Rapid Detection of Carbapenem-Resistant Klebsiella pneumoniae Using Machine Learning and MALDI-TOF MS Platform.

Background: Rapid detection of carbapenem-resistant Klebsiella pneumoniae (CRKP) is essential for specific antimicrobial therapy. Machine learning techniques combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used as a rapid, reliable, sensitive, and low-cost species identification method. Methods: Clinically collected K. pneumoniae were subjected to MALDI-TOF MS analysis. A random forest (RF) algorithm and non-linear support vector machine (SVM) were used to construct the RF, SVM, and dimension reduction (SVM-K) models, and their performance was assessed for accuracy, sensitivity, specificity, and area under the subject worker curve (AUC). Results: The RF, SVM and SVM-K models showed good classification performance with 0.88, 0.88, and 0.91 accuracy, 0.82, 0.85, and 0.89 sensitivity, 0.93, 0.92, and 0.94 specificity with an AUC of 0.9013, 0.9298, and 0.9356, respectively. For the SVM-K model, the optimal dimension reduction was 105 to 153, and the average accuracy was >0.9. The top 10 peak features of significance according to the RF algorithm with 6515 Da appeared in 56.8% of CRKP isolates and 5.3% of CSKP isolates, which indicated the best classification performance. Conclusion: The three RF, SVM, and SVM-K models showed excellent classification performance differentiating the CRKP from CSKP; the SVM-K model was the best. Data analysis with machine learning combined with MALDI-TOF MS can be employed as a rapid and inexpensive alternative to existing detection methods.

An innovative method to obtain porous porcine aorta scaffolds for tissue engineering.

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum-freeze-thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum-freeze-thawing significantly improved the decellularization efficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum-freeze-thawing into the decellularization process may produce a promising vascular graft candidate for tissue engineering application.

Evidence of Osteogenic Regulation in Calcific Porcine Aortic Valves.

BACKGROUND: Chemically cross-linked animal tissues, such as porcine aortic valves (PAVs) have many documented advantages over mechanical valves. However, calcification is the major underlying pathologic process that results in bioprosthetic valve failure. Recently, several reports described the expression of noncollagenous bone matrix proteins in bioprosthetic valves and suggested an actively regulated process of tissue repair. METHODS: Thirty-one explanted PAVs with evidence of calcification were collected and examined for the protein expression implicated in myofibroblast activation, osteoblast differentiation, and bone matrix deposition by using immunohistochemistry. RESULTS: The mean duration that PAVs were implanted was 11.5 ± 5.6 years, ranging from 12 months to 28 years. Pearson correlation analysis showed a significant relationship between the duration and valvular calcification (r = 0.3818, P = .034). The number of vimentin-positive mesenchymal cells in explanted PAVs was significantly lower than that of unused PAVs (P < .01). However, increased expression of α-smooth muscle actin (α-SMA) (P < .01), proliferating cell nuclear antigen (PCNA, P < .01), Cbfa1/Runx2 (P < .01), osterix (P = .0126), bone sialoprotein (BSP, P < .01), osteocalcin (P < .01), and osteopontin (P < .01) was found in explanted PAVs. Immunohistochemical staining of alkaline phosphatase (ALP) and osteocalcin was negative in the unused PAVs. In explanted PAVs, the expression level of these 2 proteins was also significantly increased. CONCLUSIONS: Our results support the view that PAV calcification is an actively regulated process with osteogenic signaling activation.

Comparison of detergent-based decellularization protocols for the removal of antigenic cellular components in porcine aortic valve.

BACKGROUND: Various detergent-based protocols are used to remove cells and cellular debris in porcine aortic valve (PAV). However, the removal of antigenic cellular components has not been thoroughly elucidated to date. In this study, we used 4 detergent-based protocols to decellularize PAVs and aimed to evaluate their effects on removing antigenic cellular components. METHODS: Porcine aortic valves were decellularized using sodium dodecyl sulfate (SDS), SDS in combination with sodium deoxycholate (SDS/SD), Triton X-100, and Triton X-100 in combination with SD (Triton X-100/SD), respectively. Untreated PAVs were used as controls. Immunohistochemical and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed to determine the removal of antigenic protein components. Histological, biochemical, and biomechanical analyses were performed to determine the preservation and mechanical properties of the extracellular matrix. PAV tissues were implanted subcutaneously in Sprague Dawley rats to evaluate the host immune response. Implanted PAVs were taken out at the indicated time points for histological and immunohistochemical examinations. RESULTS: All 4 protocols effectively removed the membrane antigenic proteins major histocompatibility complex I molecule and galactose-α-1,3 galactose. The SDS/SD protocol was the most effective method to remove the cytoplasmic cytoskeletal proteins, vimentin and α-SMA, and SDS alone partly removed vimentin protein. The SDS/SD protocol was the most effective method to remove nuclear DNA with residual DNA below 50 ng/mg, followed by the SDS protocol with residual DNA of 74.9 ng/mg. The SDS protocol was the most effective method to remove proteins ranged from 10 to 55 kDa, followed by the SDS/SD protocol. SDS and SDS/SD PAV implants attracted fewer neutrophils in vivo on postoperative day 3. The infiltration of macrophages and T lymphocytes was significantly lower in SDS and SDS/SD implants on days 14 and day 28. All of the decellularized protocols that were examined greatly reduced the contents of collagen, elastin, and glycosaminoglycan compared to controls. Biomechanical analysis revealed significant differences in ultimate tensile strength and Young's modulus between control PAVs and decellularized PAVs generated using SDS, SDS/SD, Triton X-100, or Triton X-100/SD. CONCLUSIONS: These results indicated that SDS-based protocols more effectively removed antigenic cellular components compared to Triton X-100-based protocols. These results are clinically significant because complete removal of antigenic determinants is critical to decrease adverse immune-mediated and inflammatory responses to a PAV when used in xenogeneic application.

Efficient decellularization for bovine pericardium with extracellular matrix preservation and good biocompatibility.

OBJECTIVES: In this study, we sought to explore an efficient decellularization protocol for bovine pericardia with better extracellular matrix preservation and good biocompatibility. METHODS: Bovine pericardia were decellularized by sodium dodecyl sulphate (SDS), SDS + sodium deoxycholate (SD), Triton X-100 (TX), TX + SD (TS), freeze-thaw cycles + SDS + SD (FSS) and freeze-thaw cycles + TX + SD (FTS), respectively. Untreated pericardia were used as native control. Histological examination, residual cellular content analysis, biochemical and biomechanical evaluations and cytotoxicity assay were performed to investigate decellularization efficiency, xenoantigens removal, extracellular matrix preservation and biocompatibility. In vivo biocompatibility was evaluated using a subcutaneous implantation method in rats. RESULTS: Among these protocols, FSS and FTS protocols were the most effective methods to remove both the DNA material and the galactose-α-1,3-galactose antigen. TX, TS and FTS bovine pericardia maintained the collagen content and had no cytotoxicity to human umbilical vein endothelial cells. The contents of elastin and glycosaminoglycan were lost to different degrees after decellularization, with the highest content of preservation with TX, followed by TS and FTS. Consistently, no significant difference was found between native bovine pericardia and TX, TS or FTS bovine pericardia. In vivo, FTS implants had minimal infiltration of macrophages and T-lymphocytes, with no histological evidence of peri-implant necrosis and calcification. CONCLUSIONS: These results suggested that the FTS protocol showed optimal decellularization results with better extracellular matrix preservation and good biocompatibility. It may be a suitable protocol for producing a suitable scaffold for heart tissue engineering.

A single nucleotide polymorphism in primary-microRNA-146a reduces the expression of mature microRNA-146a in patients with Alzheimer's disease and is associated with the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common form of dementia among the aging population. Although the incidence of the disease continues to increase, no cure has been developed. Effective treatment is restricted not only due to the lack of curative medicine, but also due to limited understanding of the underlying mechanisms and the difficulties in accurately diagnosing AD in its earliest stages prior to clinical symptoms. Micro (mi) RNAs (miR) have gained increasing attention in the investigation of neurodegenerative diseases. Previous reports have demonstrated that deregulation of miR­146a­5p is associated with the pathogenesis of human AD. In the present study, the coding region of primary (pri)­miR­146a in patients with AD was scanned and the rare C allele of rs2910164 was found to be associated with AD. Using reverse transcription quantitative polymerase chain reaction, it was demonstrated that site variation reduced the expression of mature miR­146a­5p. Notably, a reduction in the expression of miR­146a­5p led to less efficient inhibition of target genes, including Toll­like receptor (TLR)2, which is important in the pathogenesis of AD. Biological function investigations in RAW264.7 cells indicated that, compared with the G allele, the rare C allele upregulated the expression of tumor necrosis factor­α following stimulation with ß­amyloid. These findings suggested that one common polymorphism in pri­miR­146a may contribute to the genetic predisposition to AD by disrupting the production of miR­146a­5p and affecting the expression and function of TLR2.

Identification of key pathways and transcription factors related to Parkinson disease in genome wide.

Parkinson disease (PD) is a common neurodegenerative disease. Most people with PD are idiopathic, with no specific known cause. Recently, several studies have indicated small proportion of PD cases may result from a mutation in some specific genes. However, the involved pathways of these genes and the co-expression patterns of associated pathways still remain unclear. Here, we aimed to systematically investigate PD related pathways by using microarray dataset GSE7621 from the public database library of gene expression omnibus and gene set enrichment analysis on the datasets. Furthermore, candidate transcription factors were also explored by distant regulatory elements software. As a result, 11 up-regulated pathways (such as glycosaminoglycan degradation) and 24 down-regulated pathways (such as ErbB signaling pathway and Long-term depression) were identified as PD related. Most of them were classified into the maps of human diseases, organismal system, and metabolism with no previous reports. Finally, we constructed co-expression networks of related pathways with the significant core genes and transcription factors, such as OCT and HNF3. All of these may be helpful to better understand the molecular mechanisms of human PD in genome wide.

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.