Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Acute Respiratory Distress Syndrome Associated with Multisystem Inflammatory Syndrome in a Child with Covid-19 and Diabetic Ketoacidosis: A Case Report.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or coronavirus disease 2019 (Covid-19), has uncontrollable effects on many organs. A great number of previously published scientific reports have revealed that patients with diabetes mellitus face a more severe form of Covid-19 with a higher death rate. Here we present the case of a 13-year-old unvaccinated boy who was admitted to an intensive care unit (ICU) with a history of fever, cough, dyspnea, throat pain, nausea, and confusion that progressed to lethargy after 24 h. On clinical examination, he was in a coma with Kussmaul's breathing, and was anuric. His blood biochemical analysis demonstrated hyperglycemia, severe metabolic acidosis, kidney failure, electrolyte disturbances, and inflammation. Chest x-ray showed pneumonia and a pleural effusion. The results of the SARS-CoV-2 real-time polymerase chain reaction were positive. The patient was diagnosed with Covid-19-induced acute respiratory distress syndrome associated with multisystem inflammatory syndrome in children secondary to his acute respiratory failure, acute kidney injury, and new-onset type 1 diabetes mellitus with diabetic ketoacidosis. He was intubated for invasive mechanical ventilation and received a normal saline infusion and continuous insulin infusion (0.1 IU/kg/h) for the treatment of his diabetic ketoacidosis. He was also treated with methylprednisolone, aspirin, and heparin, and underwent continuous renal replacement therapy for acute renal failure for 9 days. The patient was discharged from ICU on day 16 and was followed up regularly as an outpatient with daily treatment, including subcutaneous insulin injection (30 IU/day) and a calcium channel blocker for hypertension (nifedipine 20 mg/day).

First field evaluation of the optimized CE marked Abbott protocol for HIV RNA testing on dried blood spot in a routine clinical setting in Vietnam.

BACKGROUND: Viral load (VL) monitoring of HIV-infected patients in decentralized areas is limited due to logistic constraints. Dried Blood Spots (DBS) offer the opportunity to collect samples in remote area which can be easily transferred and tested at a central laboratory. The MOVIDA (Monitoring Of Viral load In Decentralized Area) project evaluated the performance of VL measurements on DBS using the new CE marked optimized Abbott protocol. METHODS: HIV-1 infected adults from three outpatient clinics in Hanoi (Vietnam) were enrolled into the study between 1 March and 13 April 2017. VL was measured on DBS using the optimized protocol provided by the manufacturer and compared to plasma VL as reference method on the Abbott m2000rt RealTime HIV-1 platform. Sensitivity was defined as the ability for DBS samples to correctly identify VL failure at the threshold of 1000 copies/mL of plasma, while specificity represented the ability to identify patients with a plasma HIV-RNA VL of <1000 copies/mL. RESULTS: A total of 203 patients were enrolled in the study, of which 152 (75%) were male. Median age was 38 [inter quartile range: 34-43] years. Of these patients, 37 were untreated, 38 on ART for <6 months and 117 were on ART for ≥6 months. A strong correlation between VL results in plasma and from DBS was observed (ρ = 0.95; p<0.001). Plasma VL was ≥1000 copies/mL in 71 patients. The sensitivity of DBS was 90.1% (95% confidence interval [CI]: 80.7-95.9) and the specificity was 96.2% (95% CI: 91.4-98.8). CONCLUSIONS: The new optimized Abbott DBS protocol performed well in this study, meeting the WHO performance criteria for the use of DBS for HIV VL monitoring. Scaling up VL monitoring using DBS can be used to reach the last 90 in the UNAIDS targets of 90-90-90 to help end the AIDS epidemics. However, sensitivity remains the main challenge for manufacturers to prevent maintaining patients in virological failure on inefficient ART.

Ex vivo multiplex profiling of protein tyrosine kinase activities in early stages of human lung adenocarcinoma.

Despite constant improvement in existing therapeutic efforts, the overall survival rate of lung cancer patients remains low. Enzyme activities may identify new therapeutically targetable biomarkers and overcome the marked lack of correlation between cellular abundance of translated proteins and corresponding mRNA expression levels. We analysed tyrosine kinase activities to classify lung adenocarcinoma (LuAdCa) resection specimens based on their underlying changes in cellular processes and pathways that are agents of or result from malignant transformation. We characterised 71 same-patient pairs of early-stage LuAdCa and non-neoplastic LuAdCa resection specimen lysates in the presence or absence of a tyrosine kinase inhibitor. We performed ex vivo multiplex tyrosine phosphorylation assays using 144 selected microarrayed kinase substrates. The obtained 76 selected phosphotyrosine signature peptides were subsequently analysed in terms of follow-up treatments and outcomes recorded in the patient files. For tumour, node, metastasis (TNM) stage 1 LuAdCa patients, we noticed a larger tyrosine kinase inhibitor-induced decrease in tyrosine phosphorylation for long-term as opposed to short-term disease survivors, for which 26 of 76 selected peptides were significantly (p < 0.01, FDR < 3%) more inhibited in the long-term survivors. Using statistical class prediction analysis, we obtained a 'prognostic-signature' for long- versus short-term disease survivors and correctly predicted the survival status of 73% of our patients. Our translational approach may assist clinical disease management after surgical resection and may help to direct patients for an optimal treatment strategy.