Cephem-Pyrazinoic Acid Conjugates: Circumventing Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. ß-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of ß-lactamases that destroy the electrophilic ß-lactam warhead. We have developed novel ß-lactam conjugates, which exploit this inherent ß-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the ß-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.

Synthesis and biological evaluation of orally active prodrugs and analogs of para-aminosalicylic acid (PAS).

Tuberculosis (TB) is one of the world's most deadly infectious diseases resulting in nearly 1.3 million deaths annually and infecting nearly one-quarter of the population. para-Aminosalicylic acid (PAS), an important second-line agent for treating drug-resistant Mycobacterium tuberculosis, has moderate bioavailability and rapid clearance that necessitate high daily doses of up to 12 g per day, which in turn causes severe gastrointestinal disturbances presumably by disruption of gut microbiota and host epithelial cells. We first synthesized a series of alkyl, acyloxy and alkyloxycarbonyloxyalkyl ester prodrugs to increase the oral bioavailability and thereby prevent intestinal accumulation as well as undesirable bioactivation by the gut microbiome to non-natural folate species that exhibit cytotoxicity. The pivoxyl prodrug of PAS was superior to all of the prodrugs examined and showed nearly quantitative absorption. While the conceptually simple prodrug approach improved the oral bioavailability of PAS, it did not address the intrinsic rapid clearance of PAS mediated by N-acetyltransferase-1 (NAT-1). Thus, we next modified the PAS scaffold to reduce NAT-1 catalyzed inactivation by introduction of groups to sterically block N-acetylation and fluorination of the aryl ring of PAS to attenuate N-acetylation by electronically deactivating the para-amino group. Among the mono-fluorinated analogs prepared, 5-fluoro-PAS, exhibited the best activity and an 11-fold decreased rate of inactivation by NAT-1 that translated to a 5-fold improved exposure as measured by area-under-the-curve (AUC) following oral dosing to CD-1 mice. The pivoxyl prodrug and fluorination at the 5-position of PAS address the primary limitations of PAS and have the potential to revitalize this second-line TB drug.

Numbers matter: how irruptive bark beetles initiate transition to self-sustaining behavior during landscape-altering outbreaks.

Irruptive forest insects such as bark beetles undergo intermittent outbreaks that cause landscape-scale tree mortality. Despite their enormous economic and ecological impacts, we still have only limited understanding of the dynamics by which populations transition from normally stable endemic to irruptive densities. We investigated density-dependent changes in mountain pine beetle reliance on stressed hosts, host selection, spatial configuration of attacks, and the interaction of host selection and spatial configuration by performing a complete census of lodgepole pine across six stands and 6 years. In addition, we compared the dynamics of mountain pine beetle with those of other bark beetles. We found that as population size increased, reliance on stressed trees decreased and new attacks shifted to larger trees with thicker phloem and higher growth rates that can support higher offspring production. Moreover, the spatial configuration of beetle-attacked trees shifted from random to spatially aggregated. Further, we found evidence that beetle utilization of larger trees was related to aggregation behavior as the size of tree attacked was positively correlated at 10-25 m, within the effective distance of pheromone-mediated signaling. In contrast, non-irruptive bark beetle species did not exhibit such density-dependent spatial aggregation at the stand scale or switches in host selection behavior. These results identify how density-dependent linkages between spatial configuration and host utilization can converge to drive population transitions from endemic to irruptive phases. Specifically, a combination of stand-level spatial aggregation, behavioral shifts, and higher quality of attainable hosts defines a critical threshold beyond which continual population growth becomes self-driving.

When discretionary boundary spanning relationships cease becoming discretionary: The impact of closed ties on informal leadership perceptions.

Organizations have recognized that effective informal leadership is a source of competitive advantage and invest heavily in leadership development efforts. Moreover, because of historical shifts in the nature of work, this informal leadership often takes the form of inter-unit boundary spanning. Because of these two developments, discretionary boundary spanning (DBS) between units has increasingly become a critical, dynamic, bottom-up activity where individuals lacking formal authority step up and take on informal leadership responsibilities. In this study, we draw upon Simmelian Tie Theory (STT) to examine the relationship between different types of DBS and formal leaders' perceptions of a subordinate's informal leadership and performance. We empirically document that a small number of closed task-oriented and closed friendship-oriented DBSs are instrumental in helping individuals demonstrate informal leadership. However, we also show that DBS places constraints on informal leadership when closed ties become too numerous. This results in an inverted-U relationship between the number of closed DBS ties and perceptions of leadership where the apex (i.e., point of over-embeddedness) emerges at a smaller number for friendship-oriented DBS relative to the apex for task-oriented DBSs. We discuss the theoretical implications of these results, as well as the practical implications for managers of organizations. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Climate-induced outbreaks in high-elevation pines are driven primarily by immigration of bark beetles from historical hosts.

Warming temperatures are allowing native insect herbivores to expand into regions that previously exceeded their thermal tolerance, encounter new host species, and pose significant threats to native communities. However, the dynamics of these expansions remain poorly understood, particularly in the extent to which outbreaks remain reliant on emigration from historical hosts or are driven by local reproduction within novel hosts in the expanded range. We tested these non-mutually exclusive hypotheses using spatially explicit data on mountain pine beetle (Dendroctonus ponderosae), which historically undergoes intermittent outbreaks in low-elevation lodgepole pine (Pinus contorta), but is now causing severe mortality in a high-elevation endangered species, whitebark pine (Pinus albicaulis). We compiled data from 2000 to 2019 across British Columbia, Canada, at 1-km2 resolution, and analyzed spatiotemporal patterns of beetle infestations, lodgepole pine distributions, expansion into habitats dominated by whitebark pine, and the likelihood of future outbreaks in all pine communities under simulated conditions. Overall, we found strong support for the hypothesis of emigration from the historical host species continuing to be a major driver of outbreaks in the more recently accessed host. First, beetle population pressure was consistently the best predictor of infestation severity in both lodgepole and whitebark pine, and appeared to be mostly unidirectional from lodgepole to whitebark pine. Second, infestations in lodgepole pine were of a longer duration than those in whitebark pine, which appeared too brief to sustain transitions from endemic to eruptive dynamics. Furthermore, resource depletion appears to drive emigration from lodgepole pine, whereas in whitebark pine drought appears to favor establishment of immigrants although bioclimatic factors and stand structure preclude self-sustaining outbreaks. Finally, we project that most pine in British Columbia will be at risk in the event of a new major outbreak. We describe implications for conserving and protecting whitebark pine and to other climate-driven range expansions.

Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry.

Precision antimicrobials aim to kill pathogens without damaging commensal bacteria in the host, and thereby cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of a synthetic host defence peptide that targets a specific pathogen by mimicking key molecular features of the pathogen's channel-forming membrane proteins. By exploiting physical and structural vulnerabilities within the pathogen's cellular envelope, we designed a peptide sequence that undergoes instructed tryptophan-zippered assembly within the mycolic acid-rich outer membrane of Mycobacterium tuberculosis to specifically kill the pathogen without collateral toxicity towards lung commensal bacteria or host tissue. These mycomembrane-templated assemblies elicit rapid mycobactericidal activity and enhance the potency of antibiotics by improving their otherwise poor diffusion across the rigid M. tuberculosis envelope with respect to agents that exploit transmembrane protein channels for antimycobacterial activity. This biomimetic strategy may aid the design of other narrow-spectrum antimicrobial peptides.

Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis.

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

The formyl peptide receptor agonist FPRa14 induces differentiation of Neuro2a mouse neuroblastoma cells into multiple distinct morphologies which can be specifically inhibited with FPR antagonists and FPR knockdown using siRNA.

The N-formyl peptide receptors (FPRs) have been identified within neuronal tissues and may serve as yet undetermined functions within the nervous system. The FPRs have been implicated in the progression and invasiveness of neuroblastoma and other cancers. In this study the effects of the synthetic FPR agonist FPRa14, FPR antagonists and FPR knockdown using siRNA on mouse neuroblastoma neuro2a (N2a) cell differentiation plus toxicity were examined. The FPRa14 (1-10µM) was found to induce a significant dose-dependent differentiation response in mouse neuroblastoma N2a cells. Interestingly, three distinct differentiated morphologies were observed, with two non-archetypal forms observed at the higher FPRa14 concentrations. These three forms were also observed in the human neuroblastoma cell-lines IMR-32 and SH-SY5Y when exposed to 100µM FPRa14. In N2a cells combined knockdown of FPR1 and FPR2 using siRNA inhibited the differentiation response to FPRa14, suggesting involvement of both receptor subtypes. Pre-incubating N2a cultures with the FPR1 antagonists Boc-MLF and cyclosporin H significantly reduced FPRa14-induced differentiation to near baseline levels. Meanwhile, the FPR2 antagonist WRW4 had no significant effect on FPRa14-induced N2a differentiation. These results suggest that the N2a differentiation response observed has an FPR1-dependent component. Toxicity of FPRa14 was only observed at higher concentrations. All three antagonists used blocked FPRa14-induced toxicity, whilst only siRNA knockdown of FPR2 reduced toxicity. This suggests that the toxicity and differentiation involve different mechanisms. The demonstration of neuronal differentiation mediated via FPRs in this study represents a significant finding and suggests a role for FPRs in the CNS. This finding could potentially lead to novel therapies for a range of neurological conditions including neuroblastoma, Alzheimer's disease, Parkinson's disease and neuropathic pain. Furthermore, this could represent a potential avenue for neuronal regeneration therapies.

Spirocyclic and Bicyclic 8-Nitrobenzothiazinones for Tuberculosis with Improved Physicochemical and Pharmacokinetic Properties.

8-Nitrobenzothiazinones (BTZs) typified by the second-generation analogue PBTZ169 are a new class of antitubercular agents. The activity of BTZs and lipophilicity are tightly coupled since the molecular target DprE1 is located in the mycobacterial cell envelope. A series of analogues was designed to address the notorious insolubility of the BTZs while preserving the required lipophilicity. This was accomplished by decreasing the molecular planarity and symmetry through bioisosteric replacement of the piperazine moiety of PBTZ169 with spirocyclic and bicyclic diamines. Several promising compounds with improved aqueous solubilities were identified with potent antitubercular activity. Compound 5 was identified as the most promising candidate based on its excellent antitubercular activity (MIC of 32 nM), more than 1000-fold improvement in solubility, 2-fold lower clearance in mouse and human microsomes relative to PBTZ169, and promising pharmacokinetic parameters.

Investigation of ( S)-(-)-Acidomycin: A Selective Antimycobacterial Natural Product That Inhibits Biotin Synthase.

The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of ( S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 µM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 µM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a Ki of approximately 1 µM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5'-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.

General mental ability and goal type as antecedents of recurrent adaptive task performance.

Even though considerable work has demonstrated a robust positive relationship between general mental ability (GMA) and task performance, recent work indicates that the expected relationship may not hold in the context of adaptive performance. By integrating the concept of choking, or performing worse than expected, with goal theory, the present work advances a theoretical framework aimed at furthering our understanding of how and when GMA is most likely to meaningfully impact performance. Drawing on this perspective, we propose that the relationship between GMA and adaptive performance is uniquely dependent on the type of goal individuals are striving to achieve. Additionally, we note that the nature of this relationship may evolve as people gain experience dealing with unexpected changes. Results of a discontinuous growth model fit to data obtained from a stock market exercise generally indicate that compared with performance goals, do-your-best and learning goals strengthen the relationship between GMA and adaptive performance. Further, we find that performance goals seem to effectively neutralize the GMA-adaptive performance relationship by benefiting those lower on GMA while simultaneously hindering those with higher levels. In contrast, the relationship is largely positive when either a do-your-best or a learning goal is being pursued, particularly after individuals are exposed to a second change. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Wearable bluetooth sensors for capturing relational variables and temporal variability in relationships: A construct validation study.

The advent of wearable sensor technologies has the potential to transform organizational research by offering the unprecedented opportunity to collect continuous, objective, highly granular data over extended time periods. Recent evidence has demonstrated the potential utility of Bluetooth-enabled sensors, specifically, in identifying emergent networks via colocation signals in highly controlled contexts with known distances and groups. Although there is proof of concept that wearable Bluetooth sensors may be able to contribute to organizational research in highly controlled contexts, to date there has been no explicit psychometric construct validation effort dedicated to these sensors in field settings. Thus, the two studies described here represent the first attempt to formally evaluate longitudinal Bluetooth data streams generated in field settings, testing their ability to (a) show convergent validity with respect to traditional self-reports of relational data; (b) display discriminant validity with respect to qualitative differences in the nature of alternative relationships (i.e., advice vs. friendship); (c) document predictive validity with respect to performance; (d) decompose variance in network-related measures into meaningful within- and between-unit variability over time; and (e) complement retrospective self-reports of time spent with different groups where there is a "ground truth" criterion. Our results provide insights into the validity of Bluetooth signals with respect to capturing variables traditionally studied in organizational science and highlight how the continuous data collection capabilities made possible by wearable sensors can advance research far beyond that of the static perspectives imposed by traditional data collection strategies. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Methionine Antagonizes para-Aminosalicylic Acid Activity via Affecting Folate Precursor Biosynthesis in Mycobacterium tuberculosis.

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as, para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter, designated MetM, play crucial roles in this process. Disruption of metM by transposon insertion resulted in a ≥30-fold decrease in uptake of methionine in M. bovis BCG, indicating that metM is the major facilitator of methionine transport. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.

Pine Engravers Carry Bacterial Communities Whose Members Reduce Concentrations of Host Monoterpenes With Variable Degrees of Redundancy, Specificity, and Capability.

Bark beetles are eruptive forest insects that have the potential to cause landscape level mortality to conifer forests. The pine engraver, Ips pini (Say) (Coleoptera: Curculionidae), is the predominant pest of mature red pine (Pinus resinosa Aiton) plantations throughout the Great Lakes region of North America. Pine engraver attack elicits a localized response by host trees in which concentrations of terpenes rapidly exceed the tolerance levels of beetles and their fungal associates. We considered how bacterial associates degrade these toxins from the perspective of the symbiont communities of individual beetles. We demonstrate that 1) most pine engravers harbor bacterial communities that reduce monoterpene concentrations in vivo; 2) several individual bacterial isolates can reduce monoterpenes even at high concentrations; and 3) bacteria isolated from pine engravers are similar to those found in other bark beetles. Bacteria isolated from pine engravers decreased concentrations of (-)-α-pinene, myrcene, and 3-carene. Most beetles carried at least one bacterial isolate that reduced concentrations of at least one monoterpene. Different bacteria vary in the uppermost concentrations at which they can degrade monoterpenes. The community of bacteria associated with an individual beetle appears to have some manner of functional redundancy that could collectively increase the likelihood of successful host colonization.

Imagining tomorrow's university in an era of open science.

As part of a recent workshop entitled "Imagining Tomorrow's University", we were asked to visualize the future of universities as research becomes increasingly data- and computation-driven, and identify a set of principles characterizing pertinent opportunities and obstacles presented by this shift. In order to establish a holistic view, we take a multilevel approach and examine the impact of open science on individual scholars as well as on the university as a whole. At the university level, open science presents a double-edged sword: when well executed, open science can accelerate the rate of scientific inquiry across the institution and beyond; however, haphazard or half-hearted efforts are likely to squander valuable resources, diminish university productivity and prestige, and potentially do more harm than good. We present our perspective on the role of open science at the university.

Personality similarity in negotiations: Testing the dyadic effects of similarity in interpersonal traits and the use of emotional displays on negotiation outcomes.

We build on the small but growing literature documenting personality influences on negotiation by examining how the joint disposition of both negotiators with respect to the interpersonal traits of agreeableness and extraversion influences important negotiation processes and outcomes. Building on similarity-attraction theory, we articulate and demonstrate how being similarly high or similarly low on agreeableness and extraversion leads dyad members to express more positive emotional displays during negotiation. Moreover, because of increased positive emotional displays, we show that dyads with such compositions also tend to reach agreements faster, perceive less relationship conflict, and have more positive impressions of their negotiation partner. Interestingly, these results hold regardless of whether negotiating dyads are similar in normatively positive (i.e., similarly agreeable and similarly extraverted) or normatively negative (i.e., similarly disagreeable and similarly introverted) ways. Overall, these findings demonstrate the importance of considering the dyad's personality configuration when attempting to understand the affective experience as well as the downstream outcomes of a negotiation. (PsycINFO Database Record

How functional traits, herbivory, and genetic diversity interact in Echinacea: implications for fragmented populations.

Habitat fragmentation produces small, spatially isolated populations that promote inbreeding. Remnant populations often contain inbred and outbred individuals, but it is unclear how inbreeding relative to outbreeding affects the expression of functional traits and biotic interactions such as herbivory. We measured a suite of 12 functional traits and herbivore damage on three genotypic cross types in the prairie forb, Echinacea angustifolia: inbred, and outbred crosses resulting from matings within and between remnant populations. Inbreeding significantly affected the expression of all 12 functional traits that influence resource capture. Inbred individuals had consistently lower photosynthetic rates, water use efficiencies, specific leaf areas, and had higher trichome numbers, percent C, and percent N than outbred individuals. However, herbivore damage did not differ significantly among the cross types and was not correlated with other leaf functional traits. Leaf architecture and low physiological rates of the inbred compared to outbred individuals imply poorer capture or use of resources. Inbred plants also had lower survival and fitness relative to outbred plants. Our results show that inbreeding, a phenomenon predicted and observed to occur in fragmented populations, influences key functional traits such as plant structure, physiology and elemental composition. Because of their likely role in fitness of individuals and ecological dynamics plant functional traits can serve as a bridge between evolution and community or ecosystem ecology.

Underutilization of Radial Access in Patients Undergoing Percutaneous Coronary Intervention for ST-Segment-Elevation Myocardial Infarction: Insights From the Blue Cross Blue Shield of Michigan Cardiovascular Consortium.

BACKGROUND: The purpose of this study was to evaluate the frequency and temporal trends in use of transradial access (TRA) for percutaneous coronary intervention (PCI) in ST-segment-elevation myocardial infarction (STEMI). The use of TRA has been associated with less bleeding and improved clinical outcomes in patients undergoing PCI for STEMI. METHODS AND RESULTS: The frequency of TRA compared with transfemoral access for patients undergoing PCI for STEMI or other indications (non-ST-segment-elevation myocardial infarction, unstable angina, and non-acute coronary syndrome) in The Blue Cross Blue Shield of Michigan Cardiovascular Consortium database between 2010 and 2013 was evaluated. Propensity matching was used to assess the relationship of TRA with in-hospital clinical end points of major bleeding, transfusion, and death. The TRA cohort of patients was stratified into deciles based on their predicted bleeding risk and compared with PCI indication. Of 122,728 PCI procedures, 17,912 (14.6%) were via TRA. Among patients with STEMI cases, 8.3% of the PCI cases were performed via TRA. The use of TRA increased over the study period although the growth was slower for STEMI than for other indications, P<0.001. The use of TRA for PCI in STEMI was associated with a lower rate of bleeding (11.7% versus 20.0%; P<0.001) and vascular complications (0.7% versus 2.6%; P=0.001), but no mortality difference (1.25% versus 2.33%; P=0.175). There was a strong negative association between the predicted risk of bleeding and the use of TRA (P<0.001). CONCLUSIONS: The use of radial access for PCI in STEMI is increasing but at a slower pace than for patients with other indications. TRA was associated with a reduction in bleeding and transfusion, but there is a strong negative correlation between the predicted risk of bleeding and actual use of TRA in STEMI.

Cor triatriatum: a reversible cause of severe pulmonary hypertension.

Cor triatriatum sinister is a congenital heart disorder that can lead to progressive dyspnea, pulmonary hypertension, and ultimately right ventricular (RV) failure. We report a case in which symptoms of progressive pulmonary hypertension were initially attributed to asthma, leading to a delayed diagnosis that resulted in suprasystemic pulmonary pressures and RV dysfunction. Rapid symptomatic and hemodynamic improvement was observed after surgical repair, with normalization of pulmonary artery pressures and RV function.

Small sample mediation testing: misplaced confidence in bootstrapped confidence intervals.

Bootstrapping is an analytical tool commonly used in psychology to test the statistical significance of the indirect effect in mediation models. Bootstrapping proponents have particularly advocated for its use for samples of 20-80 cases. This advocacy has been heeded, especially in the Journal of Applied Psychology, as researchers are increasingly utilizing bootstrapping to test mediation with samples in this range. We discuss reasons to be concerned with this escalation, and in a simulation study focused specifically on this range of sample sizes, we demonstrate not only that bootstrapping has insufficient statistical power to provide a rigorous hypothesis test in most conditions but also that bootstrapping has a tendency to exhibit an inflated Type I error rate. We then extend our simulations to investigate an alternative empirical resampling method as well as a Bayesian approach and demonstrate that they exhibit comparable statistical power to bootstrapping in small samples without the associated inflated Type I error. Implications for researchers testing mediation hypotheses in small samples are presented. For researchers wishing to use these methods in their own research, we have provided R syntax in the online supplemental materials.