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Human milk oligosaccharides (HMOs) ensure proper infant gut microbiome establishment. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigated microbiome responses to 2’-fucosyllactose (2’FL), a prevalent HMO and infant formula additive, by establishing individualized microbiomes using fecal samples from three different infants as the inocula.Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2’FL. Using metagenomic data, we predicted that extracellular fucosidases encoded by co-existing members such asRuminococcus gnavusinitiate 2’FL breakdown, thus critical forB. breve’sgrowth. Using both targeted co-cultures and by supplementation ofR. gnavusinto one microbiome, we show thatR. gnavuscan promote extensive growth ofB. brevethrough the release of lactose from 2’FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrated that HMO utilization can vary with an individual’s microbiome.

The special issue editors selected us to form an "adversarial collaboration" because our publications and teaching encompass both supportive and critical attitudes toward the Rorschach and its recently developed system for use, the Rorschach Performance Assessment System (R-PAS). We reviewed the research literature and case law to determine if the Rorschach and specifically R-PAS meet legal standards for admissibility in court. We included evidence on norms, reliability, validity, utility, general acceptance, forensic evaluator use, and response style assessment, as well as United States and selected European case law addressing challenges to mental examination motions, admissibility, and weight. Compared to other psychological tests, the Rorschach is not challenged at unusually high rates. Although the recently introduced R-PAS is not widely referenced in case law, evidence suggests that information from it is likely to be ruled admissible when used by a competent evaluator and selected variables yield scores that are sufficiently reliable and valid to evaluate psychological processes that inform functional psycholegal capacities. We identify effective and ethical but also inappropriate uses (e.g., psychological profiling) of R-PAS in criminal, civil, juvenile, and family court. We recommend specific research to clarify important aspects of R-PAS and advance its utility in forensic mental health assessment.

Invasive insects facilitate secondary invasive species by altering forest structure and function. Specifically, invasive insect herbivores may promote the establishment and growth of invasive plants by creating canopy gaps. Such secondary invasions may be influenced by ecological integrity — the degree to which ecosystem composition, structure, and function deviate from their natural or historical range of variation. Here we investigate (i) whether emerald ash borer (Agrilus planipennis; EAB) induced ash (Fraxinus spp.) mortality facilitates European buckthorn (Rhamnus cathartica) — an invasive, shade-tolerant shrub, and (ii) the role of ecological integrity in this relationship. We use a principal component analysis (PCA) to calculate an index of ecological integrity and a zero-altered negative binomial generalized linear mixed model (GLMM) to describe European buckthorn occurrence and abundance. European buckthorn occurrence is influenced by canopy gaps, independent of EAB-induced gap formation. Ecological integrity and EAB-induced ash mortality interact to control European buckthorn abundance, with high ecological integrity limiting EAB-facilitated buckthorn invasion. This is the first evidence for EAB-facilitated buckthorn invasion and for an interaction between a secondary invasion and ecological integrity. Thus, ecological integrity plays an important role in the EAB–buckthorn system and may be used to manage the impacts of secondary invasions.

In fall, alate males of the potato aphid

ObjectivesThe use of external control arms to study treatment effects is growing in interest among drug sponsors and regulators. However, experience with performing these kinds of studies for complex, immune-mediated diseases is limited. We sought to analyze a retrospective cohort of Crohn’s patients to predict the outcome of a prospective cohort.MethodsWe queried electronic health records databases and screened records at the University of California, San Francisco to identify patients meeting the eligibility criteria of TRIDENT, a concurrent trial involving ustekinumab as a reference arm. Timepoints were defined to balance the tradeoff between missing disease activity and bias. We compared two imputation models by their impacts on cohort membership and outcomes. We compared the results of ascertaining disease activity using structured data algorithms against manual review. We used these data to estimate ustekinumab’s real-world effectiveness.ResultsScreening identified 183 patients. 30% of the cohort had missing baseline data. Two imputation models were tested and had similar effects on cohort definition and outcomes. Algorithms for ascertaining non-symptom-based elements of disease activity were similar in accuracy to manual review. The final cohort consisted of 56 patients. 34% of the cohort was in steroid-free clinical remission by week 24.ConclusionsWe predict that a third of the ustekinumab-treated cohort in TRIDENT will be in steroid-free remission by week 24. However, our prediction is limited by substantial missing data. Efforts to improve real-world data capture and align trial design with clinical practice may enable more robust future studies and improve trial efficiency.STUDY HIGHLIGHTSWHAT IS KNOWNExternal control arm studies are receiving growing interest from drug sponsors and regulators as a potential source of real-world evidenceHowever, the feasibility and robustness of this approach is currently limitedUstekinumab is an FDA-approved treatment of moderately to severely active Crohn’s diseaseWHAT IS NEW HEREWe derived a retrospective cohort of patients designed to resemble the participants of TRIDENT, a concurrent phase 2b trial using ustekinumab as a reference armWe predict that about 34% of the ustekinumab-assigned participants in TRIDENT will be in steroid-free clinical remission by week 24.EHR structured data algorithms may be an accurate and less laborious alternative to manual abstraction of non-symptom-based components of the Crohn’s Disease Activity IndexReal-world practice differs from controlled studies in important ways, including the treatment goals and the timing of encountersThese differences can pose challenges to the feasibility of external control arm studies, and must be addressed to enable this novel study designThe methods, data, and code used in this pilot study are shared here for reproducibility and enhancement by others

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here, we describe a generalizable strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

OBJECTIVEA paucity of studies have examined neurosurgical interventions in the mild traumatic brain injury (mTBI) population with intracranial hemorrhage (ICH). Furthermore, it is not understood how the dimensions of an ICH relate to the risk of a neurosurgical intervention. These limitations contribute to a lack of treatment guidelines. Isolated subdural hematomas (iSDHs) are the most prevalent ICH in mTBI, carry the highest neurosurgical intervention rate, and account for an overwhelming majority of all neurosurgical interventions. Decision criteria in this population could benefit from understanding the risk of requiring neurosurgical intervention. The aim of this study was to quantify the risk of neurosurgical intervention based on the dimensions of an iSDH in the setting of mTBI.METHODSThis was a 3.5-year, retrospective observational cohort study at a Level I trauma center. All adult (≥ 18 years) trauma patients with mTBI and iSDH were included in the study. Maximum length and thickness (in mm) of acute SDHs, the presence of acute-on-chronic (AOC) SDH, mass effect, and other hemorrhage-related variables were double–data entered; discrepant results were adjudicated after a maximum of 4 reviews. Patients with coagulopathy, skull fractures, no acute hemorrhage, a non-SDH ICH, or who did not undergo imaging on admission were excluded. Tentorial SDHs were not measured. The primary outcome was neurosurgical intervention (craniotomy, burr holes, intracranial pressure monitor placement, shunt, ventriculostomy, or SDH evacuation). Multivariate stepwise logistic regression was used to identify significant covariates, to assess interactions, and to create the scoring system.RESULTSThere were a total of 176 patients included in our study: 28 patients did and 148 did not receive a neurosurgical intervention. There were no significant differences between neurosurgical intervention groups in 11 demographic and 22 comorbid variables. Patients with neurosurgical intervention had significantly longer and thicker SDHs than nonsurgical controls. Logistic regression identified thickness and AOC hemorrhage as being the most important variables in predicting neurosurgical intervention; SDH length was not. Risk of neurosurgical intervention was calculated based on the SDH thickness and presence of an AOC hemorrhage from a multivariable logistic regression model (area under the receiver operating characteristic curve 0.94, 95% CI 0.90–0.97; p < 0.001). With a decision point of 2.35% risk, we predicted neurosurgical intervention with 100% sensitivity, 100% negative predictive value, and 53% specificity.CONCLUSIONSThis is the first study to quantify the risk of neurosurgical intervention based on hemorrhage characteristics in patients with mTBI and iSDH. Once validated in a second population, these data can be used to inform the necessity of interhospital transfers and neurosurgical consultations.

OBJECTIVEIsolated subdural hematomas (iSDHs) are one of the most common intracranial hemorrhage (ICH) types in the population with mild traumatic brain injury (mTBI; Glasgow Coma Scale score 13–15), account for 66%–75% of all neurosurgical procedures, and have one of the highest neurosurgical intervention rates. The objective of this study was to examine how quantitative hemorrhage characteristics of iSDHs in patients with mTBI at admission are associated with subsequent neurosurgical intervention.METHODSThis was a 3.5-year, retrospective observational cohort study at a Level I trauma center. All adult trauma patients with mTBI and iSDHs were included in the study. Maximum length and thickness (in mm) of acute SDHs, the presence of acute-on-chronic SDH, mass effect, and other hemorrhage-related variables were double–data entered; discrepant results were adjudicated after a maximum of 4 reviews. Patients with coagulopathy, skull fractures, no acute hemorrhage, a non-SDH ICH, or who did not undergo imaging on admission were excluded. The primary outcome was neurosurgical intervention (craniotomy, burr hole, catheter drainage of SDH, placement of intracranial pressure monitor, shunt, or ventriculostomy). Multivariate stepwise logistic regression was used to identify significant covariates and to assess interactions.RESULTSA total of 176 patients were included in our study: 28 patients did and 148 patients did not receive a neurosurgical intervention. Increasing head Abbreviated Injury Scale score was significantly associated with neurosurgical interventions. There was a strong correlation between the first 3 reviews on maximum hemorrhage length (R2 = 0.82) and maximum hemorrhage thickness (R2 = 0.80). The neurosurgical intervention group had a mean maximum SDH length and thickness that were 61 mm longer and 13 mm thicker than those of the nonneurosurgical intervention group (p < 0.001 for both). After adjusting for the presence of an acute-on-chronic hemorrhage, for every 1-mm increase in the thickness of an iSDH, the odds of a neurosurgical intervention increase by 32% (95% CI 1.16–1.50). There were no interventions for any SDH with a maximum thickness ≤ 5 mm on initial presenting scan.CONCLUSIONSThis is the first study to quantify the odds of a neurosurgical intervention based on hemorrhage characteristics in patients with an iSDH and mTBI. Once validated in a second population, these data can be used to better inform patients and families of the risk of future neurosurgical intervention, and to evaluate the necessity of interhospital transfers.

Life’s most dramatic innovations, from the emergence of self-replicating molecules to highly-integrated societies, often involve increases in biological complexity. Some groups traverse different levels of complexity, providing a framework to identify key factors shaping these evolutionary transitions. Halictid bees span the transition from individual to group reproduction, with repeated gains and losses of eusociality. We generated chromosome-length genome assemblies for 17 species and searched for genes that both experienced positive selection when eusociality arose and relaxed selection when eusociality was secondarily lost. Loci exhibiting these complementary evolutionary signatures are predicted to carry costs outweighed by their importance for traits in eusocial lineages. Strikingly, these loci included two proteins that bind and transport juvenile hormone (JH) – a key regulator of insect development and reproduction. Though changes in JH abundance are frequently associated with polymorphisms, the mechanisms coupling JH to novel phenotypes are not well understood. Our results suggest novel links between JH and eusociality arose in halictids by altering transport and availability of JH in a tissue-specific manner, including in the brain. Through genomic comparisons of species encompassing both the emergence and breakdown of eusociality, we provide insights into the mechanisms targeted by selection to shape a key evolutionary transition.

Knowledge of microbial gene functions comes from manipulating the DNA of individual species in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here we describe a generalizable methodology for editing the genomes of specific organisms within a complex microbial community. First, we identified genetically tractable bacteria within a community using a new approach, Environmental Transformation Sequencing (ET-Seq), in which non-targeted transposon integrations were mapped and quantified following community delivery. ET-Seq was repeated with multiple delivery strategies for both a nine-member synthetic bacterial community and a ~200-member microbial bioremediation community. We achieved insertions in 10 species not previously isolated and identified natural competence for foreign DNA integration that depends on the presence of the community. Second, we developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. These results demonstrate a strategy for targeted genome editing of specific organisms within microbial communities, establishing a new paradigm for microbial manipulation relevant to research and applications in human, environmental, and industrial microbiomes.

OBJECTIVEPosttraumatic hydrocephalus (PTH) is a frequent sequela of traumatic brain injury (TBI) and complication of related cranial surgery. The roles of PTH and the timing of cerebrospinal fluid (CSF) shunt placement in TBI outcome have not been well described. The goal of this study was to assess the impact of hydrocephalus and timing of ventriculoperitoneal (VP) shunt placement on outcome during inpatient rehabilitation after TBI.METHODSIn this cohort study, all TBI patients admitted to Craig Hospital between 2009 and 2013 were evaluated for PTH, defined as ventriculomegaly, and hydrocephalus symptoms, delayed or deteriorating recovery, or elevated opening pressure on lumbar puncture. Extent of ventriculomegaly was quantified by the Evans index from CT scans. Outcome measures were emergence from and duration of posttraumatic amnesia (PTA) and functional status as assessed by means of the Functional Independence Measure (FIM). Findings in this group were compared to findings in a group of TBI patients without PTH (controls) who were admitted for inpatient rehabilitation during the same study period and met specific criteria for inclusion.RESULTSA total of 701 patients were admitted with TBI during the study period. Of these patients, 59 (8%) were diagnosed with PTH and were included in this study as the PTH group, and 204 who were admitted for rehabilitation and met the criteria for inclusion as controls constituted the comparison group (no-PTH group). PTH was associated with initial postinjury failure to follow commands, midline shift or cistern compression, subcortical contusion, and craniotomy or craniectomy. In multivariable analyses, independent predictors of longer PTA duration and lower FIM score at rehabilitation discharge were PTH, emergency department Glasgow Coma Scale motor score < 6, and longer time from injury to rehabilitation admission. PTH accounted for a 51-day increase in PTA duration and a 29-point reduction in discharge FIM score. In 40% of PTH patients with preshunt CT brain imaging analyzed, ventriculomegaly (Evans index > 0.3) was observed 3 or more days before VP shunt placement (median 10 days, range 3–102 days). Among PTH patients who received a VP shunt, earlier placement was associated with better outcome by all measures assessed and independently predicted better FIM total score and shorter PTA duration.CONCLUSIONSPosttraumatic hydrocephalus predicts worse outcome during inpatient rehabilitation, with poorer functional outcomes and longer duration of PTA. In shunt-treated PTH patients, earlier CSF shunting predicted improved recovery. These results suggest that clinical vigilance for PTH onset and additional studies on timing of CSF diversion are warranted.

Animals select resources to maximize fitness but associated costs and benefits are spatially and temporally variable. Differences in wetland management influence resource availability for ducks and mortality risk from duck hunting. The local distribution of the Mallard (Anas platyrhynchos) is affected by this resource heterogeneity and variable risk from hunting. Regional conservation strategies primarily focus on how waterfowl distributions are affected by food resources during the nonbreeding season. To test if Mallard resource selection was related to the abundance of resources, risks, or a combination, we studied resource selection of adult female Mallards during autumn and winter. We developed a digital spatial layer for Lake St. Clair, Ontario, Canada, that classified resources important to Mallards and assigned these resources a risk level based on ownership type and presumed disturbance from hunting. We monitored 59 individuals with GPS back‐pack transmitters prior to, during, and after the hunting season and used discrete choice modeling to generate diurnal and nocturnal resource selection estimates. The model that classified available resources and presumed risk best explained Mallard resource selection strategies. Resource selection varied within and among seasons. Ducks selected for federal, state and private managed wetland complexes that provided an intermediate or relatively greater amount of refuge and foraging options than public hunting areas. Across all diel periods and seasons, there was selection for federally managed marshes and private supplemental feeding refuges that prohibited hunting. Mallard resource selection demonstrated trade‐offs related to the management of mortality risk, anthropogenic disturbances, and foraging opportunities. Understanding how waterfowl respond to heterogeneous landscapes of resources and risks can inform regional conservation strategies related to waterfowl distribution during the nonbreeding season.

Objectives We assessed the efficacy and risks of diagnostic bilateral intracranial electroencephalography (bICEEG) in patients with treatment-resistant epilepsy (TRE) with poorly lateralized epileptogenic zone on noninvasive studies as reflected by progress to resection, Engel outcome, and complication rate. Methods This is a retrospective chart review of 199 patients with TRE who had diagnostic bICEEG at New York University Medical Center between 1994 and 2013. Study end points were progress to resection, surgical outcome, and perioperative complications. Univariate analysis was performed with analysis of variance, t test, or Fisher exact test; multivariable analysis was performed using discriminant function analysis. Results bICEEG lateralized the epileptogenic zone and the patient had resection in 60.3% of cases. The number of depth electrodes used was positively correlated with resection, and surgical complications during bICEEG negatively correlated. Vagal nerve stimulators were implanted in 58.2% of patients who did not undergo resection and 20.7% of those who did. Among the 87 patients who progressed to resection and had more than 1-year follow-up, 47.1% were seizure free compared with 12.7% of the 55 who did not. Male sex correlated with good postoperative seizure control. The most common complication was infection requiring debridement, occurring in 3.1% of admissions (9 of 290). Conclusions At our center, 60% of patients undergoing bICEEG progress to resection and 57% of these had more than 90% reduction in seizures. We conclude that bICEEG allows the benefits of epilepsy surgery to be extended to patients with poorly lateralized and localized TRE.

Objective: Undergoing mild traumatic brain injury (mTBI) increases mortality risk, but it is unclear what drives this finding. This study explored associations with mortality in patients with mTBI.Methods: This was a retrospective study of patients with mTBI and controls admitted to six level 1 trauma centers in 1/1/2009-12/31/2013. Mortality data were from the CDC National Death Index. Patients with mTBI were identified by ICD-9 code, Glasgow Coma Scale 13-15, Injury Severity Score (ISS)

Background: Behavior reflects an organism's health status. Many organisms display a generalized suite of behaviors that indicate infection or predict infection susceptibility. We apply this concept to honey bee aggression, a behavior that has been associated with positive health outcomes in previous studies. We sequenced the transcriptomes of the brain, fat body, and midgut of adult sibling worker bees who developed as pre-adults in relatively high versus low aggression colonies. Previous studies showed that this pre-adult experience impacts both aggressive behavior and resilience to pesticides. We performed enrichment analyses on differentially expressed genes to determine whether variation in aggression resembles the molecular response to infection. We further assessed whether the transcriptomic signature of aggression in the brain is similar to the neuromolecular response to acute predator threat, exposure to a high-aggression environment as an adult, or adult behavioral maturation. Results: Across all three tissues assessed, genes that are differentially expressed as a function of aggression significantly overlap with genes whose expression is modulated by a variety of pathogens and parasitic feeding. In the fat body, and to some degree the midgut, our data specifically support the hypothesis that low aggression resembles a diseased or parasitized state. However, we find little evidence of active infection in individuals from the low aggression group. We also find little evidence that the brain molecular signature of aggression is enriched for genes modulated by social cues that induce aggression in adults. However, we do find evidence that genes associated with adult behavioral maturation are enriched in our brain samples. Conclusions: Results support the hypothesis that low aggression resembles a molecular state of infection. This pattern is most robust in the peripheral fat body, an immune responsive tissue in the honey bee. We find no evidence of acute infection in bees from the low aggression group, suggesting the physiological state characterizing low aggression may instead predispose bees to negative health outcomes when they are exposed to additional stressors. The similarity of molecular signatures associated with the seemingly disparate traits of aggression and disease suggests that these characteristics may, in fact, be intimately tied.

Background Orthopaedic surgeons are confronted with a difficult dilemma: managing acute pain postoperatively and balancing the risk of prescription opioid use. To our knowledge, a prospective performance-improvement project providing opioid-prescription recommendations based on the actual amounts of usual and customary medication consumed following simple knee meniscectomy has not been described. Methods One hundred and two patients undergoing arthroscopic knee meniscectomy prospectively recorded postoperative pain medications in a pain journal. Arthroscopic procedures were performed at 2 centers by 9 fellowship-trained senior surgeons. Various usual and customary prescribing protocols were observed, and the amount of medication consumed was recorded. Prescription and over-the-counter pain medication, quantity, frequency, and visual analog scale (VAS) pain scores were collected. Results One hundred and two patients filled a prescription opioid medication and were included in the study. A total of 3,765 pills were prescribed, and a total of 573.5 were consumed. For the 102 patients who filled a prescription, the average time consuming opioid medication was 2 ± 2 days (range, 0 to 13 days) postoperatively. No cases of persistent use were recorded. Of the 102 patients who filled a prescription, 29.4% did not take any prescription opioids postoperatively. A total of 3,191.5 pills (or 22,183.75 morphine milligram equivalents [MME]) were unused and were potentially available to the community. Conclusions Following simple knee arthroscopy, the amount of prescribed opioid medication exceeds the need for postoperative pain management. In general, 68% of patients require a maximum of 13 pills postoperatively for 6 days. Surgeons should adjust prescribing standards accordingly to limit the amount of prescription opioids available to the community. Furthermore, a comprehensive response to include increased patient screening and monitoring as well as opioid use and disposal education is recommended.

OBJECT Intrathecal baclofen (ITB) is a valuable therapeutic option for patients with spasticity and dystonia. The techniques that place an ITB pump catheter into the subcutaneous fat of a lumbar incision are well described. Because patients who require ITB often have low body fat content, they may be predisposed to catheter-related complications. The senior author used a novel technique to place the catheter in a paraspinal subfascial fashion, and the short-term results were previously published. That study demonstrated no development of hardware erosions, catheter migrations, or CSF leaks within an average follow-up of 5 months. This study followed up on those initial findings by looking at the long-term outcomes since this technique was introduced. METHODS Using the institutional review board-approved protocol, the electronic medical records were reviewed retrospectively for all patients who underwent paraspinal subfascial catheter placement by the senior author. Patients received follow-up with the surgeon at 2 weeks postoperatively and were followed routinely by their physiatrist thereafter. RESULTS Of the 43 patients identified as having undergone surgery by the senior author using the paraspinal subfascial technique between July 2010 and February 2014, 12 patients (27.9%) required reoperation. There were 5 patients (11.6%) who had complications related to the catheter or lumbar incision. No hardware erosions or CSF leaks were identified. These patients received a median follow-up of 3.0 years, with 30 of 43 patients receiving follow-up over 2.0 years. CONCLUSION This follow-up study suggests that the technique of paraspinal subfascial catheter placement translates to long-term decreases in CSF leakage and complications from erosion, infection, and also catheter malfunctions. It does not seem to affect the overall rate of complications.

Cyanobacteria are photosynthetic prokaryotes that are influential in global geochemistry and are promising candidates for industrial applications. Because the livelihood of cyanobacteria is directly dependent upon light, a comprehensive understanding of metabolism in these organisms requires taking into account the effects of day–night transitions and circadian regulation. These events synchronize intracellular processes with the solar day. Accordingly, metabolism is controlled and structured differently in cyanobacteria than in heterotrophic bacteria. Thus, the approaches applied to engineering heterotrophic bacteria will need to be revised for the cyanobacterial chassis. Here, we summarize important findings related to diurnal metabolism in cyanobacteria and present open questions in the field.

Weather has been recognized as a density independent factor influencing the abundance, distribution, and behavior of vertebrates. Male wild turkeys’ (Meleagris gallopavo) breeding behavior includes vocalizations and courtship displays to attract females, the phenology of which can vary with latitude. State biologists design spring turkey-hunting season frameworks centered on annual vocalization patterns to maximize hunter engagement. The Mississippi Department of Wildlife, Fisheries, and Parks has traditionally instituted a statewide, 7-week, spring harvest season. However, hunters routinely argue that different peaks in gobbling activity across the state exist. The objective of this study was to determine whether differences in peak gobbling activity existed across a latitudinal gradient of Mississippi and assess the effect of weather on gobbling. During 2008 and 2009, we conducted a statewide gobbling survey. We used generalized additive mixed models to describe the probability and frequency of gobbling activity within northern and southern regions of the state. We also investigated the effect of daily weather conditions on gobbling activity. Our results revealed an approximate 10–14-day difference in peak gobbling activity between southern and northern Mississippi. The majority of all gobbling activity occurred within the current spring harvest framework. Perhaps more importantly, gobbling activity was more prevalent on days of regionally dry conditions (i.e., less humid) according to the Spatial Synoptic Classification. Our results provide information on gobbling activity phenology relative to hunting-season dates and weather-response information. Our approach may be particularly applicable in states with relatively shorter seasons or highly variable daily weather conditions that moderate gobbling frequency.

The recurrent pattern of light and darkness generated by Earth’s axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses, but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a night-time metabolic program that is essential for S. elongatus to survive in the dark.SignificanceUnderstanding how photosynthetic bacteria respond to and anticipate natural light–dark cycles is necessary for predictive modeling, bioengineering, and elucidating metabolic strategies for diurnal growth. Here, we identify the genetic components that are important specifically under light-dark cycling conditions and determine how a properly functioning circadian clock prepares metabolism for darkness, a starvation period for photoautotrophs. This study establishes that the core circadian clock protein KaiA is necessary to enable rhythmic de-repression of a night-time circadian program.

The broadly conserved signaling nucleotide cyclic di-adenosine monophosphate (c-di-AMP) is essential for viability in most bacteria where it has been studied. However, characterization of the cellular functions and metabolism of c-di-AMP has largely been confined to the class Bacilli, limiting our functional understanding of the molecule among diverse phyla. We identified the cyclase responsible for c-di-AMP synthesis and characterized the molecule’s role in survival of darkness in the model photosynthetic cyanobacterium Synechococcus elongatus PCC 7942. In addition to the use of traditional genetic, biochemical, and proteomic approaches, we developed a high-throughput genetic interaction screen (IRB-Seq) to determine pathways where the signaling nucleotide is active. We found that in S. elongatus c-di-AMP is produced by an enzyme of the diadenylate cyclase family, CdaA, which was previously unexplored experimentally. A cdaA-null mutant experiences increased oxidative stress and death during the nighttime portion of day-night cycles, in which potassium transport is implicated. These findings suggest that c-di-AMP is biologically active in cyanobacteria and has non-canonical roles in the phylum including oxidative stress management and day-night survival. The pipeline and analysis tools for IRB-Seq developed for this study constitute a quantitative high-throughput approach for studying genetic interactions., Author summary Cyclic di-adenosine monophosphate (c-di-AMP) is a molecule that has significant roles in many microorganisms. This work shows the existence of c-di-AMP for the first time in photosynthetic microorganisms, cyanobacteria, and demonstrates its role in survival during the light-to-dark shifts that occur in day-night cycles. Despite the obvious importance of adaptation to these daily cycles for organisms that are fundamentally reliant on light, such as cyanobacteria, understanding of diurnal physiology is lacking because most cyanobacterial research is conducted during growth in constant light. To identify other players in c-di-AMP’s function we developed a low-cost and efficient method for finding interactions between genes. The technique combines one mutation, in this case for the gene that encodes the enzyme for synthesis of c-di-AMP, with thousands of other individual mutations to find pairwise interactions that affect fitness of the resulting mutants. Mutants are tagged with DNA barcodes to allow their survival to be easily tracked in a population of cells. The method enables us to place the function of c-di-AMP within the context of pathways previously known to be involved in day-night survival. Taken together, this work expands the known roles of c-di-AMP, improves our understanding of cyanobacterial survival in day-night cycles, and presents an improved approach for determining genetic interactions.

A characteristic of eusocial bees is a reproductive division of labor in which one or a few queens monopolize reproduction, while her worker daughters take on reproductively altruistic roles within the colony. The evolution of worker reproductive altruism involves indirect selection for the coordinated expression of genes that regulate personal reproduction, but evidence for this type of selection remains elusive. In this study, we tested whether genes coexpressed under queen‐induced worker sterility show evidence of adaptive organization within a model brain transcriptional regulatory network (TRN). If so, this structured pattern would imply that indirect selection on nonreproductive workers has influenced the functional organization of genes within the network, specifically to regulate the expression of sterility. We found that literature‐curated sets of candidate genes for sterility, ranging in size from 18 to 267, show strong evidence of clustering within the three‐dimensional space of the TRN. This finding suggests that our candidate sets of genes for sterility form functional modules within the living bee brain's TRN. Moreover, these same gene sets colocate to a single, albeit large, region of the TRN's topology. This spatially organized and convergent pattern contrasts with a null expectation for functionally unrelated genes to be haphazardly distributed throughout the network. Our meta‐genomic analysis therefore provides first evidence for a truly “social transcriptome” that may regulate the conditional expression of honeybee worker sterility.

We examined testing's ability to enhance adult spelling acquisition, relative to copying and reading. Across 3 experiments in which testing with feedback was compared with copying, the spelling improvement after testing matched that following the same amount of time spent copying. A potent testing advantage, however, was observed for spelling words free-recalled. In the fourth experiment, a large testing advantage for both word free recall and spelling was observed, versus reading. Subjects also generally preferred testing and rated it as more effective than copying or reading. The equivalent performance of testing and copying for spelling contrasts with prior work involving children and suggests that retrieval practice may not be the only effective mechanism for spelling skill acquisition. Rather, we suggest that the critical learning event for spelling is focused study on phoneme-to-grapheme mappings for previously unlearned letter sequences. For adults with extensive spelling expertise, focused study is more automatic during both copying and testing with feedback than for individuals with beginning spelling skills. Reading, however, would not be expected to produce efficient focused study of phoneme-to-grapheme mappings, regardless of expertise level. Overall, adult spelling skill acquisition benefits both from testing and copying, and substantially less from reading.

A systematic approach to knee problems by anatomical location of symptoms helps the physician make a specific diagnosis. The aim is to keep the runner running, so treatment is conservative whenever possible.

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light-dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.

The model cyanobacterium, Synechococcus elongatus PCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism in S. elongatus using random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism. The model explicitly describes photon absorption and accounts for shading, resulting in the characteristic linear growth curve of photoautotrophs. GEM predictions of gene essentiality were compared with data obtained from recent dense-transposon mutagenesis experiments. This dataset allowed major improvements to the accuracy of the model. Furthermore, discrepancies between GEM predictions and the in vivo dataset revealed biological characteristics, such as the importance of a truncated, linear TCA pathway, low flux toward amino acid synthesis from photorespiration, and knowledge gaps within nucleotide metabolism. Coupling of strong experimental support and photoautotrophic modeling methods thus resulted in a highly accurate model of S. elongatus metabolism that highlights previously unknown areas of S. elongatus biology.

SummaryThe function of nearly half of all protein-coding genes identified in bacterial genomes remains unknown. To systematically explore the functions of these proteins, we generated saturated transposon mutant libraries from 25 diverse bacteria and we assayed mutant phenotypes across hundreds of distinct conditions. From 3,903 genome-wide mutant fitness assays, we obtained 14.9 million gene phenotype measurements and we identified a mutant phenotype for 8,487 proteins with previously unknown functions. The majority of these hypothetical proteins (57%) had phenotypes that were either specific to a few conditions or were similar to that of another gene, thus enabling us to make informed predictions of protein function. For 1,914 of these hypothetical proteins, the functional associations are conserved across related proteins from different bacteria, which confirms that these associations are genuine. This comprehensive catalogue of experimentally-annotated protein functions also enables the targeted exploration of specific biological processes. For example, sensitivity to a DNA-damaging agent revealed 28 known families of DNA repair proteins and 11 putative novel families. Across all sequenced bacteria, 14% of proteins that lack detailed annotations have an ortholog with a functional association in our data set. Our study demonstrates the utility and scalability of high-throughput genetics for large-scale annotation of bacterial proteins and provides a vast compendium of experimentally-determined protein functions across diverse bacteria.

The need to assess and manage airway issues is not an infrequent event in the ICU. Acute or progressive respiratory distress or respiratory failure in the decompensating patient often requires special equipment, medications, skills, and knowledge to successfully manage. The most common events that require intervention include cardiac or pulmonary events, bleeding, airway edema, hypoventilation, and hypercapnia. These events often require different skills and equipment in the treatment of the incident.

Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼ 250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA(Leu), which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism's physiology and defines the essential gene set required for the growth of a photosynthetic organism.

© 2015, National Academy of Sciences. All rights reserved. Synechococcus elongatus PCC 7942 is a genetically tractable model cyanobacterium that has been engineered to produce industrially relevant biomolecules and is the best-studied model for a prokaryotic circadian clock. However, the organism is commonly grown in continuous light in the laboratory, and data on metabolic processes under diurnal conditions are lacking. Moreover, the influence of the circadian clock on diurnal metabolism has been investigated only briefly. Here, we demonstrate that the circadian oscillator influences rhythms of metabolism during diurnal growth, even though light-dark cycles can drive metabolic rhythms independently. Moreover, the phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Although RpaA activity is important for carbon degradation at night, KaiC is dispensable for those processes. Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies a ΔrpaA strain. Inhibition of RpaA by the oscillator in the morning suppresses metabolic processes that normally are active at night, and kaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.