Does Gratitude Promote Resilience During a Pandemic? An Examination of Mental Health and Positivity at the Onset of COVID-19.

Researchers have emphasized the detrimental effects of COVID-19 on mental health, but less attention has been given to personal strengths promoting resilience during the pandemic. One strength might be gratitude, which supports wellbeing amidst adversity. A two-wave examination of 201 college students revealed anxiety symptom severity increased to a lesser extent from pre-COVID (January-March 2020) to onset-COVID (April 2020) among those who reported greater pre-COVID gratitude. A similar trend appeared for depression symptom severity. Gratitude was also correlated with less negative changes in outlook, greater positive changes in outlook, and endorsement of positive experiences resulting from COVID-19. Thematic analysis showed "strengthened interpersonal connections" and "more time" were the most commonly reported positive experiences. Overall findings suggest gratitude lessened mental health difficulties and fostered positivity at the onset of the pandemic, but more research is needed to determine whether gratitude and other strengths promote resilience as COVID-19 continues.

Programmable siRNA pro-drugs that activate RNAi activity in response to specific cellular RNA biomarkers.

Since Paul Ehrlich's introduction of the "magic bullet" concept in 1908, drug developers have been seeking new ways to target drug activity to diseased cells while limiting effects on normal tissues. In recent years, it has been proposed that coupling riboswitches capable of detecting RNA biomarkers to small interfering RNAs (siRNAs) to create siRNA pro-drugs could selectively activate RNA interference (RNAi) activity in specific cells. However, this concept has not been achieved previously. We report here that we have accomplished this goal, validating a simple and programmable new design that functions reliably in mammalian cells. We show that these conditionally activated siRNAs (Cond-siRNAs) can switch RNAi activity against different targets between clearly distinguished OFF and ON states in response to different cellular RNA biomarkers. Notably, in a rat cardiomyocyte cell line (H9C2), one version of our construct demonstrated biologically meaningful inhibition of a heart-disease-related target gene protein phosphatase 3 catalytic subunit alpha (PPP3CA) in response to increased expression of the pathological marker atrial natriuretic peptide (NPPA) messenger RNA (mRNA). Our results demonstrate the ability of synthetic riboswitches to regulate gene expression in mammalian cells, opening a new path for development of programmable siRNA pro-drugs.

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications.

In-Play Cooling Interventions for Simulated Match-Play Tennis in Hot/Humid Conditions.

PURPOSE: This study aimed to assess the efficacy of different in-play cooling strategies for mitigating heat strain during simulated tennis match-play activity in a hot/humid environment representing the most extreme conditions during the US Open (36°C, 50% relative humidity). METHODS: On three occasions, nine males completed an intermittent treadmill protocol with an exercise intensity and activity profile simulating a four-set tennis match, with 90-s breaks between odd-numbered games and 120-s breaks between sets, according to International Tennis Federation rules. During breaks, 1) the currently used cooling strategy-an ice-filled damp towel around the neck and a cold-damp towel on the head and thighs (ICE); 2) wetting of arms, neck, face, and lower legs with a sponge in front of an electric fan (FANwet); or 3) no cooling (CON) were applied. Rectal (Tre) and mean skin (Tsk) temperature and HR were measured throughout. Thermal sensation and RPE were assessed during breaks. Trials were terminated upon reaching a Tre ≥ 39.5°C or volitional exhaustion. RESULTS: Seven, five, and one participant completed FANwet, ICE, and CON, respectively. By end set 1, ΔTre was lower in FANwet (0.92°C ± 0.15°C) compared with CON (1.09°C ± 0.09°C, P = 0.01), and by end set 2, ΔTre was lower (P < 0.001) in FANwet (1.55°C ± 0.23°C) and ICE (1.59°C ± 0.17°C) compared with CON (1.99°C ± 0.19°C). Mean RPE (FANwet = 13.9 ± 2.2, ICE = 13.6 ± 1.8, CON = 16.6 ± 1.8), HR (FANwet = 163 ± 21, ICE 164 ± 22, CON = 175 ± 19 bpm), Tsk (FANwet = 36.56°C ± 0.69°C, ICE 36.12°C ± 0.44°C, CON = 37.21°C ± 0.42°C), and thermal sensation were lower in FANwet and ICE (P < 0.05) compared with CON by end set 2. CONCLUSIONS: The currently recommended ICE strategy successfully mitigates thermal strain during simulated tennis match play in hot/humid conditions. The FANwet intervention is an equally effective alternative that may be more practical in limited resource settings.

Optimized lentiviral vectors for HIV gene therapy: multiplexed expression of small RNAs and inclusion of MGMT(P140K) drug resistance gene.

Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMT(P140K) marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

Interplay between HIV-1 infection and host microRNAs.

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

Ex vivo gene therapy for HIV-1 treatment.

Until recently, progress in ex vivo gene therapy (GT) for human immunodeficiency virus-1 (HIV-1) treatment has been incremental. Long-term HIV-1 remission in a patient who received a heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5(-/-) donor, even after discontinuation of conventional therapy, has energized the field. We review the status of current approaches as well as future directions in the areas of therapeutic targets, combinatorial strategies, vector design, introduction of therapeutics into stem cells and enrichment/expansion of gene-modified cells. Finally, we discuss recent advances towards clinical application of HIV-1 GT.

Optimization and characterization of tRNA-shRNA expression constructs.

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNA(Lys3)-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3' tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.

Targeting cellular genes with PCR cassettes expressing short interfering RNAs.

The synthesis and transfection of PCR short interfering/short hairpin RNA (si/shRNA) expression cassettes described in this chapter can be used to rapidly test siRNA targeting and function in cells. One critical element in the design of effective siRNAs is the selection of siRNA-target sequence combinations that yield the best inhibitory activity. This can be accomplished by using synthetic siRNAs and transfection procedures, but these can be costly and time consuming. By using the PCR strategy, it is possible to create several expression cassettes and simultaneously screen for the best target sites on any given mRNA. This PCR strategy allows a rapid and inexpensive approach for intracellular expression of si/shRNAs and subsequent testing of target site sensitivity to downregulation by RNA interference (RNAi).

Rapid assessment of anti-HIV siRNA efficacy using PCR-derived Pol III shRNA cassettes.

Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.

Recent applications of RNAi in mammalian systems.

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incorporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new therapeutic modality.

Approaches for the sequence-specific knockdown of mRNA.

Over the past 25 years there have been thousands of published reports describing applications of antisense nucleic acid derivatives for targeted inhibition of gene function. The major classes of antisense agents currently used by investigators for sequence-specific mRNA knockdowns are antisense oligonucleotides (ODNs), ribozymes, DNAzymes and RNA interference (RNAi). Whatever the method, the problems for effective application are remarkably similar: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. These challenges have been in existence from the first attempts to use antisense research tools, and need to be met before any antisense molecule can become widely accepted as a therapeutic agent.

Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.

We previously demonstrated that chimeric tRNA(Lys3)-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA(Lys3) promoter variants and compared their expression levels from the internal tRNA(Lys3) promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA(Lys3)-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts.