Highly Sensitive and Cost-Effective Portable Sensor for Early Gastric Carcinoma Diagnosis.

Facile and efficient early detection of cancer is a major challenge in healthcare. Herein we developed a novel sensor made from a polycarbonate (PC) membrane with nanopores, followed by sequence-specific Oligo RNA modification for early gastric carcinoma diagnosis. In this design, the gastric cancer antigen CA72-4 is specifically conjugated to the Oligo RNA, thereby inhibiting the electrical current through the PC membrane in a concentration-dependent manner. The device can determine the concentration of cancer antigen CA72-4 in the range from 4 to 14 U/mL, possessing a sensitivity of 7.029 µAU-1mLcm-2 with a linear regression (R2) of 0.965 and a lower detection limit of 4 U/mL. This device has integrated advantages including high specificity and sensitivity and being simple, portable, and cost effective, which collectively enables a giant leap for cancer screening technologies towards clinical use. This is the first report to use RNA aptamers to detect CA72-4 for gastric carcinoma diagnosis.

Differential Tropism of SARS-CoV and SARS-CoV-2 in Bat Cells.

Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.

A novel incompatibility group X3 plasmid carrying blaNDM-1 encodes a small RNA that regulates host fucose metabolism and biofilm formation.

The emergence of New Delhi metallo-beta-lactamase (NDM-1) has become a major health threat to clinical managements of gram-negative bacteria infections. A novel incompatibility group X3 plasmid (IncX3) pNDM-HN380 carrying blaNDM-1 has recently been found to epidemiologically link with multiple geographical areas in China. In this paper, we studied the metabolic responses of host bacteria E. coli J53 upon introduction of pNDM-HN380. A reduction of bacterial motility was observed in J53/pNDM-HN380. We profiled the RNA repertoires of the transconjugants and found a downregulation of genes involved in flagella and chemotaxis metabolic pathways at logarithmic (log) phase. We also identified a novel intragenic region (IGR) small RNA plas2. The plasmid-transcribed sRNA IGR plas2 was further characterized as a regulator of fucRwhich controls the fucose metabolism. By knockdown of IGR plas2 using an antisense decoy, we managed to inhibit the formation of bacterial biofilm of the host. Our study demonstrated a potential way of utilizing plasmid-transcribed sRNA against infectious bacteria.

Identification of a Novel Betacoronavirus (Merbecovirus) in Amur Hedgehogs from China.

While dromedaries are the immediate animal source of Middle East Respiratory Syndrome (MERS) epidemic, viruses related to MERS coronavirus (MERS-CoV) have also been found in bats as well as hedgehogs. To elucidate the evolution of MERS-CoV-related viruses and their interspecies transmission pathway, samples were collected from different mammals in China. A novel coronavirus related to MERS-CoV, Erinaceus amurensis hedgehog coronavirus HKU31 (Ea-HedCoV HKU31), was identified from two Amur hedgehogs. Genome analysis supported that Ea-HedCoV HKU31 represents a novel species under Merbecovirus, being most closely related to Erinaceus CoV from European hedgehogs in Germany, with 79.6% genome sequence identity. Compared to other members of Merbecovirus, Ea-HedCoV HKU31 possessed unique non-structural proteins and putative cleavage sites at ORF1ab. Phylogenetic analysis showed that Ea-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012 were closely related to NeoCoV and BatCoV PREDICT from African bats in the spike region, suggesting that the latter bat viruses have arisen from recombination between CoVs from hedgehogs and bats. The predicted HKU31 receptor-binding domain (RBD) possessed only one out of 12 critical amino acid residues for binding to human dipeptidyl peptidase 4 (hDPP4), the MERS-CoV receptor. The structural modeling of the HKU31-RBD-hDPP4 binding interphase compared to that of MERS-CoV and Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) suggested that HKU31-RBD is unlikely to bind to hDPP4. Our findings support that hedgehogs are an important reservoir of Merbecovirus, with evidence of recombination with viruses from bats. Further investigations in bats, hedgehogs and related animals are warranted to understand the evolution of MERS-CoV-related viruses.

Circulating miR-99b-5p as a novel predictor of erosion progression on high-resolution peripheral quantitative computed tomography in early rheumatoid arthritis: A prospective cohort study.

OBJECTIVES: To compare micro RNA (miRNA) expression: (a) between healthy individuals and early rheumatoid arthritis (ERA) patients with and without erosion on high-resolution peripheral quantitative computed tomography (HR-pQCT) at baseline; and (b) to explore whether these miRNAs could inform a signature predictive of erosion progression despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs). METHODS: The second metacarpophalangeal head (MCP2) was scanned by HR-pQCT at baseline and 1 year in 117 ERA patients. We performed global profiling of 377 miRNAs in 10 ERA patients with and without erosion on HR-pQCT at baseline and six healthy controls. Validation of the miRNAs of interest were conducted using TaqMan® quantitative real-time polymerase chain reaction in the validation ERA cohort (n = 117) at baseline. Correlation between the candidate miRNAs and erosion progression over 1 year were also assessed. RESULTS: In the 377 screened miRNAs, 94 (60.6%) miRNAs were upregulated in patients with erosions, with 13 (8.4%) upregulated more than 2-fold. Sixty-one (39.4%) miRNAs were downregulated in patients with erosions, with 6 (3.9%) downregulated more than 2-fold. Expression of miR-143-3p, miR-145-5p and miR-99b-5p were significantly higher in the plasma of ERA patients with erosions compared with those without erosions. Logistic regression analysis revealed that the baseline expression of miR-99b-5p was an independent predictor of erosion progression at month 12 (Exp [B] = 4.257, 95% CI 1.178-15.386, P = 0.027). CONCLUSIONS: Differential expressions of circulating miR-143-3p, miR-145-5p and miR-99b-5p in the plasma of ERA patients may characterize a severe form of the disease. MiR-99b-5p, in particular, may serve as a possible predictor for erosion progression.

Identification of Plasmid-Encoded sRNAs in a blaNDM-1-Harboring Multidrug-Resistance Plasmid pNDM-HK in Enterobacteriaceae.

Small RNAs (sRNAs) play significant roles in regulating gene expression post-transcriptionally in response to environmental changes in bacteria. In this work, we identified and characterized six novel sRNAs from an emerging multidrug-resistance (MDR) plasmid pNDM-HK, a New Delhi metallo-ß-lactamase 1 gene (blaNDM-1)-carrying IncL/M plasmid that has caused worldwide threat in recent years. These sRNAs are located at different regions of pNDM-HK, such as replication, stability, and variable regions. Moreover, one of the plasmid-encoded sRNAs (NDM-sR3) functions in an Hfq-dependent manner and possibly plays roles in the fitness of pNDM-HK carrying bacteria. In addition, we attempted to construct the phylogenetic tree based on these novel sRNAs and surprisingly, the sRNA-phylogenetic tree provided significant information about the evolutionary pathway of pNDM-HK, including possible gene acquisition and insertion from relevant plasmids. Moreover, the sRNA-phylogenetic tree can specifically cluster the IncM2 type and distinguish it from other IncL/M subtypes. In summary, this is the first study to systematically identify and characterize sRNAs from clinically-isolated MDR plasmids. We believe that these newly found sRNAs could lead to further understanding and new directions to study the evolution and dissemination of the clinically MDR bacterial plasmids.

Receptor Usage of a Novel Bat Lineage C Betacoronavirus Reveals Evolution of Middle East Respiratory Syndrome-Related Coronavirus Spike Proteins for Human Dipeptidyl Peptidase 4 Binding.

Although bats are known to harbor Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses, the role of bats in the evolutionary origin and pathway remains obscure. We identified a novel MERS-CoV-related betacoronavirus, Hp-BatCoV HKU25, from Chinese pipistrelle bats. Although it is closely related to MERS-CoV in most genome regions, its spike protein occupies a phylogenetic position between that of Ty-BatCoV HKU4 and Pi-BatCoV HKU5. Because Ty-BatCoV HKU4 but not Pi-BatCoV HKU5 can use the MERS-CoV receptor human dipeptidyl peptidase 4 (hDPP4) for cell entry, we tested the ability of Hp-BatCoV HKU25 to bind and use hDPP4. The HKU25-receptor binding domain (RBD) can bind to hDPP4 protein and hDPP4-expressing cells, but it does so with lower efficiency than that of MERS-RBD. Pseudovirus assays showed that HKU25-spike can use hDPP4 for entry to hDPP4-expressing cells, although with lower efficiency than that of MERS-spike and HKU4-spike. Our findings support a bat origin of MERS-CoV and suggest that bat CoV spike proteins may have evolved in a stepwise manner for binding to hDPP4.

Molecular Evolution of MERS Coronavirus: Dromedaries as a Recent Intermediate Host or Long-Time Animal Reservoir?

While dromedary camels are the immediate animal source of MERS coronavirus (MERS-CoV) infection, the evolutionary origin of MERS-CoV remains obscure. We analyzed 219 camel and human MERS-CoV genome sequences available in GenBank. Phylogenetic analysis showed that 5 and 214 strains belong to clade A and B, respectively, with clade A further divided into lineage A1 (3 human strains) and lineage A2 (2 camel strains), and clade B divided into B1 to B6 (each containing both human and camel strains). Recombination analysis showed potential recombination events in five strains from dromedaries in Saudi Arabia, with recombination between lineage B5 and B3 in four strains, and between lineage B3 and B4 in one strain. The spike protein showed the highest number of amino acid substitutions, especially between A2 and other lineages, and contained positively selected codons. Notably, codon 1020 was positively selected among B and B5 strains, and can distinguish between clade A (Q1020) and B (R1020/H1020) strains, suggesting that this residue may play a role in the evolution of S protein during divergence of different lineages. The time of the most recent common ancestor of all MERS-CoV was dated to approximately 2010. The implications on the role of camels in the evolution of MERS-CoV are discussed.

The CTX-M-14 plasmid pHK01 encodes novel small RNAs and influences host growth and motility.

The dissemination of extended-spectrum ß-lactamases (ESBLs) genes among bacteria is commonly achieved by plasmid conjugation. In the last decade, the CTX-M type enzyme was the most widespread and prevalent ESBLs in the world. In Hong Kong and mainland China, among the commonly found CTX-M-carrying plasmids were pHK01 and pHK01-like plasmids, which belong to incompatibility group FII (IncFII). In this work, we studied the physiological effect caused by the pHK01 plasmid in bacterial host Escherichia coli J53. The plasmid did not affect cell growth of the host but reduced their motility. The reduction of host motility was attributed to downregulation of genes that encode the flagellar system. We also identified several plasmid-encoded sRNAs, and showed that the overexpression of one of them, AS-traI, in the presence of pHK01 plasmid shortened the lag phase of host growth. In addition to the study of pHK01 in bacteria, we also developed a fast and incompatibility group-specific curing method using countertranscribed RNA, which could be of general usage for studying plasmid-host interaction in clinical aspects.

Familial young-onset diabetes, pre-diabetes and cardiovascular disease are associated with genetic variants of DACH1 in Chinese.

In Asia, young-onset type 2 diabetes (YOD) is characterized by obesity and increased risk for cardiovascular disease (CVD). In a genome-wide association study (GWAS) of 99 Chinese obese subjects with familial YOD diagnosed before 40-year-old and 101 controls, the T allele of rs1408888 in intron 1 of DACH1(Dachshund homolog 1) was associated with an odds ratio (OR) of 2.49(95% confidence intervals:1.57-3.96, P = 8.4 × 10(-5)). Amongst these subjects, we found reduced expression of DACH1 in peripheral blood mononuclear cells (PBMC) from 63 cases compared to 65 controls (P = 0.02). In a random cohort of 1468 cases and 1485 controls, amongst top 19 SNPs from GWAS, rs1408888 was associated with type 2 diabetes with a global P value of 0.0176 and confirmation in a multiethnic Asian case-control cohort (7370/7802) with an OR of 1.07(1.02-1.12, P(meta)  = 0.012). In 599 Chinese non-diabetic subjects, rs1408888 was linearly associated with systolic blood pressure and insulin resistance. In a case-control cohort (n = 953/953), rs1408888 was associated with an OR of 1.54(1.07-2.22, P = 0.019) for CVD in type 2 diabetes. In an autopsy series of 173 non-diabetic cases, TT genotype of rs1408888 was associated with an OR of 3.31(1.19-9.19, P = 0.0214) and 3.27(1.25-11.07, P = 0.0184) for coronary heart disease (CHD) and coronary arteriosclerosis. Bioinformatics analysis revealed that rs1408888 lies within regulatory elements of DACH1 implicated in islet development and insulin secretion. The T allele of rs1408888 of DACH1 was associated with YOD, prediabetes and CVD in Chinese.

Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates.

A total of 1878 non-duplicate clinical Escherichia coli isolates (comprising 1711 urinary isolates and 167 blood-culture isolates), which were collected from multiple centres in Hong Kong during 1996-2008, were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin (fos) resistance genes. Eighteen of the 1878 clinical E. coli isolates were fosfomycin resistant, of which six were fosA3 positive and two were positive for another fosA variant (designated fosKP96). No isolates had the fosC2 gene. The clones of the eight isolates were diverse: sequence type (ST) 95 (n = 2), ST118 (n = 1), ST131 (n = 1), ST617 (n = 1), ST648 (n = 1), ST1488 (n = 1) and ST2847 (n = 1). In the isolates, fosA3 and blaCTX-M genes were co-harboured on conjugative plasmids with F2:A-:B- (n = 2), N (n = 1), F-:A-:B1 and N (n = 1) and untypable (n = 2) replicons. Both fosKP96-carrying plasmids belonged to replicon N. RFLP analysis showed that the two F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes shared the same pattern. Complete sequencing of one of the two F2:A-:B- plasmids, pFOS-HK151325 (69 768 bp) demonstrated it to be >99 % identical to the previously sequenced plasmid pHK23a originating from a pig E. coli isolate in the same region. This study demonstrated the dissemination of fosA3 genes in diverse E. coli clones on multiple blaCTX-M-carrying plasmid types, of which F2:A-:B- plasmids closely related to pHK23a were shared by isolates from human and animal sources.

CCR5 antagonist TD-0680 uses a novel mechanism for enhanced potency against HIV-1 entry, cell-mediated infection, and a resistant variant.

Regardless of the route of transmission, R5-tropic HIV-1 predominates early in infection, rendering C-C chemokine receptor type 5 (CCR5) antagonists as attractive agents not only for antiretroviral therapy but also for prevention. Here, we report the specificity, potency, and underlying mechanism of action of a novel small molecule CCR5 antagonist, TD-0680. TD-0680 displayed the greatest potency against a diverse group of R5-tropic HIV-1 and SIV strains when compared with its prodrug, TD-0232, the Food and Drug Administration-approved CCR5 antagonist Maraviroc, and TAK-779, with EC(50) values in the subnanomolar range (0.09-2.29 nm). Importantly, TD-0680 was equally potent at blocking envelope-mediated cell-cell fusion and cell-mediated viral transmission as well as the replication of a TAK-779/Maraviroc-resistant HIV-1 variant. Interestingly, TD-0232 and TD-0680 functioned differently despite binding to a similar transmembrane pocket of CCR5. Site-directed mutagenesis, drug combination, and antibody blocking assays identified a novel mechanism of action of TD-0680. In addition to binding to the transmembrane pocket, the unique exo configuration of this molecule protrudes and sterically blocks access to the extracellular loop 2 (ECL2) region of CCR5, thereby interrupting the interaction between virus and its co-receptor more effectively. This mechanism of action was supported by the observations of similar TD-0680 potency against CD4-dependent and -independent SIV strains and by molecular docking analysis using a CCR5 model. TD-0680, therefore, merits development as an anti-HIV-1 agent for therapeutic purposes and/or as a topical microbicide for the prevention of sexual transmission of R5-tropic HIV-1.

Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis.

OBJECTIVES: Ninety-six percent of rifampicin resistance in Mycobacterium tuberculosis was shown to be associated with mutations inside the 81 bp rifampicin resistance-determining region (RRDR) located in the centre of the rpoB gene. The detection of rifampicin resistance by targeting the RRDR failed to match with a resistant phenotype in 4% of all cases. Our study aims to identify the mutations outside the RRDR that are associated with rifampicin resistance in M. tuberculosis. METHODS AND RESULTS: Among 50 rifampicin-resistant and 20 rifampicin-susceptible clinical isolates of M. tuberculosis, 2 of the rifampicin-resistant isolates did not harbour any known mutations in the RRDR. Sequencing analysis of the whole rpoB gene identified two rare mutations, V146F and I572F. A molecular structure model based on Thermus thermophilus RpoB revealed that both these substituted amino acids are located in close proximity to the rifampicin-binding pocket of the ß-subunit. Substitutions of simple amino acids for bulky ones are likely to affect the protein-drug interaction. Cloning and transformation of the mutated rpoB gene into wild-type Mycobacterium smegmatis and M. tuberculosis successfully elevated the MIC of rifampicin and conferred the rifampicin resistance phenotype. CONCLUSIONS: Our study showed that amino acid positions 146 and 572 are associated with rifampicin resistance in M. tuberculosis in addition to the RRDR. Molecular assays for identifying rifampicin-resistant M. tuberculosis might be improved in terms of accuracy by including these two positions.

Molecular characterization of fluoroquinolone resistance in Mycobacterium tuberculosis: functional analysis of gyrA mutation at position 74.

A PCR-sequencing assay was evaluated for direct detection of mutations in the quinolone resistance-determining region (QRDR) of gyrase A (gyrA) gene in fluoroquinolone-resistant Mycobacterium tuberculosis in respiratory specimens. As determined by gyrA QRDR analysis, complete concordance of genotypic and phenotypic fluoroquinolone resistance was demonstrated. Our results indicate that the assay is a rapid and reliable method for the diagnosis of fluoroquinolone-resistant tuberculosis, facilitating timely clinical management and public health control. Using the assay, we detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from sputum specimens. The functional effect of the Ala74Ser mutant was verified through the study of the DNA supercoiling inhibitory activity of fluoroquinolones against the recombinant gyrase. The drug-mediated gyrase-DNA cleavage complex model suggests perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative quinolone binding pocket and leads to the reduction of the drug binding affinity. A number of gyrA mutations (Glu21Gln, Ser95Thr, and Gly668Asp) were also characterized to be natural polymorphisms not associated with fluoroquinolone resistance.