Micrococcin cysteine-to-thiazole conversion through transient interactions between the scaffolding protein TclI and the modification enzymes TclJ and TclN.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCE: Thiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

Homophily and social mixing in a small community: Implications for infectious disease transmission.

Community mixing patterns by sociodemographic traits can inform the risk of epidemic spread among groups, and the balance of in- and out-group mixing affects epidemic potential. Understanding mixing patterns can provide insight about potential transmission pathways throughout a community. We used a snowball sampling design to enroll people recently diagnosed with SARS-CoV-2 in an ethnically and racially diverse county and asked them to describe their close contacts and recruit some contacts to enroll in the study. We constructed egocentric networks of the participants and their contacts and assessed age-mixing, ethnic/racial homophily, and gender homophily. The total size of the egocentric networks was 2,544 people (n = 384 index cases + n = 2,160 recruited peers or other contacts). We observed high rates of in-group mixing among ethnic/racial groups compared to the ethnic/racial proportions of the background population. Black or African-American respondents interacted with a wider range of ages than other ethnic/racial groups, largely due to familial relationships. The egocentric networks of non-binary contacts had little age diversity. Black or African-American respondents in particular reported mixing with older or younger family members, which could increase the risk of transmission to vulnerable age groups. Understanding community mixing patterns can inform infectious disease risk, support analyses to predict epidemic size, or be used to design campaigns such as vaccination strategies so that community members who have vulnerable contacts are prioritized.

Social networks and HIV treatment adherence among people with HIV initiating treatment in rural Uganda and peri-urban South Africa.

Timely initiation of and adherence to antiretroviral therapy (ART) is critical for improving HIV outcomes and reducing HIV transmissibility. Social networks, or the social relationships individuals have with each other, have been linked with positive health outcomes, but less is known about the extent to which social network composition and structure are associated with improved ART adherence among people living with HIV (PLWH). We conducted an ego-centric network study among 828 previously ART-naïve PLWH presenting for ART initiation at 11 clinics in Mbarara, Uganda (rural population) and Gugulethu, South Africa (peri-urban population). We collected social network data using name generator and name interpreter questions. ART adherence was monitored over 12 months using wireless monitors (Wisepill). Our primary outcome of interest was ART adherence during the 12-month follow-up period. We used generalized linear models to estimate the associations between network measures and ART adherence. PLWH at the Uganda site (compared with the South Africa site) were less isolated, had larger social networks, and had more social ties providing sufficient social support; they were also more likely to bridge different social groups whereby not all social ties were connected to each other. In Uganda, social isolation was associated with a 5.5 percentage point reduction in ART adherence (95% confidence interval [CI] -9.95 to -1.13; p = 0.014), while having more same gender social ties was associated with higher ART adherence (b = 0.13, 95% CI 0.02-0.25, p = 0.025). In South Africa, there was no association between social isolation and ART adherence, and having more friendship ties (vs. family ties) was associated with lower ART adherence (b = -2.20, 95% CI -3.56 to -0.84; p = 0.002). Identifying and supporting PLWH who are isolated may facilitate optimal adherence, but understanding how networks differentially affect ART adherence by country context is important.

Micrococcin cysteine-to-thiazole conversion through transient interactions between a scaffolding protein and two modification enzymes.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core.

Increasing the bulk of the 1TEL-target linker and retaining the 10×His tag in a 1TEL-CMG2-vWa construct improves crystal order and diffraction limits.

TELSAM-fusion crystallization has the potential to become a revolutionary tool for the facile crystallization of proteins. TELSAM fusion can increase the crystallization rate and enable crystallization at low protein concentrations, in some cases with minimal crystal contacts [Nawarathnage et al. (2022), Open Biol. 12, 210271]. Here, requirements for the linker composition between 1TEL and a fused CMG2 vWa domain were investigated. Ala-Ala, Ala-Val, Thr-Val and Thr-Thr linkers were evaluated, comparing metrics for crystallization propensity and crystal order. The effect on crystallization of removing or retaining the purification tag was then tested. It was discovered that increasing the linker bulk and retaining the 10×His purification tag improved the diffraction resolution, likely by decreasing the number of possible vWa-domain orientations in the crystal. Additionally, it was discovered that some vWa-domain binding modes are correlated with scrambling of the 1TEL polymer orientation in crystals and an effective mitigation strategy for this pathology is presented.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1 ubiquitin-associated (UBA) domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. No experimentally determined molecular structure of this unusual UBA domain is available. We fused the UBA domain to the 1TEL variant of the translocation ETS leukemia protein sterile alpha motif (TELSAM) crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and crystallize at protein concentrations as low as 0.2 mg/mL. Our studies support a mechanism of 1TEL fusion crystallization and show that 1TEL fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1-UBA domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. Sequence analysis suggests an unusual architecture for the TNK1 UBA domain, but an experimentally-validated molecular structure is undetermined. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. A 1TEL search model enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our studies support a mechanism of TELSAM fusion crystallization and show that TELSAM fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Our Friends Keep Us Together: The Stability of Adolescents' Cross-Race Friendships.

Substantive racial integration depends on both access to cross-race friendship opportunities (demographic integration) and the development of stable and rewarding social relations (social integration). Yet, we know little about the relative stability of cross-race friendship nominations over time. Cross-race friendships are also experienced within social contexts, where other individual, dyadic, and contextual factors may simultaneously affect whether such ties persist. Based on longitudinal network data on over 2,000 students in multiple communities, we test whether cross-race friendships are more or less stable than same-race friendships. We find that cross-race friendships at first glance appear less likely to persist than same-race friendships, but cross-race ties become no less stable than same-race ties after accounting for other social factors, including reciprocity and shared friends. This pattern suggests a threshold process where strong, socially recognized ties embedded among peers face less threat to maintaining friendship stability.

Decreasing the flexibility of the TELSAM-target protein linker and omitting the cleavable fusion tag improves crystal order and diffraction limits.

TELSAM crystallization promises to become a revolutionary tool for the facile crystallization of proteins. TELSAM can increase the rate of crystallization and form crystals at low protein concentrations without direct contact between TELSAM polymers and, in some cases, with very minimal crystal contacts overall (Nawarathnage et al ., 2022). To further understand and characterize TELSAM-mediated crystallization, we sought to understand the requirements for the composition of the linker between TELSAM and the fused target protein. We evaluated four different linkers Ala-Ala, Ala-Val, Thr-Val, and Thr-Thr, between 1TEL and the human CMG2 vWa domain. We compared the number of successful crystallization conditions, the number of crystals, the average and best diffraction resolution, and the refinement parameters for the above constructs. We also tested the effect of the fusion protein SUMO on crystallization. We discovered that rigidification of the linker improved diffraction resolution, likely by decreasing the number of possible orientations of the vWa domains in the crystal, and that omitting the SUMO domain from the construct also improved the diffraction resolution. Synopsis: We demonstrate that the TELSAM protein crystallization chaperone can enable facile protein crystallization and high-resolution structure determination. We provide evidence to support the use of short but flexible linkers between TELSAM and the protein of interest and to support the avoidance of cleavable purification tags in TELSAM-fusion constructs.

Socioeconomic disparities in the prevalence of multidrug resistance in Enterobacterales.

We examined the association between multidrug resistance and socioeconomic status (SES), analyzing microbiological and ZIP-code-level socioeconomic data. Using generalized linear models, we determined that multidrug resistance is significantly and persistently more prevalent in samples taken from patients residing in low-income ZIP codes versus high-income ZIP codes in North Carolina.

Scalable Strategies to Increase Efficiency and Augment Public Health Activities During Epidemic Peaks.

OBJECTIVE: Scalable strategies to reduce the time burden and increase contact tracing efficiency are crucial during early waves and peaks of infectious transmission. DESIGN: We enrolled a cohort of SARS-CoV-2-positive seed cases into a peer recruitment study testing social network methodology and a novel electronic platform to increase contact tracing efficiency. SETTING: Index cases were recruited from an academic medical center and requested to recruit their local social contacts for enrollment and SARS-CoV-2 testing. PARTICIPANTS: A total of 509 adult participants enrolled over 19 months (384 seed cases and 125 social peers). INTERVENTION: Participants completed a survey and were then eligible to recruit their social contacts with unique "coupons" for enrollment. Peer participants were eligible for SARS-CoV-2 and respiratory pathogen screening. MAIN OUTCOME MEASURES: The main outcome measures were the percentage of tests administered through the study that identified new SARS-CoV-2 cases, the feasibility of deploying the platform and the peer recruitment strategy, the perceived acceptability of the platform and the peer recruitment strategy, and the scalability of both during pandemic peaks. RESULTS: After development and deployment, few human resources were needed to maintain the platform and enroll participants, regardless of peaks. Platform acceptability was high. Percent positivity tracked with other testing programs in the area. CONCLUSIONS: An electronic platform may be a suitable tool to augment public health contact tracing activities by allowing participants to select an online platform for contact tracing rather than sitting for an interview.

Computational engineering of previously crystallized pyruvate formate-lyase activating enzyme reveals insights into SAM binding and reductive cleavage.

Radical S-adenosyl-l-methionine (SAM) enzymes are ubiquitous in nature and carry out a broad variety of difficult chemical transformations initiated by hydrogen atom abstraction. Although numerous radical SAM (RS) enzymes have been structurally characterized, many prove recalcitrant to crystallization needed for atomic-level structure determination using X-ray crystallography, and even those that have been crystallized for an initial study can be difficult to recrystallize for further structural work. We present here a method for computationally engineering previously observed crystallographic contacts and employ it to obtain more reproducible crystallization of the RS enzyme pyruvate formate-lyase activating enzyme (PFL-AE). We show that the computationally engineered variant binds a typical RS [4Fe-4S]2+/+ cluster that binds SAM, with electron paramagnetic resonance properties indistinguishable from the native PFL-AE. The variant also retains the typical PFL-AE catalytic activity, as evidenced by the characteristic glycyl radical electron paramagnetic resonance signal observed upon incubation of the PFL-AE variant with reducing agent, SAM, and PFL. The PFL-AE variant was also crystallized in the [4Fe-4S]2+ state with SAM bound, providing a new high-resolution structure of the SAM complex in the absence of substrate. Finally, by incubating such a crystal in a solution of sodium dithionite, the reductive cleavage of SAM is triggered, providing us with a structure in which the SAM cleavage products 5'-deoxyadenosine and methionine are bound in the active site. We propose that the methods described herein may be useful in the structural characterization of other difficult-to-resolve proteins.

Demography's Changing Intellectual Landscape: A Bibliometric Analysis of the Leading Anglophone Journals, 1950-2020.

Much of what we know about the intellectual landscape of anglophone demography comes from two sources: subjective narratives authored by leaders in the field, whose reviews and observations are derived from their research experience and field-specific knowledge; and professional histories covering the field's foundational controversies, which tend to focus on individuals, institutions, and influence. Here we use bibliographic information from all articles published in the three leading journals of anglophone demography-Demography, Population Studies, and Population and Development Review-to survey the changing contours of anglophone demography's key research areas over the past 70 years. We characterize the field of demography by applying a two-pronged, data-grounded approach from the sociology of science. The first uses natural language processing that lets the substance of the field emerge from the contents of publication records and applies social network analyses to identify groups of papers that talk about the same thing. The second uses bibliometric tools to capture the "conversations" of demography with other disciplines. Our goals are to (1) identify the primary topics of demography since the discipline first gained prominence as an organized field; (2) assess changes in the field's intellectual cohesion and the topical areas that have grown or shrunk; and (3) examine how demographers place their work in relationship to other disciplines, the visibility and influence of demographic research in the broader scientific literature, and the cross-disciplinary translational reach of demographic research. Results provide a dynamic view of the field's scientific development in the second half of the twentieth century and the first two decades of the twenty-first century.

Associations of peer generational status on adolescent weight across Hispanic immigrant generations: A social network analysis.

BACKGROUND: Childhood obesity disproportionately impacts Hispanics in the United States (US), the nation's largest ethnic minority population. However, even among Hispanic children, those born in the US are at increased risk of developing obesity than those not born in the US (i.e. first-generation Hispanics). The objective of this study is to assess whether ethnic and generational differences in the friend networks of Hispanic adolescents moderate the association between immigrant generation and weight. METHODS: We analyzed data from first-generation, second-generation, and third-generation Hispanic 12 to 19 year-old participants in Wave 1 of the Longitudinal Study of Adolescent to Adult Health (Add Health). Using multivariable linear regression, we examined the association between generational status and body mass index (BMI), and whether the ethnic and generational composition of friends moderated that association. RESULTS: Higher generational status was associated with higher BMI. The ethnic and generational composition of friends was not independently associated with BMI among Hispanic adolescents. However, a social network with a greater proportion of second-generation Hispanics was positively associated with BMI among first-generation Hispanics, and negatively associated with BMI among second-generation Hispanics. CONCLUSIONS: The generational status of peers in Hispanic adolescents' social networks, particularly the proportion that are second-generation Hispanic, moderates the positive association between immigrant generation and BMI. Moreover, this moderation effect is different across immigrant generations so that the proportion of second-generation adolescents within a social network is associated with higher BMI in first-generation Hispanic adolescents, but with lower BMI among those who are second-generation. These results were confirmed in sensitivity analyses. Our findings suggest that the generational composition of social networks alters the association between the generational status and weight of Hispanic adolescents, and thus that social factors within those networks may contribute to those associations.

Sociodemographic Variables Can Guide Prioritized Testing Strategies for Epidemic Control in Resource-Limited Contexts.

BACKGROUND: Targeted surveillance allows public health authorities to implement testing and isolation strategies when diagnostic resources are limited, and can be implemented via the consideration of social network topologies. However, it remains unclear how to implement such surveillance and control when network data are unavailable. METHODS: We evaluated the ability of sociodemographic proxies of degree centrality to guide prioritized testing of infected individuals compared to known degree centrality. Proxies were estimated via readily available sociodemographic variables (age, gender, marital status, educational attainment, household size). We simulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemics via a susceptible-exposed-infected-recovered individual-based model on 2 contact networks from rural Madagascar to test applicability of these findings to low-resource contexts. RESULTS: Targeted testing using sociodemographic proxies performed similarly to targeted testing using known degree centralities. At low testing capacity, using proxies reduced infection burden by 22%-33% while using 20% fewer tests, compared to random testing. By comparison, using known degree centrality reduced the infection burden by 31%-44% while using 26%-29% fewer tests. CONCLUSIONS: We demonstrate that incorporating social network information into epidemic control strategies is an effective countermeasure to low testing capacity and can be implemented via sociodemographic proxies when social network data are unavailable.

Predicting primate-parasite associations using exponential random graph models.

Ecological associations between hosts and parasites are influenced by host exposure and susceptibility to parasites, and by parasite traits, such as transmission mode. Advances in network analysis allow us to answer questions about the causes and consequences of traits in ecological networks in ways that could not be addressed in the past. We used a network-based framework (exponential random graph models or ERGMs) to investigate the biogeographic, phylogenetic and ecological characteristics of hosts and parasites that affect the probability of interactions among nonhuman primates and their parasites. Parasites included arthropods, bacteria, fungi, protozoa, viruses and helminths. We investigated existing hypotheses, along with new predictors and an expanded host-parasite database that included 213 primate nodes, 763 parasite nodes and 2319 edges among them. Analyses also investigated phylogenetic relatedness, sampling effort and spatial overlap among hosts. In addition to supporting some previous findings, our ERGM approach demonstrated that more threatened hosts had fewer parasites, and notably, that this effect was independent of hosts also having a smaller geographic range. Despite having fewer parasites, threatened host species shared more parasites with other hosts, consistent with loss of specialist parasites and threat arising from generalist parasites that can be maintained in other, non-threatened hosts. Viruses, protozoa and helminths had broader host ranges than bacteria, or fungi, and parasites that infect non-primates had a higher probability of infecting more primate species. The value of the ERGM approach for investigating the processes structing host-parasite networks provided a more complete view on the biogeographic, phylogenetic and ecological traits that influence parasite species richness and parasite sharing among hosts. The results supported some previous analyses and revealed new associations that warrant future research, thus revealing how hosts and parasites interact to form ecological networks.

Quantifying In Situ Structural Stabilities of Human Blood Plasma Proteins Using a Novel Iodination Protein Stability Assay.

Many of the diseases that plague society today are driven by a loss of protein quality. One method to quantify protein quality is to measure the protein folding stability (PFS). Here, we present a novel mass spectrometry (MS)-based approach for PFS measurement, iodination protein stability assay (IPSA). IPSA quantifies the PFS by tracking the surface-accessibility differences of tyrosine, histidine, methionine, and cysteine under denaturing conditions. Relative to current methods, IPSA increases protein coverage and granularity to track the PFS changes of a protein along its sequence. To our knowledge, this study is the first time the PFS of human serum proteins has been measured in the context of the blood serum (in situ). We show that IPSA can quantify the PFS differences between different transferrin iron-binding states in near in vivo conditions. We also show that the direction of the denaturation curve reflects the in vivo surface accessibility of the amino acid residue and reproducibly reports a residue-specific PFS. Along with IPSA, we introduce an analysis tool Chalf that provides a simple workflow to calculate the residue-specific PFS. The introduction of IPSA increases the potential to use protein structural stability as a structural quality metric in understanding the etiology and progression of human disease. Data is openly available at Chorusproject.org (project ID 1771).

The role of informational support from women's social networks on antenatal care initiation: qualitative evidence from pregnant women in Uganda.

BACKGROUND: Early and appropriate use of antenatal care services is critical for reducing maternal and neonatal mortality and morbidity. Yet most women in sub-Saharan Africa, including Uganda, do not seek antenatal care until later during pregnancy. This qualitative study explored pregnant women's reliance on social ties for information about initiation of antenatal care. METHODS: We conducted semi-structured, in-depth interviews with 30 pregnant women seeking their first antenatal care visit at Kawempe Referral Hospital in Kampala, Uganda. Recruitment was done purposively to obtain variation by parity and whether women currently had a male partner. Study recruitment occurred from August 25th 2020 - October 26th, 2020. We used thematic analysis following a two-stage coding process, with both deductive and inductive codes. Deductive codes followed the key domains of social network and social support theory. RESULTS: We found that the most important source of information about antenatal care initiation was these women's mothers. Other sources included their mothers-in-law, female elders including grandmothers, and male partners. Sisters and female friends were less influential information sources about antenatal initiation. One of the primary reasons for relying on their own mothers, mothers-in-law, and elder women was due to these women's lived experience with pregnancy and childbirth. Trust in the relationship was also an important factor. Some pregnant women were less likely to rely on their sisters or female friends, either due to lack of trust or these women's lack of experience with pregnancy and childbirth. The advice that pregnant women received from their mothers and others on the ideal timing for antenatal care initiation varied significantly, including examples of misinformation about when to initiate antenatal care. Pregnant women seemed less likely to delay care when more than one social tie encouraged early antenatal care. CONCLUSIONS: Educating women's social networks, especially their mothers, mothers-in-law, and community elders, about the importance of early antenatal care initiation is a promising avenue for encouraging pregnant women to seek care earlier in pregnancy.

Visualizing COVID Restrictions: Activity Patterns Before, During, and After COVID-19 Lockdowns in Uttar Pradesh, India.

Globally, restrictions implemented to limit the spread of COVID-19 have highlighted deeply rooted social divisions, raising concerns about differential impacts on members of different groups. Inequalities among households of different castes are ubiquitous in certain regions of India. Drawing on a novel data set of 8,564 households in Uttar Pradesh, the authors use radar plots to examine differences between castes in rates of activity for several typical behaviors before, during, and upon lifting strict lockdown restrictions. The visualization reveals that members of all castes experienced comparable reductions in activity rates during lockdown and recovery rates following it. Nonetheless, members of less privileged castes procure water outside the household more often than their more privileged peers, highlighting an avenue of improvement for future public health efforts.

Targeted maximum likelihood estimation of causal effects with interference: A simulation study.

Interference, the dependency of an individual's potential outcome on the exposure of other individuals, is a common occurrence in medicine and public health. Recently, targeted maximum likelihood estimation (TMLE) has been extended to settings of interference, including in the context of estimation of the mean of an outcome under a specified distribution of exposure, referred to as a policy. This paper summarizes how TMLE for independent data is extended to general interference (network-TMLE). An extensive simulation study is presented of network-TMLE, consisting of four data generating mechanisms (unit-treatment effect only, spillover effects only, unit-treatment and spillover effects, infection transmission) in networks of varying structures. Simulations show that network-TMLE performs well across scenarios with interference, but issues manifest when policies are not well-supported by the observed data, potentially leading to poor confidence interval coverage. Guidance for practical application, freely available software, and areas of future work are provided.