Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Dynamics of Cas10 Govern Discrimination between Self and Non-self in Type III CRISPR-Cas Immunity.

Adaptive immune systems must accurately distinguish between self and non-self in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNA to recognize complementary RNA targets, which triggers the degradation of both the invader's transcripts and their template DNA. These systems can broadly eliminate foreign targets with multiple mutations but circumvent damage to the host genome. To explore the molecular basis for these features, we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10-the DNase effector of the complex-displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Target mutations differentially modulate Cas10 dynamics and tune the CRISPR interference activity in vivo. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self versus non-self discrimination and target specificity.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

Measuring mantled howler monkey (Alouatta palliata) testes via parallel laser photogrammetry: Expanding the use of noninvasive methods.

Parallel laser photogrammetry (PLP), which consists of attaching two or three parallel laser beams at a known inter-beam distance to a camera, can be used to collect morphological measurements of organisms noninvasively. The lasers project onto the photo being taken, and because the inter-beam distance is known, they act as a scale for image analysis programs like ImageJ. Traditionally, this method has been used to measure larger morphological traits (e.g., limb length, crown-rump length) to serve as proxies for overall body size, whereas applications to smaller anatomical features remain limited. To that end, we used PLP to measure the testes of 18 free-living mantled howler monkeys (Alouatta palliata) at La Selva Biological Station, Costa Rica. We tested whether this method could reliably measure this relatively small and globular morphology, and whether it could detect differences among individuals. We tested reliability in three ways: within-photo (coefficient of variation [CV] = 4.7%), between-photo (CV = 5.5%), and interobserver (intraclass correlation = 0.92). We found an average volume of 36.2 cm3 and a range of 16.4-54.4 cm3, indicating variation in testes size between individuals. Furthermore, these sizes are consistent with a previous study that collected measurements by hand, suggesting that PLP is a useful method for making noninvasive measurements of testes.

Associations between faecal chemical pollutants and hormones in primates inhabiting Kibale National Park, Uganda.

While anthropogenic pollutants are known to be a threat to primates, our understanding of exposure to pollutants in situ and their sub-lethal effects is still limited. We used non-invasive biomonitoring to examine associations between faecal concentrations of 97 chemical pollutants and faecal hormone metabolites of cortisol and oestradiol in four primate species inhabiting Kibale National Park, Uganda (chimpanzees-Pan troglodytes, olive baboons-Papio anubis, red colobus-Piliocolobus tephrosceles and red-tailed monkeys-Cercopithecus ascanius). Across all species (n = 71 samples), results demonstrated positive associations of organochlorine pesticides (OCPs) (ß = 0.143, p = 0.020) and organophosphate esters (ß = 0.112, p = 0.003) with cortisol in adult females. Additionally, we observed positive associations of OCPs (ß = 0.192, p = 0.013) and brominated flame retardants (ß = 0.176, p = 0.004) with cortisol in juveniles. Results suggest that cumulative pesticides and flame retardants are disruptive to endocrine function in these populations, which could have implications for development, metabolism and reproduction. Our study further demonstrates that faeces can be an important, non-invasive matrix for examining pollutant-hormone associations in wild primates and other critical wildlife populations.

Using an on-site laboratory for fecal steroid analysis in wild white-faced capuchins.

Hormone laboratories located "on-site" where field studies are being conducted have a number of advantages. On-site laboratories allow hormone analyses to proceed in near-real-time, minimize logistics of sample permits/shipping, contribute to in-country capacity-building, and (our focus here) facilitate cross-site collaboration through shared methods and a shared laboratory. Here we provide proof-of-concept that an on-site hormone laboratory (the Taboga Field Laboratory, located in the Taboga Forest Reserve, Costa Rica) can successfully run endocrine analyses in a remote location. Using fecal samples from wild white-faced capuchins (Cebus imitator) from three Costa Rican forests, we validate the extraction and analysis of four steroid hormones (glucocorticoids, testosterone, estradiol, progesterone) across six assays (DetectX® and ISWE, all from Arbor Assays). Additionally, as the first collaboration across three long-term, wild capuchin field sites (Lomas Barbudal, Santa Rosa, Taboga) involving local Costa Rican collaborators, this laboratory can serve as a future hub for collaborative exchange.

Efficacy of Fluoxetine for Post-Ischemic Stroke Depression in Tanzania.

OBJECTIVE: Post-stroke fluoxetine trials are primarily conducted in high-income countries. We characterize post-ischemic stroke depression in fluoxetine-treated and -untreated study participants in urban Tanzania. METHODS: Adults (>18 years old) within 14 days of CT-confirmed acute ischemic stroke onset were enrolled at Muhimbili National Hospital, Tanzania. The fluoxetine-treated group took 20mg fluoxetine daily for 90 days in a phase II trial and were compared to fluoxetine-untreated historical controls. The primary outcome was depression at 90 days, measured by the Patient Health Questionnaire-9 (PHQ-9). PHQ-9 scores were compared between fluoxetine-treated and -untreated groups. A score >=9 points was considered to reflect depression. A multivariable linear regression model assessed associations with post-stroke PHQ-9 scores. RESULTS: Of the fluoxetine-treated (n=27) and -untreated (n=32) participants, the average age was 56.8 years old (39% women, 100% Black/African). The average presentation NIHSS score was 12.1 points and modified Rankin Scale (mRS) score was 3.5. The average mRS score at 90-day follow-up was 2.3. There was no significant difference between 90-day PHQ-9 scores in the fluoxetine-treated (mean=4.1 points, standard deviation=3.2; 11% depression) and untreated (mean=4.4, standard deviation=4.8; 19% depression) groups, p=.69. In the multivariable analysis, older age (ß=0.08, p=.03) and higher NIHSS score (ß=0.15, p=.04), but neither fluoxetine (ß=0.57, p=.59) nor sex (ß=-0.51, p=.63), were significantly associated with more depressive symptoms. CONCLUSIONS: Our findings parallel results from trials from higher income settings that fluoxetine does not significantly improve post-ischemic stroke depression, although our sample size was small. More work is needed to depict the longitudinal nature and treatment of post-stroke depression in Sub-Saharan Africa.

Ecological and evolutionary significance of primates' most consumed plant families.

Angiosperms have been essential components of primate diets for millions of years, but the relative importance of different angiosperm families remains unclear. Here, we assess the contribution and ecological and evolutionary significance of plant families to diets of wild primates by compiling an unprecedented dataset of almost 9000 dietary records from 141 primary sources covering 112 primate species. Of the 205 angiosperm plant families recorded in primate diets, only 10 were consumed by more than half of primate species. Plants of the Moraceae and Fabaceae families were the most widely and frequently consumed, and they likely represent keystone resources for primates. Over 75% of species fed on these two families, and together they made up a median of approximately 13% of primate diets. By analysing the relative proportion of different plant parts consumed, we found that Moraceae was mainly eaten as fruit and Fabaceae as non-fruit parts, with the consumption of these two families not showing a significant phylogenetic signal across primate species. Moraceae consumption was associated with small home range sizes, even though more frugivorous primates tended to have larger home ranges compared to more folivorous species, possibly due to the year-round availability of moraceous fruits and the asynchrony in their phenology. Our results suggest that primates may be intricately and subtly shaped by the plant families that they have consumed over millions of years, and highlight the importance of detailed dietary studies to better understand primate ecology and evolution.

Notification System for Overdue Radiology Recommendations Improves Rates of Follow-Up and Diagnosis.

OBJECTIVE. The purpose of this study was to quantify improved rates of follow-up and additional important diagnoses made after notification for overdue workups recommended by radiologists. MATERIALS AND METHODS. Standard reports from imaging studies performed at our institution from October through November 2016 were searched for the words "recommend" or "advised," yielding 9784 studies. Of these, 5245 were excluded, yielding 4539 studies; reports for 1599 of these 4539 consecutive studies were reviewed to identify firm or soft recommendations or findings requiring immediate management. If recommended follow-ups were incomplete within 1 month of the advised time, providers were notified. Compliance was calculated before and after notification and was compared using a one-sample test of proportion. RESULTS. Of 1599 patients, 92 were excluded because they had findings requiring immediate management, and 684 were excluded because of soft recommendations, yielding 823 patients. Of these patients, 125 were not yet overdue for follow-up and were excluded, and 18 were excluded because of death or transfer to another institution. Of the remaining 680 patients, follow-up was completed for 503 (74.0%). A total of 177 (26.0%) of the 680 patients were overdue for follow-up, and providers were notified. Of these 177 patients, 36 (20.3%) completed their follow-ups after notification, 34 (19.2%) had follow-up designated by the provider as nonindicated, and 107 (60.5%) were lost to follow-up, yielding four clinically important diagnoses: one biopsy-proven malignancy, one growing mass, and two thyroid nodules requiring biopsy. The rate of incomplete follow-ups after communication decreased from 26.0% (177/680) to 20.7% (141/680) (95% CI, 17.7-23.9%; p = .002), with a 20.4% reduction in relative risk of noncompliance, and 39.5% (70/177) of overdue cases were resolved when nonindicated studies were included. CONCLUSION. Notification of overdue imaging recommendations reduces incomplete follow-ups and yields clinically important diagnoses.

Feces are Effective Biological Samples for Measuring Pesticides and Flame Retardants in Primates.

The habitats of wild primates are increasingly threatened by surrounding anthropogenic pressures, but little is known about primate exposure to frequently used chemicals. We applied a novel method to simultaneously measure 21 legacy pesticides (OCPs), 29 current use pesticides (CUPs), 47 halogenated flame retardants (HFRs), and 19 organophosphate flame retardants in feces from baboons in the U.S.A., howler monkeys in Costa Rica, and baboons, chimpanzees, red-tailed monkeys, and red colobus in Uganda. The most abundant chemicals were α-hexachlorocyclohexane (α-HCH), ß-hexachlorocyclohexane (ß-HCH), and hexachlorobenzene among OCPs across all sites, chlorpyrifos among CUPs in Costa Rica and Indiana, decabromodiphenylethane (DBDPE) in Costa Rica and Indiana and 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47) in Uganda as HFRs, and tris(2-butoxyethyl) phosphate (TBOEP) as OPFRs across all sites. The detected chemical concentrations were generally higher in red-tailed monkeys and red colobus than in chimpanzees and baboons. Our methods can be used to examine the threat of chemical pollutants to wildlife, which is critical for endangered species where only noninvasive methods can be used.

Chimpanzee (Pan troglodytes schweinfurthii) Population Spans Multiple Protected Areas in the Albertine Rift.

We used mitochondrial DNA to examine gene flow in a region of western Uganda that has received little attention regarding chimpanzee population dynamics. The area is critical to gene flow between isolated Democratic Republic of Congo populations and the rest of East Africa. None of the chimpanzees in each of the 4 protected areas under consideration (Toro-Semliki Wildlife Reserve, Semuliki National Park, Rwenzori Mountains National Park and Itwara Central Forest Reserve) are fully habituated. Therefore, it is not clear whether one or more populations have historically used this fragmented landscape for (1) regular ranging and/or (2) infrequent dispersal. We incorporated the published sequences of the first hypervariable region of the D-loop of the mitochondrial genome from 3 previously sampled sites (n = 39) while also contributing the first extensive genetic sampling of chimpanzees in Toro-Semliki (n = 80). Our goal was to generate a historical baseline model of metapopulation dynamics in this region and determine which, if any, of these protected areas forms a fragmented landscape for a single chimpanzee population. According to a discriminant analysis of principal components, the haplotypes at Toro-Semliki form a central cluster, and Itwara is its nearest genetic neighbor. Rwenzori Mountains National Park is the most distant neighbor of all protected areas. We performed an analysis of molecular variance for 14 different population models that divided the samples from the 4 protected areas into 2, 3 or 4 populations. The best fit model included 3 populations: Toro-Semliki Wildlife Reserve and Itwara Forest Reserve comprised a single population; Semuliki National Park and Rwenzori Mountains National Park formed 2 additional separate populations (variance among = 9%, p = 0.014). The results indicated that some protected areas comprised distinctive populations, while others formed a fragmented landscape for a population's ranging for foraging purposes. Therefore, the edges of a protected area do not always define a chimpanzee population. We propose a closer examination of those dynamics through renewed sampling. Advances in DNA extraction and next-generation sequencing will allow us to compare thousands of single nucleotide polymorphisms in the genomes of unhabituated chimpanzees living in each of these protected areas.

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines.

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale.

Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.

Atmospheric Occurrence of Legacy Pesticides, Current Use Pesticides, and Flame Retardants in and around Protected Areas in Costa Rica and Uganda.

Protected areas have developed alongside intensive changes in land use and human settlements in the neighboring landscape. Here, we investigated the occurrence of 21 organochlorine pesticides (OCPs), 14 current use pesticides (CUPs), 47 halogenated flame retardants (HFRs), and 19 organophosphate esters (OPEs) in air around Las Cruces (LC) and La Selva (LS) Biological Stations, Costa Rica, and Kibale National Park (KNP), Uganda using passive air samplers (PAS) with polyurethane foam (PUF) discs (PAS-PUF). Significantly higher concentrations of CUPs were observed around LS, while LC had a higher concentration of OCPs. Land use analysis indicated that LS had a higher fraction of agriculture than LC (33% vs 14%), suggesting the higher CUPs concentration at LS was related to pesticide intensive crops, while higher OCPs concentration at LC may be attributed to the area's long agricultural history characterized by small-scale subsistence farming or long-range transport. In Uganda, CUPs and OCPs were generally lower than in Costa Rica, but high concentrations of HFRs were observed inside KNP, possibly due to human activity at research camps near the protected forest. This is the first study that documented the occurrence of anthropogenic chemicals in the air at protected areas with tropical forests.

Primate microbial endocrinology: An uncharted frontier.

Gut microbial communities communicate bidirectionally with the brain through endocrine, immune, and neural signaling, influencing the physiology and behavior of hosts. The emerging field of microbial endocrinology offers innovative perspectives and methods to analyze host-microbe relationships with relevance to primate ecology, evolution, and conservation. Herein we briefly summarize key findings from microbial endocrinology and explore how applications of a similar framework could inform our understanding of primate stress and reproductive physiology and behavior. We conclude with three guiding hypotheses to further investigate endocrine signaling between gut microbes and the host: (a) host-microbe communication systems promote microbe-mediated stability, in which the microbes are using endocrine signaling from the host to maintain a functioning habitat for their own fitness, (b) host-microbe communication systems promote host-mediated stability, in which the host uses the endocrine system to monitor microbial communities and alter these communities to maintain stability, or (c) host-microbe systems are simply the product of coincidental cross-talk between the host and microbes due to similar molecules from shared ancestry. Utilizing theory and methodology for studying relationships between the microbiome, hormones, and behavior of wild primates is an uncharted frontier with many promising insights when applied to primatology.

Is genetic drift to blame for testicular dysgenesis syndrome in Semliki chimpanzees (Pan troglodytes schweinfurthii)?

BACKGROUND: We present 3 likely cases of testicular dysgenesis syndrome (TDS) within a community of chimpanzees (Pan troglodytes schweinfurthii). We tested whether genetic drift may be the culprit, as a genetic cause has been suspected to account for TDS among other wildlife. METHODS: We successfully sequenced a 367-bp segment spanning the first hypervariable region within the D-loop of the mitochondrial genome for 78 DNA samples. RESULTS: We found 24 polymorphic sequence sites consisting of 7 singletons and 17 parsimony informative sites. This sample contained 9 haplotypes with a diversity index of 0.78 (SD = 0.03). All tests against the null hypothesis of neutral polymorphisms were non-significant (P > .10). The mismatch distribution of pairwise differences does not fit a Poisson's curve (raggedness index = 0.166; SSD = 0.12; P = 1). CONCLUSIONS: Thus, we found no significant signs of genetic isolation, population expansion, or genetic bottleneck. Alternative causes of TDS and how they might pertain to this population are discussed.

Multidetector CT of Surgically Proven Blunt Bowel and Mesenteric Injury.

Blunt traumatic injury is one of the leading causes of morbidity and mortality in the United States. Unintentional injury represents the leading cause of death in the United States for all persons between the ages of 1 and 44 years. In the setting of blunt abdominal trauma, the reported rate of occurrence of bowel and mesenteric injuries ranges from 1% to 5%. Despite the relatively low rate of blunt bowel and mesenteric injury in patients with abdominal and pelvic trauma, delays in diagnosis are associated with increased rates of sepsis, a prolonged course in the intensive care unit, and increased mortality. During the past 2 decades, as multidetector computed tomography (CT) has emerged as an essential tool in emergency radiology, several direct and indirect imaging features have been identified that are associated with blunt bowel and mesenteric injury. The imaging findings in cases of blunt bowel and mesenteric injury can be subtle and may be seen in the setting of multiple complex injuries, such as multiple solid-organ injuries and spinal fractures. Familiarity with the various imaging features of blunt bowel and mesenteric injury, as well as an understanding of their clinical importance with regard to the care of the patient, is essential to making a timely diagnosis. Once radiologists are familiar with the spectrum of findings of blunt bowel and mesenteric injury, they will be able to make timely diagnoses that will lead to improved patient outcomes. ©RSNA, 2017.

Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

Ultra-stable organic fluorophores for single-molecule research.

Fluorescence provides a mechanism for achieving contrast in biological imaging that enables investigations of molecular structure, dynamics, and function at high spatial and temporal resolution. Small-molecule organic fluorophores have proven essential for such efforts and are widely used in advanced applications such as single-molecule and super-resolution microscopy. Yet, organic fluorophores, like all fluorescent species, exhibit instabilities in their emission characteristics, including blinking and photobleaching that limit their utility and performance. Here, we review the photophysics and photochemistry of organic fluorophores as they pertain to mitigating such instabilities, with a specific focus on the development of stabilized fluorophores through derivatization. Self-healing organic fluorophores, wherein the triplet state is intramolecularly quenched by a covalently attached protective agent, exhibit markedly improved photostabilities. We discuss the potential for further enhancements towards the goal of developing "ultra-stable" fluorophores spanning the visible spectrum and how such fluorophores are likely to impact the future of single-molecule research.

Implementation of training level-specific self-study guides for diagnostic radiology residents.

INTRODUCTION: Currently, there is no universally accepted diagnostic radiology curriculum for self-studying, so diagnostic radiology residents often have a challenging time knowing where to begin their independent studying away from work. In an effort to meet the needs of today's residents, we attempted to provide structured, comprehensive self-studying suggestions in a digestible and personalized format. MATERIALS AND METHODS: Each radiology division attempted to create an optimal learning resource form for the residents to use for self-studying while on each rotation. Each self-study guide included hyperlinks to easily accessible online resources. RESULTS: Training level-specific week-by-week recommendations were provided based on the expected types of cases a resident would typically encounter during that timeframe. All of the neuroradiology rotation forms and several of the other subspecialty forms were originally made available to the residents and faculty electronically on July 1, 2022. The forms are now distributed to newly rotating residents on a monthly basis. DISCUSSION: There is a fine balance between providing residents with comprehensive review material and promoting realistic expectations. In addition, educators must keep in mind financial limitations of their residents and institutions. Learning resources must be affordable to be accessible to all residents. As radiology and technology continuously advance, there will undoubtedly be more and more excellent resources for trainees to learn from. To optimize self-studying, retention of information, and wellness, it is imperative to provide our residents with a structured, personalized, manageable curriculum including easily accessible high-yield resources.