Gender systems in the Putin autocracy.

Over the last 23 years, Russian President Vladimir Putin's autocracy has revealed a set of interlocking gender systems that have come to the fore particularly vividly since the full-scale invasion of Ukraine on February 24, 2022. How, this article asks, have the masculinist cultural and political practices of the Putin regime undermined democratic practices and engagement broadly speaking? How have they organized Russian state and society in ways that have led to today's war in Ukraine with its massive destruction, violence, and brutality? And have there been earlier signals that should have warned observers that this regime might undertake such a war of aggression? Drawing on public, mass media data, this article analyzes the gendered structures of power in Russia that have contributed to the degeneration of democracy in three main areas: (1) male-on-male domination in discourse and practice that supports Putin's personal rule and emasculates his enemies; (2) the elevation of male power clans, including the President's personal praetorian guard and the Russian private military companies; and (3) the overall taming and emasculation of the Russian Parliament combined with the elevation of tough women deputies, whom I call the Baba Commissars. These female MPs support the President's domination by creating an appearance of a threatening outside world that needs to be kept at bay. At the same time, they support a neo-traditional gender order with women managing the house under the direction of the patriarchal male leader.

Controlling genome topology with sequences that trigger post-replication gap formation during replisome passage: the E. coli RRS elements.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, $ \le$2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

Controlling Genome Topology with Sequences that Trigger Post-replication Gap Formation During Replisome Passage: The E. coli RRS Elements.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, ≤2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

Identification of recG genetic interactions in Escherichia coli by transposon sequencing.

IMPORTANCE: DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

RecF protein targeting to post-replication (daughter strand) gaps II: RecF interaction with replisomes.

The bacterial RecF, RecO, and RecR proteins are an epistasis group involved in loading RecA protein into post-replication gaps. However, the targeting mechanism that brings these proteins to appropriate gaps is unclear. Here, we propose that targeting may involve a direct interaction between RecF and DnaN. In vivo, RecF is commonly found at the replication fork. Over-expression of RecF, but not RecO or a RecF ATPase mutant, is extremely toxic to cells. We provide evidence that the molecular basis of the toxicity lies in replisome destabilization. RecF over-expression leads to loss of genomic replisomes, increased recombination associated with post-replication gaps, increased plasmid loss, and SOS induction. Using three different methods, we document direct interactions of RecF with the DnaN ß-clamp and DnaG primase that may underlie the replisome effects. In a single-molecule rolling-circle replication system in vitro, physiological levels of RecF protein trigger post-replication gap formation. We suggest that the RecF interactions, particularly with DnaN, reflect a functional link between post-replication gap creation and gap processing by RecA. RecF's varied interactions may begin to explain how the RecFOR system is targeted to rare lesion-containing post-replication gaps, avoiding the potentially deleterious RecA loading onto thousands of other gaps created during replication.

Spatiotemporal Dynamics of Single-stranded DNA Intermediates in Escherichia coli.

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

RecA and SSB genome-wide distribution in ssDNA gaps and ends in Escherichia coli.

Single-stranded DNA (ssDNA) gapped regions are common intermediates in DNA transactions. Using a new non-denaturing bisulfite treatment combined with ChIP-seq, abbreviated 'ssGap-seq', we explore RecA and SSB binding to ssDNA on a genomic scale in E. coli in a wide range of genetic backgrounds. Some results are expected. During log phase growth, RecA and SSB assembly profiles coincide globally, concentrated on the lagging strand and enhanced after UV irradiation. Unexpected results also abound. Near the terminus, RecA binding is favored over SSB, binding patterns change in the absence of RecG, and the absence of XerD results in massive RecA assembly. RecA may substitute for the absence of XerCD to resolve chromosome dimers. A RecA loading pathway may exist that is independent of RecBCD and RecFOR. Two prominent and focused peaks of RecA binding revealed a pair of 222 bp and GC-rich repeats, equidistant from dif and flanking the Ter domain. The repeats, here named RRS for replication risk sequence, trigger a genomically programmed generation of post-replication gaps that may play a special role in relieving topological stress during replication termination and chromosome segregation. As demonstrated here, ssGap-seq provides a new window on previously inaccessible aspects of ssDNA metabolism.

Interaction with the carboxy-terminal tip of SSB is critical for RecG function in E. coli.

In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.

Global health-based virtual exchange to improve intercultural competency in students: Long-lasting impacts and areas for improvement.

Introduction: As public health expands its role in global settings, the need to develop intercultural competency for public health students also grows. One initiative being applied to promote global awareness is the use of virtual exchange (VE) programs. VE programs promote collaborative online international learning (COIL) and allow students from different countries to connect and work together on projects related to their field of study; however, there is little research around the long-term impacts of these programs. Methods: Undergraduate pre-health students from the United States who participated in a VE program a year prior were interviewed about their experiences engaging with undergraduate medical students in Egypt. They were asked if the experience impacted their current behaviors, skills, or knowledge, and what improvements could be made to the program. Mezirow's Transformative Learning Theory (TLT) served as the theoretical framework, grounding interview instrument development and directed content analysis procedures. Researchers also engaged in inductive analysis to capture other salient themes. Results: Ten students were interviewed with a majority engaging in either of the two final stages of Mezirow's TLT: "building of self-confidence and self-competence" (60%) and "reintegration" (50%). Other salient themes found were intercultural interactions, VE appreciation, and VE improvements. When describing their experience in one word, students overwhelmingly provided words with positive connotations (80%), with the negative responses being explained by the structure and presentation of the VE. Discussion: Students were able to apply lessons they learned during the VE within a 1-year follow-up period. This is beneficial, as health professionals require intercultural competency to promote and provide improved health outcomes. Results from this study indicate the need for structure when conducting a VE, addressing the need to increase the number of direct interactions and thereby promoting more intercultural exchanges. Likewise, the interviews demonstrated that changes in course instruction need to be implemented gradually to allow for students to adjust to unfamiliar teaching methods.

Transforming Perspectives Through Virtual Exchange: A US-Egypt Partnership Part 1.

With more classrooms within higher education mobilizing strategies for internationalization, collaborative online international learning (COIL), also referred to as virtual exchange, is an effective approach at offering intercultural competence through experiential learning. This strategy provides students who face barriers to international travel the opportunity to engage with students from other countries in meaningful ways, while enhancing and reinforcing course content. Grounded in the transformative learning theory, this study evaluates the effectiveness of a virtual exchange that was implemented within an undergraduate global public health course. The virtual exchange connected students from the University of Florida (within the US) with medical students in a microbiology course at Ain Shams University in Cairo, Egypt. Using adapted reflection prompts, we assessed the students' knowledge and learning before, during, and after the virtual exchange. This was coupled with a final paper to capture how personal backgrounds and experiences may contribute to their perception of the virtual exchange, as well as if they felt their global perspective had changed or shifted during the experience. Using directed content analysis for each of the measurements, two researchers coded the data independently to then present agreed upon salient themes to the larger group. Of the 28 randomly sampled students who participated in the virtual exchange, seven major themes emerged from the data: Connectedness; Openness; Acquisition of Knowledge and Skills; Communication; Cultural Identity; Anticipation of Options for New Roles, Relationships, and Actions; and Absence of Change. Through this evaluation it was clear there was a variance of different perspectives with many sampled students having diverse lived experiences that influenced their worldview prior to the virtual exchange. Despite course-related barriers, students acknowledged several facilitating factors that improved their intercultural competence and knowledge of course content. The integration of a virtual exchange within the classroom, with careful design and implementation, can provide a unique experience for students and an inclusive approach to learning.

Transforming Perspectives Through Virtual Exchange: A US-Egypt Partnership Part 2.

Collaborative online international learning programs, such as virtual exchange, that utilize telecollaborative activities have been integrated into more classrooms within the higher education setting. These programs provide students exposure to international cultures, perspectives, and ideas is no longer considered "value added", but a prerequisite to entering many workforces. These programmatic objectives compliment Mezirow's Transformative Learning Theory, that substantiates two major elements of transformative learning are critical reflection and dialectical discourse. This study presents the second half of a qualitative inquiry into the prominent themes that arose during a virtual exchange that was conducted in March 2021 between students in the United States (US) enrolled in a global public health course and Egyptian microbiology students. This study sought to expand upon the Transformative Learning Theory through inductive analysis procedures to offer a modernized adaptation of the theoretical framework within international learning environments. Student responses enrolled in an undergraduate global public health course were collected and analyzed by two coders using inductive/open coding to identify salient codes. These codes were then summarized into categories and subsequently defined. Resulting themes include Connectedness, Openness, Acquisition of Knowledge and Skills, Communication, Cultural Identity, Anticipation of Options for New Roles, Relationships, and Actions, and Absence of Change. Several themes have corresponding categories and subcategories. Adult learning environments such as the modern college classroom have changed with the introduction and reliance upon online learning domains, as well as the diversification of higher education student demographics, accentuating the need to inductively analyze student learning processes and outcomes. In doing so, our findings provide a modernized adaptation of the Transformative Learning Theory that allows for adult learning theorists, researchers, and scholars to integrate tenets of transformative learning more appropriately. As such, this provides an opportunity for educators to coalesce the identified mechanisms (e.g., openness, cultural background, anticipation of roles and relationships) to bolster student's willingness and ability to engage in transformative critical reflections. By capitalizing on students' innate characteristics, such as open-mindedness predispositions and cultural background, educators are able to augment transformative learning strategies through tailored assignments and course activities.

Design and implementation of an integrated data model to support clinical and translational research administration.

OBJECTIVE: Both academic medical centers and biomedical research sponsors need to understand impact of scientific funding to determine value. For the National Institutes of Health (NIH) Clinical and Translational Science Award (CTSA) hubs, tracking research activities can be complex, often involving multiple institutions and continually changing federal reporting requirements. Existing research administrative systems are institution-specific and tend to focus only on parts of a greater whole. The goal of this case report is to describe a comprehensive data model that addresses this gap. MATERIALS AND METHODS: Web-based Center Administrative Management Program (WebCAMP) has been developed over a period of over 15 years in the context of CTSA hubs, with the recent addition of T32 programs. Its data model centers around the key concepts of people, projects, resources (inputs), and outcomes (outputs). RESULTS: The WebCAMP data model and associated toolset for biomedical research administration integrates multiple components of the research enterprise, has been used by our CTSA hub for over 15 years and has been adopted by more than 20 other CTSA hubs. DISCUSSION: To the best of our knowledge, this study is among the first to describe a comprehensive data model for biomedical research administration. Opportunities for future work include improved grant tracking through the development of a universal identifier that spans public and private funders, and a more generic outcomes tracking model able to rapidly incorporate new outcome types. CONCLUSION: We propose that the WebCAMP data model, or a derivative of it, could serve as a future standard for research administrative data warehousing.

The Escherichia coli serS gene promoter region overlaps with the rarA gene.

Deletion of the entire gene encoding the RarA protein of Escherichia coli results in a growth defect and additional deficiencies that were initially ascribed to a lack of RarA function. Further work revealed that most of the effects reflected the presence of sequences in the rarA gene that affect expression of the downstream gene, serS. The serS gene encodes the seryl aminoacyl-tRNA synthetase. Decreases in the expression of serS can trigger the stringent response. The sequences that affect serS expression are located in the last 15 nucleotides of the rarA gene.

The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ.

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

Exploring the differences between men's and women's perceptions of gender-based violence in rural Tajikistan: a qualitative study.

BACKGROUND: Most women living in rural provinces of Tajikistan, specifically Khatlon, experience little to no opportunities for education and economic growth, making them vulnerable to gender-based violence (GBV). Unfortunately due to social norms that are bolstered by a patriarchal society, GBV has become tolerated and even normalized in rural areas. This study looks to investigate the differences in perceptions of violence as it relates to empowerment among men and women in rural Tajikistan. METHODS: Data collection was done through participatory workshops and semi-structured interviews (SSI) that were led by Extension Home Economists, which lectured on gender, violence, and empowerment. Community Empowerment Drawings is a novel tool that has been used to further gather sensitive information that was not previously discussed. During this process, participant groups were asked to draw their perception of empowered versus disempowered women, later explaining the different characteristics and traits of both. Random participants across both genders were later contacted for a SSI to triangulate the data from the participatory workshops. This qualitative study implemented qualitative content analysis to explore the data inductively. Analysis of the drawings and transcripts from the workshops and SSIs included two researchers coding through an iterative process. Themes were stratified by men's and women's perceptions and codebooks were compared to ensure consensus. RESULTS: Men and women from 12 villages participated in the Community Empowerment Drawings within each workshop, with 234 participants total. Results were stratified into two categories which were later broken down into notable themes: education, employment, decision-making, marital status, relationship wellness and respect, violence, mental health, and substance abuse. Major findings illustrated how disempowered women were perceived to have more exposure to men who experience alcohol abuse. This study found that differences in perceptions of empowerment between men and women remain-with men still holding onto the traditional power structure within a household and women challenging gender roles and mobility. CONCLUSION: Future studies may find engaging communities through drawings will yield more information regarding sensitive topics rather than traditional instruments. More support and advocacy are needed in areas of mental, neurological, and substance abuse disorders throughout rural Tajikistan.

Redox controls RecA protein activity via reversible oxidation of its methionine residues.

Reactive oxygen species (ROS) cause damage to DNA and proteins. Here, we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted four out of nine Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function, whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.

Addressing Domestic Violence as It Relates to Livelihoods in Rural Tajikistan.

This study investigates patterns of rural livelihoods in Khatlon Province, Tajikistan, as it relates to gender roles and responsibilities. This included a workshop in the train-the-trainer format for community workers to implement a violence participatory workshop among village-related women and men. Key informant interviews were performed to collect information about the perceptions of violence among village households followed by individual semistructured interviews with participants of the participatory workshops. Analysis included ethnographic content analysis using NVivo 12 software to extract salient themes. Results concluded that education and employment were essential in building empowerment among women in the villages.