Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered Peptide Tag.

Controlling enzyme orientation and location on surfaces is a critical step for their successful deployment in diverse applications from biosensors to lab-on-a-chip devices. Functional activity of the enzymes on the surface will largely depend on the spatial arrangement and orientation. Solid binding peptides have been proven to offer versatility for immobilization of biomolecules on inorganic materials including metals, oxides, and minerals. Previously, we demonstrated the utility of a gold binding peptide genetically incorporated into the enzyme putrescine oxidase (PutOx-AuBP), enabling self-enzyme assembly on gold substrates. PutOx is an attractive biocatalyst among flavin oxidases, using molecular oxygen as an electron acceptor without requiring a dissociable coenzyme. Here, we explore the selective self-assembly of this enzyme on a range of surfaces using atomic force microscopy (AFM) along with the assessment of functional activity. This work probes the differences in surface coverage, distribution, size, shape, and activity of PutOx-AuBP in comparison to those of native putrescine oxidase (PutOx) on multiple surfaces to provide insight for material-selective enzymatic assembly. Surfaces investigated include metal (templated-stripped gold (TSG)), oxide (native SiO2 on Si(111)), minerals (mica and graphite), and self-assembled monolayers (SAMs) with a range of hydrophobicity and charge. Supported by both the coverage and the dimensions of immobilized enzymes, our results indicate that of the surfaces investigated, material-selective binding takes place with orientation control only for PutOx-AuBP onto the TSG substrate. These differences are consistent with the measurements of surface-bound enzymatic activities. Substrate-dependent differences observed indicate significant variations in enzyme-surface interactions ranging from peptide-directed self-assembly to enzyme aggregation. The implications of this study provide insight for the fabrication of enzymatic patterns directed by self-assembling peptide tags onto localized surface regions. Enabling functional enzyme-based nanoscale materials offers a fascinating path for utilization of sustainable biocatalysts integrated into multiscale devices.

Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide.

Flavin oxidases are valuable biocatalysts for the oxidative synthesis of a wide range of compounds, while at the same time reduce oxygen to hydrogen peroxide. Compared to other redox enzymes, their ability to use molecular oxygen as an electron acceptor offers a relatively simple system that does not require a dissociable coenzyme. As such, they are attractive targets for adaptation as cost-effective biosensor elements. Their functional immobilization on surfaces offers unique opportunities to expand their utilization for a wide range of applications. Genetically engineered peptides have been demonstrated as enablers of the functional assembly of biomolecules at solid material interfaces. Once identified as having a high affinity for the material of interest, these peptides can provide a single step bioassembly process with orientation control, a critical parameter for functional immobilization of the enzymes. In this study, for the first time, we explored the bioassembly of a putrescine oxidase enzyme using a gold binding peptide tag. The enzyme was genetically engineered to incorporate a gold binding peptide with an expectation of an effective display of the peptide tag to interact with the gold surface. In this work, the functional activity and expression were investigated, along with the selectivity of the binding of the peptide-tagged enzyme. The fusion enzyme was characterized using multiple techniques, including protein electrophoresis, enzyme activity, and microscopy and spectroscopic methods, to verify the functional expression of the tagged protein with near-native activity. Binding studies using quartz crystal microbalance (QCM), nanoparticle binding studies, and atomic force microscopy studies were used to address the selectivity of the binding through the peptide tag. Surface binding AFM studies show that the binding was selective for gold. Quartz crystal microbalance studies show a strong increase in the affinity of the peptide-tagged protein over the native enzyme, while activity assays of protein bound to nanoparticles provide evidence that the enzyme retained catalytic activity when immobilized. In addition to showing selectivity, AFM images show significant differences in the height of the molecules when immobilized through the peptide tag compared to immobilization of the native enzyme, indicating differences in orientation of the bound enzyme when attached via the affinity tag. Controlling the orientation of surface-immobilized enzymes would further improve their enzymatic activity and impact diverse applications, including oxidative biocatalysis, biosensors, biochips, and biofuel production.

Coupling Infusion and Gyration for the Nanoscale Assembly of Functional Polymer Nanofibers Integrated with Genetically Engineered Proteins.

Nanofibers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofibers with inherent biological functions by simply adjusting the flow rate of a polymer solution. Sufficient polymer chain entanglement is obtained at Berry number > 1.6 to make bead-free fibers integrated with gold nanoparticles and proteins, in the diameter range of 117-216 nm. Integration of gold nanoparticles into the nanofiber assembly is followed using a gold-binding peptide tag genetically conjugated to red fluorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the engineered red fluorescence protein with the nanofibers. The gold nanoparticle decorated nanofibers having red fluorescence protein as an integral part keep their biological functionality including copper-induced fluorescence quenching of the DsRed protein due to its selective Cu(+2) binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofibers to generate biohybrid materials for imaging, sensing, and biomaterial applications.

Impact of the COVID-19 Pandemic on Diabetic Foot Patients: A Shift in the Infectious Agent Profile Toward Nonfermentative Gram-Negative Bacilli.

BACKGROUND: Diabetes foot infection is a very important public health problem that causes serious health problems, mortality, and high health expenditures, and is one of the most important complications of diabetes mellitus. There are concerns that approaches such as limited personal visits to doctors, avoidance of hospitals, and restrictions on nonemergency surgical procedures during the coronavirus disease of 2019 pandemic pose a threat to those with diabetic foot problems, including diabetic foot ulcers (DFUs), ischemia, and infection, resulting in increased limb loss and mortality. METHODS: This multicenter, retrospective, cross-sectional study was conducted in 14 tertiary care hospitals from various regions of Turkey. A total of 1,394 patient records were evaluated, 794 of which were between January 1, 2019, and January 30, 2020 (prepandemic [Pre-P]), and 605 of which were between February 1, 2020, and February 28, 2021 (pandemic period [PP]). RESULTS: During the PP, diabetic foot patient follow-up decreased by 23.8%. In addition, the number of hospitalizations attributable to DFU has decreased significantly during the PP (P = .035). There was no difference between the groups regarding patient demographics, medical history, DFU severity, biochemical and radiologic findings, or comorbidities, but the mean duration of diabetes mellitus years was longer in patients in the Pre-P than in those in the PP (15.1 years versus 13.7 years). There was no difference between the two groups in terms of major complications such as limb loss and mortality, but infection recurrence was higher in the PP than in the Pre-P (12.9% versus 11.4%; P < .05). The prevalence of nonfermentative gram-negative bacteria as causative agents in DFU infections increased during the PP. In particular, the prevalence of carbapenem-resistant Pseudomonas spp. increased statistically during the PP. CONCLUSIONS: The rapid adaptation to the pandemic with the measures and changes developed by the multidisciplinary diabetic foot care committees may be the reasons why there was no increase in complications because of DFU during the pandemic in Turkey.

Predictive Value of C-Reactive Protein to Albumin Ratio for Amputation Risk in Diabetic Foot Infection.

Diabetic foot infections are one of the complications of diabetes mellitus resulting in extremity amputation and mortality. This study aimed to examine the predictive value of the C-reactive protein (CRP) to albumin ratio (CAR) for amputation risk in diabetic foot infection. Data from 178 patients were retrospectively examined. We found the cut point value of 15.45 according to the receiver operating characteristic (ROC) curve to show the predictive value of CAR for amputation risk in the overall population. We then divided the patients into two groups low (<15.45, n = 96) and high risk (≥15.45, n = 82) according to their CAR value. Matching based on propensity scores produced 64 patients in each group and showed that the amputation rate was high in the high-risk groups (50 vs 25%, P = .003). In the multivariate analysis in the matching group, previous amputation, antibiotic therapy in the last 3 months, and CAR (Odds ratio [OR]: 1.30, 95%Confidence interval [CI]: 1.01-1.45, P < .001) were independent predictors of amputation. These parameters may be useful to predict amputation risk in these patient groups.

Combining C-reactive protein, procalcitonin, and serum albumin to predict long-term mortality in patients with infective endocarditis.

OBJECTIVE: To determine the predictive value of C-reactive protein (CRP) plus albumin plus procalcitonin for long-term mortality in patients with infective endocarditis. METHODS: This retrospective study included patients hospitalized with infective endocarditis between February 2008 and December 2021. CRP, procalcitonin, and albumin levels were measured within 24 h of admission and dichotomized as high or low. A CRP plus procalcitonin plus albumin points system (range, 3-6) was generated based on high or low CRP, procalcitonin, and albumin concentrations. Patients were divided into two groups: low-risk (≤4 points) and high-risk (>4 points), according to total score. The primary outcome was defined as all-cause mortality rate at long-term follow-up. RESULTS: Out of 204 patients in total, the high-risk group (n = 29) had higher procalcitonin and CRP levels versus the low-risk group (n = 175), but lower albumin level versus the low-risk group (2.7 ± 0.5 versus 3.5 ± 0.6 g/dl). Matching based on propensity scores showed a higher mortality rate in high-risk versus low-risk patients (76% versus 44%, respectively). In multivariate analysis after matching, the high-risk group was associated with increased long-term mortality (adjusted hazard ratio 2.87, 95% confidence interval 1.32, 6.26).Conclusions: A high CRP plus albumin plus procalcitonin score was associated with long-term mortality risk in patients with infective endocarditis.

Synthesis and Anticancer Activity of Novel Indole Derivatives as Dual EGFR/SRC Kinase Inhibitors.

BACKGROUND: Recent studies showed that the cooperation between c-SRC and EGFR is responsible for more aggressive phenotype in diverse tumors, including glioblastomas and carcinomas of the colon, breast, and lung. Studies show that combination of SRC and EGFR inhibitors can induce apoptosis and delay the acquired resistance to chemotherapy. Therefore, such combination may lead to a new therapeutic strategy for the treatment of EGFR-mutant lung cancer. Osimertinib was developed as a third-generation EGFR-TKI to combat the toxicity of EGFR mutant inhibitors. Due to the resistance and adverse reaction of osimertinib and other kinase inhibitors, 12 novel compounds structurally similar to osimertinib were designed and synthesized. BACKGROUND: Recent studies showed that the cooperation between c-SRC and EGFR is responsible for more aggressive phenotype in diverse tumors, including glioblastomas and carcinomas of the colon, breast, and lung. Studies show that combination of SRC and EGFR inhibitors can induce apoptosis and delay the acquired resistance to chemotherapy. Therefore, such combination may lead to a new therapeutic strategy for the treatment of EGFR-mutant lung cancer. Osimertinib was developed as a third-generation EGFR-TKI to combat the toxicity of EGFR mutant inhibitors. Due to the resistance and adverse reaction of osimertinib and other kinase inhibitors, 12 novel compounds structurally similar to osimertinib were designed and synthesized. METHODS: Compounds were synthesized by developing novel original synthesis methods and receptor interactions were evaluated through a molecular docking study. To evaluate their inhibitory activities against EGFR and SRC kinase, in vitro enzyme assays were used. Anticancer potencies were determined using lung, breast, prostate (A549, MCF6, PC3) cancer cell lines. Compounds were also tested against normal (HEK293) cell line to evaluate their cyctotoxic effects. RESULTS: Although, none of compounds showed stronger inhibition compared to osimertinib in the EGFR enzyme inhibition studies, compound 16 showed the highest efficacy with an IC50 of 1.026 µM. It also presented potent activity against SRC kinase with an IC50 of 0.002 µM. Among the tested compounds, the urea containing derivatives 6-11 exhibited a strong inhibition profile (80.12-89.68%) against SRC kinase in comparison to the reference compound dasatinib (93.26%). Most of the compounds caused more than 50% of cell death in breast, lung and prostate cancer cell lines and weak toxicity for normal cells in comparison to reference compounds osimertinib, dasatinib and cisplatin. Compound 16 showed strong cytotoxicity on lung and prostate cancer cells. Treatment of prostate cancer cell lines with the most active compound, 16, significantly increased the caspase-3 (8-fold), caspase-8 (6-fold) and Bax (5.7-fold) levels and decreased the Bcl-2 level (2.3-fold) compared to the control group. These findings revealed that the compound 16 strongly induces apoptosis in the prostate cancer cell lines. CONCLUSION: Overall kinase inhibition, cytotoxicity and apoptosis assays presented that compound 16 has dual inhibitory activity against SRC and EGFR kinases while maintaining low toxicity against normal cells. Other compounds also showed considerable activity profiles in kinase and cell culture assays.

Are bacterial coinfections really rare in COVID-19 intensive care units?

OBJECTIVES: There are limited data about nosocomial coinfections of COVID-19 cases monitored in the intensive care unit. This study aims to investigate coinfections in COVID-19 patients followed in an intensive care unit of a university hospital. METHODS: This study analyzed retrospectively the data of coinfections of 351 COVID-19 patients in the period 28.02.2020-15.01.2021 in a tertiary care intensive care unit in a university hospital. RESULTS: Bacterial coinfections were present in 216 of the 351 cases. One hundred and thirty of these cases were evaluated as nosocomial infections. On the third day the Sequential Organ Failure Assessment Score, usage of invasive mechanical ventilation and presence of septic shock were significantly higher in the coinfected group. The neutrophil/lymphocyte ratio, polymorphonuclear leukocyte count, procalcitonin, ferritin, and blood urea nitrogen values were significantly higher in the coinfection group. White blood cells (WBC) (OR: 1.075, 95% CI 1.032-1.121, p = 0.001) and ICU hospitalization day (OR: 1.114, 95% CI 1.063-1.167, p < 0.001) were found to be independent risk factors for coinfection in the multivariate logistic regression analysis. The rates of hospitalization day on the day of arrival, the 21st day, as well as total mortality (p = 0.004), were significantly higher in the coinfected group. CONCLUSION: Bacterial coinfections of COVID-19 patients in the intensive care unit remain a problem. Identifying the infectious agent, classifying colonizations and infections, and using the proper treatment of antibiotics are of great importance in the case management of COVID-19 patients in the intensive care unit.

Fabrication of hierarchical hybrid structures using bio-enabled layer-by-layer self-assembly.

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C'-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic-inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture.

Biosilver nanoparticle interface offers improved cell viability.

Silver nanoparticles (AgNP) are promising candidates for fighting drug-resistant infections because of their intrinsic antimicrobial effect. The design of high-yield antimicrobial molecules may inadvertently cause variation in host cells' biological responses. While many factors affect AgNPs' efficacy, their surface is exposed to the biological environment and thus plays a critical role in both the preservation of antimicrobial efficacy against pathogens and the modulation of host cells cytotoxicity. This work investigated an engineered biomimetic interface approach to controlling AgNP surface properties to provide them a competitive advantage in a biological environment. Here, a fusion protein featuring a silver-binding peptide (AgBP) domain was engineered to enable self-assembly and track assembly by a green fluorescent protein (GFP) reporter. Following AgNP functionalisation with GFP-AgBP, their antimicrobial and cytotoxic properties were evaluated. GFP-AgBP binding affinity to AgNPs was evaluated using localized surface plasmon resonance sensing. The GFP-AgBP biomimetic interface on AgNPs' surfaces provided sustained antibacterial efficacy at low concentrations based on bacterial growth inhibition assays. Viability and cytotoxicity measurements in fibroblast cells exposed to GFP-AgBP protein-functionalised AgNPs showed significant improvement compared to controls. Biointerface engineering offers promise towards tailoring AgNP antimicrobial efficacy while addressing safety concerns to maintain optimum cellular interactions.

Comparison of brucellar and tuberculous spondylodiscitis patients: results of the multicenter "Backbone-1 Study".

BACKGROUND CONTEXT: No direct comparison between brucellar spondylodiscitis (BSD) and tuberculous spondylodiscitis (TSD) exists in the literature. PURPOSE: This study aimed to compare directly the clinical features, laboratory and radiological aspects, treatment, and outcome data of patients diagnosed as BSD and TSD. STUDY DESIGN: A retrospective, multinational, and multicenter study was used. PATIENT SAMPLE: A total of 641 (TSD, 314 and BSD, 327) spondylodiscitis patients from 35 different centers in four countries (Turkey, Egypt, Albania, and Greece) were included. OUTCOME MEASURES: The pre- and peri- or post-treatment spinal deformity and neurologic deficit parameters, and mortality were carried out. METHODS: Brucellar spondylodiscitis and TSD groups were compared for demographics, clinical, laboratory, radiological, surgical interventions, treatment, and outcome data. The Student t test and Mann-Whitney U test were used for group comparisons. Significance was analyzed as two sided and inferred at 0.05 levels. RESULTS: The median baseline laboratory parameters including white blood cell count, C-reactive protein, and erythrocyte sedimentation rate were higher in TSD than BSD (p<.0001). Prevertebral, paravertebral, epidural, and psoas abscess formations along with loss of vertebral corpus height and calcification were significantly more frequent in TSD compared with BSD (p<.01). Surgical interventions and percutaneous sampling or abscess drainage were applied more frequently in TSD (p<.0001). Spinal complications including gibbus deformity, kyphosis, and scoliosis, and the number of spinal neurologic deficits, including loss of sensation, motor weakness, and paralysis were significantly higher in the TSD group (p<.05). Mortality rate was 2.22% (7 patients) in TSD, and it was 0.61% (2 patients) in the BSD group (p=.1). CONCLUSIONS: The results of this study show that TSD is a more suppurative disease with abscess formation requiring surgical intervention and characterized with spinal complications. We propose that using a constellation of constitutional symptoms (fever, back pain, and weight loss), pulmonary involvement, high inflammatory markers, and radiological findings will help to differentiate between TSD and BSD at an early stage before microbiological results are available.