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

Unlabelled: 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 .

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.

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.

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

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant 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 DNA.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Single-molecule imaging of transcriptionally coupled and uncoupled splicing.

Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing.

Tiny molecular beacons for in vivo mRNA detection.

The molecular beacon technology is an established approach for visualizing native mRNAs in living cells. These probes need to efficiently hybridize to accessible RNA regions in order to spatially and temporally resolve the dynamic steps of the RNA life cycle. A refined method using two computer algorithms, mfold and RNAstructure, is described for choosing shorter, more abundant target regions for molecular beacon binding.

Luminescent probes for ultrasensitive detection of nucleic acids.

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes.

Rapid universal identification of bacterial pathogens from clinical cultures by using a novel sloppy molecular beacon melting temperature signature technique.

A real-time PCR assay with the ability to rapidly identify all pathogenic bacteria would have widespread medical utility. Current real-time PCR technologies cannot accomplish this task due to severe limitations in multiplexing ability. To this end, we developed a new assay system which supports very high degrees of multiplexing.

Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes.

Background: Lyme disease, caused by Borrelia burgdorferi, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues.