Discovery of potent and selective activity-based probes (ABPs) for the deubiquitinating enzyme USP30.

Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKß-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.

Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1ß production in human macrophages and microglia.

Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labeling, affinity purification, and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-interleukin-1ß (IL-1ß) levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1ß cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1ß production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.

How does social support affect subjective well-being among Chinese widowed older adults?

Background: Social support (SS) is an important factor influencing subjective well-being (SWB) in older adults. This is especially true for the special group of widowed older adults (WOA). Widowhood means that older adults have lost their most important SS, and therefore, the search for a guardian from outside the spouse becomes a central issue in ensuring the SWB of WOA. Methods: The data for this paper were obtained from CGSS 2021, a large national social survey in China. We operationalized SWB as an individual's overall perception of his or her experience of happiness using 'affective well-being' (i.e., emphasizing an individual's positive affective experiences), and scores were calculated using a Likert scale. This study used linear regression modeling to examine the impact of SS on the SWB of WOA (aged 60 and above). Results: It was found that, first, this study presents the role of different circles of SS on the SWB of WOA, fully highlighting the importance of social context. Specifically, daughters, neighbors, and relatives constitute the guardians of the SWB for WOA in rural, whereas daughters and friends constitute the guardians of the SWB for WOA in rural. Second, the protective resources provided by the guardians not only serve as a buffer for WOA in distress but also reduce the likelihood of negative events occurring, thereby increasing WOA's SWB. Discussion: This paper partially corroborates the findings of established studies on the topic of SS and SWB among older adults and the above findings not only help us to further explain the relationship between SS and SWB theoretically but also help us to rationalize the construction of SS for WOA practically.

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

Non-Invasive Ultrasound Modulation of Solitary Tract Nucleus Exerts a Sustainable Antihypertensive Effect in Spontaneously Hypertensive Rats.

OBJECTIVE: We applied the method of non-invasive ultrasound (US) neuromodulation to regulate blood pressure (BP) by stimulating the solitary tract nucleus (NTS) of spontaneously hypertensive rats (SHRs). METHODS: The rats were exposed to US stimulation for 20 mins every day for two months. Morphology and function of the hypertensive target organs (heart and kidney) were then examined by echocardiography and immunohistochemical staining. C-fos immunofluorescence assays were used to evaluate neuronal activity in the US stimulated areas and to explore related neural pathways. Moreover, the effects of US stimulation on biochemical indicators angiotensinII (ANGII), aldosterone (Aldo), endothelin-1 (ET-1), atrial natriuretic factor (ANF), cortisol (Cor) in SHRs were detected. In addition, HE, TUNEL, and Nissl staining were performed to evaluate the safety of long-term transcranial US stimulation. RESULTS: After two months of US stimulation, systolic blood pressure (SBP) decreased from 170 ± 1.1 mmHg to 158 ± 1.8 mmHg, p < 0.01. What's more, US stimulation effectively inhibited the pathological process of target organs from both morphological and functional levels. With US stimulation, neuronal activities were also significantly enhanced in the NTS, ventrolateral periaqueductal gray (vlPAG), and the caudal ventrolateral medulla (CVLM) region. And US stimulation did not cause brain tissue damage. Meanwhile, the plasma levels of ANF, ANGII, Aldo, and Cor content were inhibited. CONCLUSION: US stimulation of the NTS could significantly lower BP in SHRs. SIGNIFICANCE: Non-invasive transcranial US stimulation acting on the NTS might be a potential therapeutic intervention due to its efficacy and safety.

The sustainable antihypertensive and target organ damage protective effect of transcranial focused ultrasound stimulation in spontaneously hypertensive rats.

OBJECTIVE: In this study, we aimed to investigate the sustainable antihypertensive effects and protection against target organ damage caused by low-intensity focused ultrasound (LIFU) stimulation and the underlying mechanism in spontaneously hypertensive rats (SHRs) model. METHODS AND RESULTS: SHRs were treated with ultrasound stimulation of the ventrolateral periaqueductal gray (VlPAG) for 20 min every day for 2 months. Systolic blood pressure (SBP) was compared among normotensive Wistar-Kyoto rats, SHR control group, SHR Sham group, and SHR LIFU stimulation group. Cardiac ultrasound imaging and hematoxylin-eosin and Masson staining of the heart and kidney were performed to assess target organ damage. The c-fos immunofluorescence analysis and plasma levels of angiotensin II, aldosterone, hydrocortisone, and endothelin-1 were measured to investigate the neurohumoral and organ systems involved. We found that SBP was reduced from 172 ±â€Š4.2 mmHg to 141 ±â€Š2.1 mmHg after 1 month of LIFU stimulation, P  < 0.01. The next month of treatment can maintain the rat's blood pressure at 146 ±â€Š4.2 mmHg at the end of the experiment. LIFU stimulation reverses left ventricular hypertrophy and improves heart and kidney function. Furthermore, LIFU stimulation enhanced the neural activity from the VLPAG to the caudal ventrolateral medulla and reduced the plasma levels of ANGII and Aldo. CONCLUSION: We concluded that LIFU stimulation has a sustainable antihypertensive effect and protects against target organ damage by activating antihypertensive neural pathways from VLPAG to the caudal ventrolateral medulla and further inhibiting the renin-angiotensin system (RAS) activity, thereby supporting a novel and noninvasive alternative therapy to treat hypertension.

Enabling Dual Phosphorescence by Locating a Flexible Ligand in Zn-Based Hybrid Frameworks.

Room-temperature phosphorescence (RTP) materials with recognizable afterglow property have gained widespread attraction. Multicolor RTP has added benefits in multiplexed biological labeling, a zero background ratiometric sensor, a multicolor display, and other fields. However, it is a great challenge to prepare multicolor RTP from a single-component compound according to Kasha's rule. Herein, we propose a strategy to design multicolor RTP in a metal-organic hybrid framework through constructing chromophores in both isolated state and dimer state using a flexible tetradentate ligand. Two compounds were synthesized that presented blue and green dual phosphorescence with different lifetimes at ambient conditions. The photoluminescence mechanism has been thoroughly studied by structure-property analysis. This study provides various possibilities to prepare high-performing RTP materials by the rational design and synthesis of similar compounds.

HDAC/MIF dual inhibitor inhibits NSCLC cell survival and proliferation by blocking the AKT pathway.

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR-/-) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

Genome-Wide Identification and Genetic Variations of the Starch Synthase Gene Family in Rice.

Starch is a major ingredient in rice, and the amylose content of starch significantly impacts rice quality. OsSS (starch synthase) is a gene family related to the synthesis of amylose and amylopectin, and 10 members have been reported. In the present study, a synteny analysis of a novel family member belonging to the OsSSIV subfamily that contained a starch synthase catalytic domain showed that three segmental duplications and multiple duplications were identified in rice and other species. Expression data showed that the OsSS gene family is involved in diverse expression patterns. The prediction of miRNA targets suggested that OsSS are possibly widely regulated by miRNA functions, with miR156s targeted to OsSSII-3, especially. Haplotype analysis exhibited the relationship between amylose content and diverse genotypes. These results give new insight and a theoretical basis for the improved amylose content and eating quality of rice.

Epigenetic Mechanism of Enrichment of A549 Lung Cancer Stem Cells with 5-Fu.

BACKGROUND: The influence of 5-fluorouracil (5-Fu) and cisplatin (CDDP) on the A549 and NCI-H226 cells was studied, and the epigenetic mechanism of enrichment of A549 lung cancer stem cells with 5-Fu was explored. MATERIALS AND METHODS: The cell proliferation of both A549 and NCI-H226 was detected by BrdU assay, and apoptosis condition was measured by flow cytometric analysis. The expressions of OCT3/4 and Nanog in cells treated with 5-Fu or CDDP were measured by immunofluorescence, Western blot and qPCR. qPCR was also performed to determine the relative expression of methyltransferase genes and miRNA. Sequencing after bisulfite treatment (BSP) was employed to detect the methylation of OCT3/4 promoter in A549 cells. And ChIP was conducted to detect the expression of H3K9Me3 and H3K9Ace. RESULTS: Both 5-Fu and CDDP result in the apoptosis of A549 and NCI-H226 cells and improve the expressions of has-miR-134 and has-miR-296. However, 5-Fu enhances the expression of OCT3/4 in A549 cells, and the change of methyltransferase genes and BSP results suggested some genetic differences between CDDP and 5-Fu treatment in A549 cells. ChIP assay showed that the expression of H3K9Me3 significantly decreased and H3K9Ace significantly increased in A549 cells. CONCLUSION: The enrichment effect of CDDP on A549 and NCI-H226 carcinoma stem cells is inconsistent with the enrichment effect of 5-Fu. The enrichment of A549 lung cancer stem cells with 5-Fu might be related to the methylation of OCT3/4 promoter and the expression of H3K9Me3 and H3K9Ace.

Biaryl sulfonamides as cisoid azosteres for photopharmacology.

Biaryl sulfonamides are excellent candidates for the azologization approach that yields photoswitchable drugs more active in their metastable cis state, compared to the stable trans state. Here we present the scope and limitations of this strategy for rational design in photopharmacology.

Functional characterization of two chalcone isomerase (CHI) revealing their responsibility for anthocyanins accumulation in mulberry.

Mulberry (Morus sp., Moraceae) is an important economic crop plant and mulberry fruits are rich in anthocyanidins. Chalcone isomerase (CHI) catalyzes the conversion of chalcones to flavanones providing precursors for biosynthesis of anthocyanidins. In this study, bona fide CHIs were cloned and characterized from different Morus species with differently colored fruits (Morus multicaulis, Mm and Morus alba variety LvShenZi, LSZ). Enzymatic assay of MmCHI1 and MmCHI2 showed that they can utilize naringenin chalcone as substrate. The catalytic efficiency of MmCHI2 and LSZCHI2 are approximately 200 and 120-fold greater than that of MmCHI1 respectively. Phylogenetic analysis showed the two mulberry CHIs belonged to different sub-clade of Type I CHI1 named type IA (CHI2) and type IB (CHI1). Type IB CHIs are mulberry specific. MmCHI1 and MmCHI2 had similar expression profiles and showed preferred expression in fruits. In addition, both mulberry CHI1 and CHI2 played roles in the response to excess zinc stress and sclerotiniose pathogen infection. Both MmCHI1 and MmCHI2 expression levels showed positive close relationship with anthocyanins content during fruit ripening process. The co-expression of MmCHI1 and MmCHI2 was observed during fruit ripening process and in transgenic mulberry. VIGS (virus induced gene silence) targeting on MmCHI1 and MmCHI2 showed significant down-regulation of MmCHI2 instead of MmCHI1 would result in significant (about 50%) decrease in anthocyanins content. MmCHI2 is the dominant CHI for anthocyanins accumulation in mulberry. The results presented in this work provided insight on bona fide CHIs in mulberry and reveal their roles in anthocyanins accumulation.

Genome-Wide Association Study of Root System Development at Seedling Stage in Rice.

Root network structure plays a crucial role in growth and development processes in rice. Longer, more branched root structures help plants to assimilate water and nutrition from soil, support robust plant growth, and improve resilience to stresses such as disease. Understanding the molecular basis of root development through screening of root-related traits in rice germplasms is critical to future rice breeding programs. This study used a small germplasm collection of 137 rice varieties chosen from the Korean rice core set (KRICE_CORE) to identify loci linked to root development. Two million high-quality single nucleotide polymorphisms (SNPs) were used as the genotype, with maximum root length (MRL) and total root weight (TRW) in seedlings used as the phenotype. Genome-wide association study (GWAS) combined with Principal Components Analysis (PCA) and Kinship matrix analysis identified four quantitative trait loci (QTLs) on chromosomes 3, 6, and 8. Two QTLs were linked to MRL and two were related to TRW. Analysis of Linkage Disequilibrium (LD) decay identified a 230 kb exploratory range for detection of candidate root-related genes. Candidates were filtered using RNA-seq data, gene annotations, and quantitative real-time PCR (qRT-PCR), and five previously characterized genes related to root development were identified, as well as four novel candidate genes. Promoter analysis of candidate genes showed that LOC_Os03g08880 and LOC_Os06g13060 contained SNPs with the potential to impact gene expression in root-related promoter motifs. Haplotype analysis of candidate genes revealed diverse haplotypes that were significantly associated with phenotypic variation. Taken together, these results indicate that LOC_Os03g08880 and LOC_Os06g13060 are strong candidate genes for root development functions. The significant haplotypes identified in this study will be beneficial in future breeding programs for root improvement.

Induced protein degradation of histone deacetylases 3 (HDAC3) by proteolysis targeting chimera (PROTAC).

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32 µM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome.

Cavitation characteristics of flowing low and high boiling-point perfluorocarbon phase-shift nanodroplets during focused ultrasound exposures.

This work investigated and compared the dynamic cavitation characteristics between low and high boiling-point phase-shift nanodroplets (NDs) under physiologically relevant flow conditions during focused ultrasound (FUS) exposures at different peak rarefactional pressures. A passive cavitation detection (PCD) system was used to monitor cavitation activity during FUS exposure at various acoustic pressure levels. Root mean square (RMS) amplitudes of broadband noise, spectrograms of the passive cavitation detection signals, and normalized inertial cavitation dose (ICD) values were calculated. Cavitation activity of low-boiling-point perfluoropentane (PFP) NDs and high boiling-point perfluorohexane (PFH) NDs flowing at in vitro mean velocities of 0-15 cm/s were compared in a 4-mm diameter wall-less vessel in a transparent tissue-mimicking phantom. In the static state, both types of phase-shift NDs exhibit a sharp rise in cavitation intensity during initial FUS exposure. Under flow conditions, cavitation activity of the PFH NDs reached the steady state less rapidly compared to PFP NDs under the lower acoustic pressure (1.35 MPa); at the higher acoustic pressure (1.65 MPa), the RMS amplitude increased more sharply during the initial FUS exposure period. In particular, the RMS-time curves of the PFP NDs shifted upward as the mean flow velocity increased from 0 to 15 cm/s; the RMS amplitude of the PFH ND solution increased from 0 to 10 cm/s and decreased at 15 cm/s. Moreover, amplitudes of the echo signal for the low boiling-point PFP NDs were higher compared to the high boiling-point PFH NDs in the lower frequency range, whereas the inverse occurred in the higher frequency range. Both PFP and PFH NDs showed increased cavitation activity in the higher frequency under the flow condition compared to the static state, especially PFH NDs. At 1.65 MPa, normalized ICD values for PFH increased from 0.93 ± 0.03 to 0.96 ± 0.04 and from 0 to 10 cm/s, then decreased to 0.86 ± 0.05 at 15 cm/s. This work contributes to our further understanding of cavitation characteristics of phase-shift NDs under physiologically relevant flow conditions during FUS exposure. In addition, the results provide a reference for selecting suitable phase-shift NDs to enhance the efficiency of cavitation-mediated ultrasonic applications.

Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing.

Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

Synthesis of Peptoid-Based Class I-Selective Histone Deacetylase Inhibitors with Chemosensitizing Properties.

There is increasing evidence that histone deacetylase (HDAC) inhibitors can (re)sensitize cancer cells for chemotherapeutics via "epigenetic priming". In this work, we describe the synthesis of a series of class I-selective HDAC inhibitors with 2-aminoanilides as zinc-binding groups. Several of the synthesized compounds revealed potent inhibition of the class I HDAC isoforms HDAC1, HDAC2, and/or HDAC3 and promising antiproliferative effects in the human ovarian cancer cell line A2780 and the human squamous carcinoma cell line Cal27. Selected compounds were investigated in a cellular model of platinum resistance. In particular, compound 2a revealed potent chemosensitizing properties and full reversal of cisplatin resistance in Cal27CisR cells. This effect is related to a synergistic increase in caspase 3/7 activation and induction of apoptosis. Thus, this work demonstrates that pan-HDAC inhibition or dual class I/class IIb inhibition is not required for full reversal of cisplatin resistance.

Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages.

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the 'lid' interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages.

The Process and Strategy for Developing Selective Histone Deacetylase 3 Inhibitors.

Histone deacetylases (HDACs) are epigenetic drug targets that have gained major scientific attention. Inhibition of these important regulatory enzymes is used to treat cancer, and has the potential to treat a host of other diseases. However, currently marketed HDAC inhibitors lack selectivity for the various HDAC isoenzymes. Several studies have shown that HDAC3, in particular, plays an important role in inflammation and degenerative neurological diseases, but the development of selective HDAC3 inhibitors has been challenging. This review provides an up-to-date overview of selective HDAC3 inhibitors, and aims to support the development of novel HDAC3 inhibitors in the future.

Using k-dependence causal forest to mine the most significant dependency relationships among clinical variables for thyroid disease diagnosis.

Numerous data mining models have been proposed to construct computer-aided medical expert systems. Bayesian network classifiers (BNCs) are more distinct and understandable than other models. To graphically describe the dependency relationships among clinical variables for thyroid disease diagnosis and ensure the rationality of the diagnosis results, the proposed k-dependence causal forest (KCF) model generates a series of submodels in the framework of maximum spanning tree (MST) and demonstrates stronger dependence representation. Friedman test on 12 UCI datasets shows that KCF has classification accuracy advantage over the other state-of-the-art BNCs, such as Naive Bayes, tree augmented Naive Bayes, and k-dependence Bayesian classifier. Our extensive experimental comparison on 4 medical datasets also proves the feasibility and effectiveness of KCF in terms of sensitivity and specificity.