Two Commercially Available Blood-Stabilization Reagents Serve as Potent Inactivators of Coronaviruses.

The continued circulation of SARS-CoV-2 and the increasing frequency of coronavirus (CoV) outbreaks over the decades demonstrates the enduring threat that the CoV family poses. There remains a significant need to develop tools to monitor and prevent the spread of these viruses. We tested blood-stabilization reagents from two commercially available blood collection tubes (BCTs) for their ability to inactivate three different coronaviruses (MHV, OC-43, and SARS-CoV-2) and stabilize their RNA. Both Cell-Free DNA BCT® (cfDNA) and Cyto-Chex® BCT (CytoChex) reagents reduced infectious virus in the buffer to below the limit of detection within 18 h of treatment, with some conditions showing this effect in as little as 3 h. CytoChex had more potent activity than cfDNA as in all cases it more rapidly reduced the actively replicating virus to the limit of detection. Despite the rapid inactivation of the virus, both reagents effectively preserved viral RNA for 7 days. Finally, both reagents accelerated viral inactivation in blood compared to the control samples. These results indicate that cfDNA and CytoChex could be used to inactivate and preserve CoV RNA for detection and further testing.

COMPARATIVE ANALYSIS OF HIP MUSCLE ACTIVATION DURING CLOSED-CHAIN REHABILITATION EXERCISES IN RUNNERS.

BACKGROUND: Increased hip adduction and internal rotation during the early stance phase of running have been linked to an increased risk of lower extremity injury. Both the gluteus maximus (GMAX) and gluteus medius (GMED) eccentrically control these motions. GMAX and GMED activation levels during commonly used rehabilitation exercises requires further exploration. HYPOTHESIS/PURPOSE: The purpose of this study was to compare peak surface electromyography (sEMG) amplitudes of GMAX and GMED between three closed-chain rehabilitation exercises: bilateral hip external rotation with resistance band (BER), forward lunge with resistance band (FL), and single-leg rotational squat (SLS). It was hypothesized that the FL would elicit greater peak amplitude in the GMAX and GMED than SLS and BER. STUDY DESIGN: Descriptive, observational cohort study. METHODS: Twenty-two healthy runners (14 male, 8 female) had sEMG electrodes placed bilaterally on GMAX and GMED. Participants completed three repetitions each of BER, FL, and SLS exercises with sEMG data normalized to the maximal amplitude recorded at each muscle during the running trial (% MRC). Seven inertial measurement units affixed to the lower extremity measured joint kinematics to enable the exercises to be split into eccentric and concentric phases respectively. RESULTS: There were no significant differences between exercises during the eccentric phases with all peak amplitudes for GMAX and GMED being less than < 30% MRC. Both the SLS (GMAX: 48.2 ± 45.2% MRC, p = 0.019; GMED: 39.3 ± 24.8% MRC, p < .001) and FL (GMAX: 65.8 ± 58.9% MRC, p < .001; GMED: 52.2 ± 34.9% MRC, p<.001) elicited significantly greater peak amplitudes than BER (GMAX: 21.7 ± 22.3% MRC; GMED: 22.8 ± 21.2% MRC) during the concentric phase. CONCLUSION: Running related injuries have been linked to deficits in GMAX and GMED activation and strength. When averaged bilaterally across 22 healthy runners, peak GMAX and GMED amplitudes during three weight bearing exercises were less than 70% MRC. All three exercises had comparable eccentric peak amplitudes; however, the BER exercise produced a significantly reduced GMAX and GMED amplitude during the concentric phase versus the FL and SLS. The FL and SLS appear equally effective at eliciting peak GMAX and GMED activation. LEVEL OF EVIDENCE: 3.

General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

Inhibition of insulin-like growth factor II (IGF-II)-dependent cell growth by multidentate pentamannosyl 6-phosphate-based ligands targeting the mannose 6-phosphate/IGF-II receptor.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) binds M6P-capped ligands and IGF-II at different binding sites within the ectodomain and mediates ligand internalization and trafficking to the lysosome. Multivalent M6P-based ligands can cross-bridge the M6P/IGF2R, which increases the rate of receptor internalization, permitting IGF-II binding as a passenger ligand and subsequent trafficking to the lysosome, where the IGF-II is degraded. This unique feature of the receptor may be exploited to design novel therapeutic agents against IGF-II-dependent cancers that will lead to decreased bioavailable IGF-II within the tumor microenvironment. We have designed a panel of M6P-based ligands that bind to the M6P/IGF2R with high affinity in a bivalent manner and cause decreased cell viability. We present evidence that our ligands bind through the M6P-binding sites of the receptor and facilitate internalization and degradation of IGF-II from conditioned medium to mediate this cellular response. To our knowledge, this is the first panel of synthetic bivalent ligands for the M6P/IGF2R that can take advantage of the ligand-receptor interactions of the M6P/IGF2R to provide proof-of-principle evidence for the feasibility of novel chemotherapeutic agents that decrease IGF-II-dependent growth of cancer cells.

PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target.

BACKGROUND: Most PCR-based diagnostics are still considered time- and labor-intensive due to disparate purification, amplification, and detection steps. Advancements in PCR enzymes and buffer chemistry have increased inhibitor tolerance, facilitating PCR directly from crude samples. Obviating the need for DNA purification, while lacking a concentration step, these direct sample methods are particularly apt for human genetic testing. However, direct PCR protocols have traditionally employed thermal cyclers with slow ramp rates and conservative hold times that significantly increase an assay's time-to-result. For this proof-of-principle study, our objective was to significantly reduce sample preparation and assay time for a PCR-based genetic test, for myotonic dystrophy type 1 (DM1), by pairing an inhibitor-resistant enzyme mix with a rapid thermal cycler to analyze samples directly in whole blood. METHODS: DM1 genetic screening was done with an adapted conventional PCR approach that employed the Streck Philisa® Thermal Cycler, the inhibitor-resistant NEBNext® High-Fidelity 2X PCR Master Mix, and agarose gel electrophoresis or an Agilent 2100 Bioanalyzer for detection. The Gene Link™ Myotonic Dystrophy Genemer™ Kit was used as a reference assay kit to evaluate the rapid assay. RESULTS: In this work, a rapid and direct PCR assay testing 10% whole blood as template has been developed as an exclusionary screening assay for DM1, a triple-repeat genetic disorder. PCR amplification was completed in 15 minutes using 30 cycles, including in situ hot-start/cell lysis. Out of the 40 donors screened, this assay identified 23 (57.5%) as DM1 negative suggesting no need for further testing. These data are 100% concordant with data collected using the commercially available Gene Link Genemer™ Kit per the kit-specific PCR protocol. CONCLUSIONS: The PCR assay described in this study amplified DM1 short tandem repeats in 15 minutes. By eliminating sample purification and slower conventional PCR protocols, we demonstrated how adaptation of current PCR technology and chemistries can produce a simple-to-use exclusionary screening assay that is independent of up-front sample prep, improving a clinical lab technician's time-to-result. We envision this direct and rapid methodology could be applied to other conventional PCR-based genetic tests and sample matrices where genomic DNA is targeted for analysis within a given molecular diagnostic platform.

Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer.

Oligomerization of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II.

A set of phosphatase-inert "molecular rulers" to probe for bivalent mannose 6-phosphate ligand-receptor interactions.

A set of bivalent mannose 6-phosphonate 'molecular rulers' has been synthesized to examine ligand binding to the M6P/IGF2R. The set is estimated to span a P-P distance range of 16-26A (MMFF energy minimization on the hydrated phosphonates). Key synthetic transformations include sugar triflate displacement for phosphonate installation and Grubbs I cross-metathesis to achieve bivalency. Relative binding affinities were tested by radioligand displacement assays versus PMP-BSA (pentamannosyl phosphate-bovine serum albumin). These compounds exhibit slightly higher binding affinities for the receptor (IC(50)'s=3.7-5 microM) than the parent, monomeric mannose 6-phosphonate ligand and M6P itself (IC(50)=11.5+/-2.5 microM). These results suggest that the use of an alpha-configured anomeric alkane tether is acceptable, as no significant thermodynamic penalty is apparently paid with this design. On the other hand, the modest gains in binding affinity observed suggest that this ligand set has not yet found true bivalent interaction with the M6P/IGF2R (i.e., simultaneous binding to two distinct M6P-binding pockets).