S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern.

The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 Are Susceptible to SARS-CoV-2 Virus Infection In Vitro.

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry, although there is limited evidence of active oral infection by SARS-CoV-2 viruses. We assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication-competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Distinct viral dynamics were seen in hTERT TIGKs compared to A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells on day 3 after infection. Analysis of oral epithelial cells infected by replication-competent SARS-CoV-2 viruses expressing GFP showed that the GFP signal and SARS-CoV-2 mRNAs were not evenly distributed. Furthermore, we found cumulative SARS-CoV-2 RNAs from released viruses in the media from oral epithelial cells on day 1 and day 2 after infection, indicating productive viral infection. Taken together, our results demonstrated that oral epithelial cells were susceptible to SARS-CoV-2 viruses despite low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for the development of future vaccines and therapeutics.

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.

Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection.

BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Development of a Novel Human CD147 Knock-in NSG Mouse Model to Test SARS-CoV-2 Viral Infection.

Background: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. Results: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD- scid IL2Rgamma null (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show 1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; 2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; 3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. Conclusions: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Multiplex PCR Assays for Identifying all Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

Variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the evolution of the virus, but is costly, slow, and not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well as other mutations of interest in the viral genome, nine mutations in total, using single-nucleotide discriminating molecular beacons. The presented variant molecular beacon assays showed a limit of detection of 50 copies of viral RNA, with 100% specificity. Twenty-six SARS-CoV-2-positive patient samples were blinded and tested using a two-tube assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26 of 26). We have used our design methodology to rapidly design an assay that detects the new omicron variant. This omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These quantitative RT-PCR assays identify all currently circulating VOCs of SARS-CoV-2, as well as other important mutations in the spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies.

SuperSelective primers, by virtue of their unique design, enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are sensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers. Herein, we provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, we provide examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non-small-cell lung cancer. The results suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual's cancer, utilizing frequent noninvasive liquid biopsies.

A Molecular-Beacon-Based Multiplex Real-Time PCR Assay To Distinguish Mycobacterium abscessus Subspecies and Determine Macrolide Susceptibility.

Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species that comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. These predominantly environmental microorganisms have emerged as life-threatening chronic pulmonary pathogens in both immunocompetent and immunocompromised patients, and their acquisition of macrolide resistance due to the erm(41) gene and mutations in the 23S rrl gene has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools to distinguish M. abscessus subspecies, and the testing for macrolide resistance is often not done. Here, we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid, and highly accurate method to both distinguish M. abscessus subspecies and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspecies sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a 2-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 h.

Rapid Pyrazinamide Drug Susceptibility Testing using a Closed-Tube PCR Assay of the Entire pncA gene.

The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis. Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2-98.6) and specificity of 97.9% (95% CI: 96.1-99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5-100) but lower specificity of 91.8% (95% CI: 87.9-95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility.

High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules.

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

Color-coded molecular beacons for multiplex PCR screening assays.

The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient's normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample.

Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays that Utilize SuperSelective Primers for the Detection of Rare Somatic Mutations.

In PCR assays designed to detect rare somatic mutations, SuperSelective primers, by virtue of their short 3'-foot sequences, selectively initiate synthesis on mutant DNA target fragments, while suppressing the synthesis of related wild-type fragments, and the resulting threshold cycle reflects the quantity of mutant targets present. However, when there are ≤10 mutant target fragments in a sample, the threshold cycle that is observed occurs so late that it can be confused with the threshold cycle that arises from samples that contain only abundant related wild-type fragments. We report here that the inclusion of the selectivity enhancing agents tetramethylammonium chloride or bis-tetramethylammonium oxalate in SuperSelective PCR assays substantially suppresses the amplification of related wild-type fragments. As a result of this selective suppression, assay sensitivity is increased to such an extent that multiplex PCR assays can be performed in which it is highly unlikely that there will be a false-positive or false-negative result. This advance provides a foundation for the development of rapid, low-cost, multiplex PCR assays for noninvasively assessing the presence of relevant mutations in cancer patients, thereby enabling individually appropriate therapy.

Detection and Differentiation of Lyme Spirochetes and Other Tick-Borne Pathogens from Blood Using Real-Time PCR with Molecular Beacons.

Real-time PCR assays have recently been implemented in diagnostics for many bacterial pathogens, allowing rapid and accurate detection, which ultimately results in improved clinical intervention. Here, we describe a sensitive method of detection for three common tick-borne pathogens Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti since coinfections with these pathogens have started occurring with increasing frequency over the last several years in both North America and Europe. A shared geographic region, the same tick vectors, and similar transmission cycle all favor simultaneous transmission of these three tick-borne pathogens. Furthermore, early symptoms of the diseases are often similar and somewhat nonspecific leading to poor clinical identification. The multiplex real-time PCR assay we describe here utilizes gene-specific primers, molecular beacon probes tagged with different fluorophores, and optimized PCR conditions to detect even small amounts of specific pathogen DNA without interference. Application of this detection method will offer better diagnostics for acute and persistent infection compared to the two-tier serological tests that are currently approved in North America and Europe, which do not necessarily detect active infection.

The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11.

To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. We report for the first time the expression patterns during oogenesis for Drongo, the fruit fly homolog of AGFG1. We find that temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. Here we show a first link between the recycling endosome pathway and Drongo, and find that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.

Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer.

We describe the use of "SuperSelective" primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long "5' anchor sequence" that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, "3' foot sequence" that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation's abundance by comparing its threshold value to the threshold value of a reference gene present in the sample.

An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease.

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg(2+) or Mn(2+) ions, which are critical for its catalytic function. There is great interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We focused here on a series of ß-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, in order to build a three-dimensional pharmacophore model. In addition, we developed a molecular beacon (MB)-based PA-Nter assay that enabled us to compare the inhibition of Mn(2+) versus Mg(2+), the latter probably being the biologically relevant cofactor. This real-time MB assay allowed us to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter, with IC50 values comparable to that of the prototype L-742,001 (i.e., below 2 µM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg(2+) or Mn(2+). Three compounds ( 10: , with a pyrrole scaffold, and 40: and 41: , with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values 64-95 µM) and were proven to affect viral RNA synthesis. Our approach of integrating complementary enzymatic, cellular, and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.

BACKGROUND: The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. RESULTS: In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients' blood samples. CONCLUSION: Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.

Tiny molecular beacons: LNA/2'-O-methyl RNA chimeric probes for imaging dynamic mRNA processes in living cells.

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides "tiny molecular beacons". We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons and the flexibility to couple them to a large variety of fluorophores and quenchers render them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disk confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons and are able to access previously inaccessible target regions.

New cross-linking quinoline and quinolone derivatives for sensitive fluorescent labeling.

A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers-compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy.