An intranasal ASO therapeutic targeting SARS-CoV-2

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 “variants of concern” tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics

A novel STING-activating vaccine for M. tuberculosis induces pathogen-specific Th17 cells and provides significant IL-17-dependent protection against infection

Before the emergence of SARS-CoV-2 and the ensuing COVID-19 pandemic, the pathogenic bacteria Mycobacterium tuberculosis was the world’s leading infectious disease killer. In 2020, an estimated 1.6 – 1.8 million people worldwide died of tuberculosis (TB) disease, comparable to the ~1.9 million lives claimed by COVID-19. However, while highly effective vaccines were rapidly developed for COVID-19, the sole approved TB vaccine, Bacillus Calmette-Guérin (BCG), is only moderately effective at blocking infection and has not prevented the TB pandemic despite 100 years of use and more widespread distribution that any vaccine in history. This highlights the urgent need for improved vaccines to prevent TB, which begins with a more complete understanding of the protective immune response to TB and the development of new strategies to effectively induce this immune response in patients. Here, we demonstrate a novel TB vaccine strategy using small molecule cyclic di-nucleotide (CDN) adjuvants to activate the STING pathway of the innate immune system. Subcutaneous administration with a CDN adjuvanted protein subunit vaccine for TB leads to protection in mice that is equivalent to that afforded by BCG. This protective efficacy is STING-dependent but Type I IFN-independent and correlates with a significant population of Th17 cells that are not present in the lungs of unvaccinated mice. Similarly, intranasal immunization leads to a multifaceted pathogen-specific CD4 T cell response characterized by both Th1 and Th17 cells, as well as a non-classical Th1-Th17 population that co-expresses IFN-γ and IL-17. Intranasally vaccinated mice also benefit from enhanced CD4 T cell localization to the lung parenchyma and homing to granulomatous lung lesions compared to unvaccinated mice. This distinct CD4 T cell response leads to protection against infection that is significantly greater than BCG and is more effective than any protein subunit vaccine tested against M. tuberculosis in mice to date. Full protective efficacy after intranasal immunization is dependent on both IFN-γ and IL-17 and, in contrast to subcutaneous administration, relies on Type I IFN signaling. Importantly, intranasal immunization with a CDN vaccine is also capable of boosting BCG-mediated immunity. These results demonstrate that stimulation of the STING pathway is sufficient to induce a protective CD4 T cell response to an intracellular pathogen and contribute to our knowledge of CD4 T cell subsets that can mediate protection against M. tuberculosis infection

LONGITUDINAL CHANGES IN SHOULDER ROM AND STRENGTH IN ASSOCIATION WITH BALL-THROWING SPEED IN ELITE YOUTH BASEBALL PITCHERS

The purpose of this prospective study was to examine the longitudinal changes in shoulder ER ROM and relative IR strength in elite youth baseball pitchers over one year and to determine their associations with changes in ball speed for throwing fastballs. One hundred and five Dutch elite youth baseball pitchers were measured three times over a period of one year. Statistical analyses of the data revealed that changes in ER ROM and relative IR strength were not significantly associated with changes in ball-throwing speed

Asymmetry and evolution over a one-year period of the upward rotation of the scapula in youth baseball pitchers

The pitching motion is an asymmetric action by which coordination of scapular rotation in the dominant arm might be affected in time and in comparison with the non-dominant arm. The study aimed to compare asymmetry and the evolution of scapular upward rotation over a one-year period. Data were collected twice, before and after a one-year period, from 92 participants (age = 15.1 SD 1.4 years, body height = 177.3 SD 10.9 cm, body weight 69.2 SD 14.5 kg). Scapular motion was tracked at different glenohumeral angles of elevation in the scapular plane: anatomical position (0°), 45°, 90° and 135°. Scapular upward rotation was calculated as the angle between the spinae scapula and the spine. Scapular upward rotation of the dominant arm was 5.1° (95% CI: 2.1°−8.1°) more compared to the non-dominant arm. Age group or glenohumeral angles of elevation did not affect this difference. Scapular upward rotation of the dominant arm decreased 1.9° (95% CI: −0.5° to 4.3°) after a one-year period, however, neither this observation, nor the interaction with age group or elevation angle was significant. These findings may indicate that pitchers could be at risk to develop shoulder injuries especially those that have been associated with scapular asymmetry

Release from UNC93B1 reinforces the compartmentalized activation of select TLRs.

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate the recognition of microbial DNA and RNA while limiting the recognition of an organism's own nucleic acids1. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory diseases2-6. Intracellular localization of these receptors is thought to be crucial for the discrimination between self and non-self7, but the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a mechanism that prevents the activation of TLR9 from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing release of TLR9 from UNC93B1, either by mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. Whereas TLR9 and TLR3 are released from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated by distinct mechanisms. This work defines a checkpoint that reinforces the compartmentalized activation of TLR9, and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated

Cross-Cultural Adaption and Validation of the Dutch Version of the Kerlan-Jobe Orthopaedic Clinic Questionnaire in Juvenile Baseball Pitchers

Monitoring the performance and functional status of baseball pitchers’ upper extremity is important in maintaining the athlete’s health and performance. This study validated a Dutch translation of the original English Kerlan-Jobe Orthopaedic Clinic (KJOC) against the previously validated Disabilities of the Arm, Shoulder and Hand (DASH) and Western Ontario Shoulder Instability Index (WOSI) questionnaires in a group of talented juvenile Dutch baseball pitchers. Three times, from 2014–2016, 107 pitchers completed the Dutch KJOC, DASH and WOSI questionnaires. Participants’ questionnaire scores were analysed for the whole group and the symptomatic player subgroup separately. Internal consistency, construct validity and ceiling and floor effects were examined. Cronbach’s alpha was consistently above 0.8 for the three time periods for the whole group, and ranged between 0.62 and 0.86 for the symptomatic subgroup. Spearman’s rank correlation coefficients ranged from 0.47 to 0.67 for the whole group and 0.32 to 0.99 for the symptomatic subgroup. No floor effects were observed in the scores of the KJOC and only a ceiling effect for the whole group (15.2%) at one time period. The Dutch version of the KJOC has shown acceptable internal consistency and construct validity and can be used to assess overhead athletes’ shoulder and elbow functionality.Biomechatronics & Human-Machine Contro

Inexpensive, versatile and open-source methods for SARS-CoV-2 detection

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited a second wave of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. To this end, we evaluated several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, we show that isopropanol precipitation provides an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, we evaluate direct addition of NP swab samples to RT-qPCR reactions without an RNA extraction step. We describe a simple, inexpensive swab collection solution suitable for direct addition, which we validate using contrived swab samples. Third, we describe an open-source master mix for RT-qPCR and show that it permits detection of viral RNA in NP swab samples. Lastly, we show that an end-point fluorescence measurement provides an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, inexpensive methods has the potential to significantly reduce the time and expense of COVID-19 testing

IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α.

The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-γ to activate bactericidal mechanisms in infected macrophages. Although IFN-γ-independent CD4 T cell based control of M. tuberculosis infection has been demonstrated in vivo it is unclear whether CD4 T cells are capable of directly activating macrophages to control infection in the absence of IFN-γ. We developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) to investigate mechanisms of CD4 dependent control of infection. We found that even in the absence of IFN-γ signaling, CD4 T cells drive macrophage activation, M1 polarization, and control of infection. This IFN-γ-independent control of infection requires activation of the transcription factor HIF-1α and a shift to aerobic glycolysis in infected macrophages. While HIF-1α activation following IFN-γ stimulation requires nitric oxide, HIF-1α-mediated control in the absence of IFN-γ is nitric oxide-independent, indicating that distinct pathways can activate HIF-1α during infection. We show that CD4 T cell-derived GM-CSF is required for IFN-γ-independent control in BMDMs, but that recombinant GM-CSF is insufficient to control infection in BMDMs or alveolar macrophages and does not rescue the absence of control by GM-CSF-deficient T cells. In contrast, recombinant GM-CSF controls infection in peritoneal macrophages, induces lipid droplet biogenesis, and also requires HIF-1α for control. These results advance our understanding of CD4 T cell-mediated immunity to M. tuberculosis, reveal important differences in immune activation of distinct macrophage types, and outline a novel mechanism for the activation of HIF-1α. We establish a previously unknown functional link between GM-CSF and HIF-1α and provide evidence that CD4 T cell-derived GM-CSF is a potent bactericidal effector

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing