Efficacy of a Lidocaine-Impregnated Elastrator Band for Castration and Tail Docking in Lambs.

The primary objective of this study was to demonstrate the non-inferiority between lidocaine-impregnated ligation bands (LLBs) and control bands (CBs) with respect to the efficacy of castration and tail docking. Secondary objectives were to compare castration and tail-docking success, evaluate local site reactions, and compare average daily gain (ADG) between the treatment groups. A total of 238 male lambs were enrolled and randomly assigned to receive LLBs or CBs on their tail and scrotum. Lambs were weighed, had a health assessment, and the band site was observed on -3, 7, 14, 21, 28, 35, and 42 days after the bands were applied. A linear regression model was built to assess average daily gain, whereas a repeated measures model was used to evaluate body weight differences at each of the measured timepoints. Furthermore, logistic regression models were used to evaluate associations with casting outcomes. Few differences were noted between treatment groups with respect to casting success for the scrotum and tail and ADG over the entire experimental period. Non-inferiority calculations demonstrated no differences in tail docking and scrotal casting success, with casting occurring for the majority of animals by d 21 and d 42 for castration and tail docking, respectively. However, lambs receiving LLBs gained more weight from d -3 to 7 (+0.03 kg/d; 95% CI: 0 to 0.07), which may be an indication of effective pain control during the first week following band application. Overall, the use of an LLB does not affect the time to successful casting of the tail and could improve short-term growth when compared to a control band. Further studies are needed to compare LLBs to multimodal methods of pain relief.

Assessment of the Effective Tissue Concentrations of Injectable Lidocaine and a Lidocaine-Impregnated Latex Band for Castration in Calves.

This study aimed to assess the effective tissue concentrations of the current standard of care for pain mitigation in calves during castration (injectable lidocaine) and to assess the ability of a lidocaine-loaded elastration band (LLB) to deliver effective concentrations into the scrotal tissue over time. This study comprised two different trials: (1) effective concentrations of injectable lidocaine in the scrotal tissue; and (2) the in vivo delivery of effective concentrations of lidocaine from LLBs placed on the calf scrotums. Sensation in the scrotal tissue was assessed by electrocutaneous stimulation. Injectable lidocaine allowed for short-term anesthesia for up to 60 min, highlighting the importance of finding additional strategies to mitigate long-term pain. An elastomeric ligation band impregnated with lidocaine could provide a suitable alternative, as it yielded tissue levels of lidocaine that approached EC50 and exceeded EC95 at 2 and 72 h following application, respectively, and remained above those levels for at least 28 days after application. Further studies are warranted to compare the use of LLBs to injectable local anesthetics.

Photodynamic Inactivation of Foodborne Bacteria: Screening of 32 Potential Photosensitizers.

The development of novel antimicrobial technologies for the food industry represents an important strategy to improve food safety. Antimicrobial photodynamic disinfection (aPDD) is a method that can inactivate microbes without the use of harsh chemicals. aPDD involves the administration of a non-toxic, light-sensitive substance, known as a photosensitizer, followed by exposure to visible light at a specific wavelength. The objective of this study was to screen the antimicrobial photodynamic efficacy of 32 food-safe pigments tested as candidate photosensitizers (PSs) against pathogenic and food-spoilage bacterial suspensions as well as biofilms grown on relevant food contact surfaces. This screening evaluated the minimum bactericidal concentration (MBC), minimum biofilm eradication concentration (MBEC), and colony forming unit (CFU) reduction against Salmonella enterica, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas fragi, and Brochothrix thermosphacta. Based on multiple characteristics, including solubility and the ability to reduce the biofilms by at least 3 log10 CFU/sample, 4 out of the 32 PSs were selected for further optimization against S. enterica and MRSA, including sunset yellow, curcumin, riboflavin-5'-phosphate (R-5-P), and erythrosin B. Optimized factors included the PS concentration, irradiance, and time of light exposure. Finally, 0.1% w/v R-5-P, irradiated with a 445 nm LED at 55.5 J/cm2, yielded a "max kill" (upwards of 3 to 7 log10 CFU/sample) against S. enterica and MRSA biofilms grown on metallic food contact surfaces, proving its potential for industrial applications. Overall, the aPDD method shows substantial promise as an alternative to existing disinfection technologies used in the food processing industry.

Assessment of the Pharmacokinetics and Pharmacodynamics of Injectable Lidocaine and a Lidocaine-Impregnated Latex Band for Castration and Tail Docking in Lambs.

The objectives of this study were to assess the pharmacokinetics and pharmacodynamics of the current standard-of-care for pain mitigation in lambs during castration and tail docking (injectable lidocaine) and assess the ability of Lidocaine-Loaded Bands (LLBs) to deliver therapeutic concentrations into the contacted tissues over time. The study was comprised of four different trials: (1) investigation of in vitro release of lidocaine from LLBs; (2) pharmacokinetics and pharmacodynamics of injectable lidocaine in scrotal and tail tissue; (3) pharmacokinetics and pharmacodynamics of in vivo delivery of lidocaine with LLBs placed on the tail and scrotum of lambs; and (4) a "proof-of-concept" study comparing the sensation of control- versus LLB-banded tail tissue over time. The use of injectable lidocaine provides effective short-term anesthesia for 120 to 180 min following the injection; however, additional strategies are needed to manage long-term pain. The use of an LLB could provide an alternative where tissue lidocaine concentrations meet or exceed the EC50 for at least 21-28 days and, based on electrostimulation data, provides local anesthesia for at least 3 days when compared to a control band. Further studies are needed to compare the use of an injectable local anesthetic to the LLBs.

Evaluation of a novel dipotassium phosphate bolus for treatment of metabolic disorders in dairy cattle.

A dipotassium phosphate bolus (K Phos-Boost) has been developed to treat both hypophosphatemia and hypokalemia, as the clinical signs of both conditions are similar and occur in the early post-partum period. The objective of this research was to evaluate the efficacy and application of the bolus for prevention and treatment of metabolic diseases that are common in dairy production systems. Study 1 (Pharmacokinetic study): Healthy post-partum cows were either untreated or received two K Phos-Boost boluses at times 0, 24, and 48 h. Blood was taken at t = 0, 2-, 4-, 6-, 8-, 10-, 24-, and 52-h post-treatment for analysis of total serum minerals. There was an increase in serum phosphorous to normal levels within 2 h of treatment with the bolus, but control cows remained hypophosphatemic. Serum potassium was significantly elevated 2 h after bolus administration relative to control, while calcium, magnesium, sodium, and chloride levels were not affected by the K Phos-Boost bolus. Study 2 (Downer Cow Treatment): K Phos-Boost boluses were provided to cows that were unresponsive to intravenous calcium therapy and had been unable to stand for over 24 h ("downer cows"). Most cows (16 of 19) treated with two boluses were standing without assistance between 1 and 24 h after treatment and the serum phosphorous was increased to normal levels in five of five tested animals. Study 3 (Ketosis Treatment): cows with clinical ketosis were provided with propylene glycol and K Phos-Boost boluses (n = 29) or only propylene glycol (n = 23). Cows treated with the K Phos-Boost bolus showed a more rapid recovery by increased milk production (3.9 kg/day) and rumination rate (97 min/day). Study 4 (Health Promotion): cows in herds with >40% post-partum hypophosphatemia received K Phos-Boost boluses (n = 130) or no treatment (n = 146) following calving. There was a trend for treated 2nd-lactation animals to have higher milk production after 30 DIM (49.1 vs. 46.2 kg/day; P = 0.09). There were no significant differences between control and bolus treated animals in the incidence of subclinical ketosis, post-calving total health events, or culling rates. The K Phos-Boost bolus is a novel product and has the potential to treat and prevent several important metabolic diseases in dairy cattle. The studies described in this paper are early investigations and further research should be conducted to demonstrate the applications of a dipotassium phosphate bolus in dairy cows.

Impact of Dystocia on Milk Production, Somatic Cell Count, Reproduction and Culling in Holstein Dairy Cows.

This study investigated the effects of dystocia on milk production, somatic cell count, reproductivity, disease, and milk production. A total of 2159 cows across 21 dairy farms in Alberta, Canada were enrolled in this study. Multivariable models were created to explore associations between outcome variables and calving ease score. In total, 89.5% of calvings were unassisted, 6.1% were an easy pull, and 4.3% were a moderate-hard pull. Cows that had a moderate-hard pull produced 4.01 kg less milk, 0.12 kg less volume of milk fat, and 0.12 kg less milk protein per day than those that had an unassisted calving. No difference was found between calving ease groups with respect to SCC. Cows with a moderate or hard pull produced 510 kg less milk per lactation than unassisted cows. Cows with a moderate to high level of assistance at birth had a higher hazard of being culled over the duration of their lactation. Cows with an easy pull had increased odds of developing a retained placenta. It is evident that assistance at calving, particularly a moderate-hard pull, is associated with significant impacts on future milk production and risk of being culled; therefore, efforts should be made to minimize dystocia and prevent these impacts.

Genetic architecture and temporal analysis of Caenorhabditis briggsae hybrid developmental delay.

Identifying the alleles that reduce hybrid fitness is a major goal in the study of speciation genetics. It is rare to identify systems in which hybrid incompatibilities with minor phenotypic effects are segregating in genetically diverse populations of the same biological species. Such traits do not themselves cause reproductive isolation but might initiate the process. In the nematode Caenorhabditis briggsae, a small percent of F2 generation hybrids between two natural populations suffer from developmental delay, in which adulthood is reached after approximately 33% more time than their wild-type siblings. Prior efforts to identify the genetic basis for this hybrid incompatibility assessed linkage using one or two genetic markers on chromosome III and suggested that delay is caused by a toxin-antidote element. Here, we have genotyped F2 hybrids using multiple chromosome III markers to refine the developmental delay locus. Also, to better define the developmental delay phenotype, we measured the development rate of 66 F2 hybrids and found that delay is not restricted to a particular larval developmental stage. Deviation of the developmental delay frequency from hypothetical expectations for a toxin-antidote element adds support to the assertion that the epistatic interaction is not fully penetrant. Our mapping and refinement of the delay phenotype motivates future efforts to study the genetic architecture of hybrid dysfunction between genetically distinct populations of one species by identifying the underlying loci.

Dissolution Rates of Calcium Boluses and Their Effects on Serum Calcium in Dairy Cattle.

PURPOSE: Calcium supplement boluses vary greatly in content and bioavailability. METHODS: In vivo dissolution and bioavailability studies were conducted to compare commercial calcium supplement boluses with various contents of calcium chloride and calcium carbonate. The products studied included: Bolus 1 (high calcium chloride, no calcium carbonate), Bolus 2 (medium calcium chloride, medium calcium carbonate), and Bolus 3 (low calcium chloride, high calcium carbonate). A bolus was placed in a pre-weighed coarse mesh net for 30, 60, 90, 120, 180, and 240 minutes to measure dissolution rates in the rumen of fistulated animals. To measure calcium uptake, 27 Holstein cows (second and third lactation) were randomly allocated to one of three oral calcium protocols: Treatment 1 (two high calcium chloride boluses at time 0); Treatment 2 (one high calcium chloride bolus at time 0 with a second bolus 12 hours later); or Treatment 3 (two high calcium carbonate boluses at time 0). Treatments were initiated within 12 hours following calving and this was considered time 0. RESULTS: Bolus 1 was the quickest to dissolve (<90 minutes), followed by Bolus 2 (<240 minutes). The high calcium carbonate bolus (Bolus 3) remained after 240 minutes in vivo with a minimum of 75% of the original bolus weight still intact. Cows with severe hypocalcemia (<1.8 mmol/L) responded with a higher serum calcium increase than cows with milder hypocalcemia (>1.8 mmol/L, <2.12 mmol/L). The high calcium carbonate bolus group (Treatment 3) did not show a rapid increase in serum calcium as compared to the high calcium chloride groups (Treatments 1 and 2). The animals receiving Treatment 1 had a greater and more persistent serum calcium response than animals receiving Treatment 2. CONCLUSION: The study outcome suggests that calcium chloride/calcium sulfate boluses are more effective at generating a serum calcium response than boluses containing high amounts of calcium carbonate and that two boluses administered rapidly after calving may be more effective than the traditional treatment of giving 2 boluses 12 hours apart.

Depletion of eukaryotic initiation factor 5B (eIF5B) reprograms the cellular transcriptome and leads to activation of endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK).

During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.

Development and Field Validation of Lidocaine-Loaded Castration Bands for Bovine Pain Mitigation.

Castration is among the most common management procedures performed in the dairy and beef cattle industries and is mainly performed by surgery or elastic banding. Despite the various benefits of castration, all methods produce pain and distress. Castration by banding is simple, inexpensive, produces fewer complications, and can be performed in a high-throughput manner. Because lidocaine, a local anesthetic, can be delivered to trauma sites topically, we have formulated lidocaine-loaded castration bands (LLBs) to deliver local pain relief to calves during banded castration. The initial lidocaine content of three band types developed was between 80 and 200 mg per band. The transfer kinetics of lidocaine into tissue was determined in vitro, indicating a rapid release for the first 30 min, followed by a slow release lasting at least 48 h. Furthermore, the lidocaine delivery and pain mitigation effects of these LLBs were compared to standard lidocaine injections in vivo. Field studies indicated that LLBs performed at least as well as lidocaine injections for short-term lidocaine delivery into tissues and pain mitigation. Moreover, LLBs significantly outperformed lidocaine injections for long-term delivery and pain mitigation. The concentrations of lidocaine in the LLB-treated tissue samples were generally in the range of 0.5-3.5 mg of lidocaine per gram of tissue and were overall highest after 6 h. Lidocaine-loaded elastration bands deliver therapeutic quantities of lidocaine into scrotal tissues over a period of at least seven days in cattle. This approach would provide long-term pain mitigation to the animals and, by avoiding surgery or the administration of injections, would also decrease the time and handling costs for the producer.

Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution.

BACKGROUND: Heteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to degeneration of the Y chromosome. The progression of degeneration is not well understood, as complete sequence assemblies of heteromorphic Y chromosomes have only been generated across a handful of taxa with highly degenerate sex chromosomes. Here, we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old and at an intermediate stage of degeneration. Our previous work identified that the non-recombining region between the X and the Y spans approximately 17.5 Mb on the X chromosome. RESULTS: We combine long-read sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome BAC clones. We find three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The threespine stickleback Y shows convergence with more degenerate sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we find no evidence for large amplicons identified in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy). CONCLUSIONS: Together, our work shows that the evolutionary forces shaping sex chromosomes can cause relatively rapid changes in the overall genetic architecture of Y chromosomes.

Comparison of the efficacy of silver-based antimicrobial burn dressings in a porcine model of burn wounds.

PURPOSE: A variety of silver-based antimicrobial dressings are available on the market and are commonly used to prevent infection. Such prophylaxis is particularly important in treating burns, yet there is a paucity of evidence confirming the efficacy of commercially available dressingsin vivo. We describe here an in vivo porcine model of burns, which we use to test the antimicrobial efficacy of three common wound dressings and a control. PROCEDURES: Domestic Yorkshire-cross pigs were medicated for pain management before inflicting burns with a heated brass rod. The wounds were artificially challenged with a mixture of two pathogens commonly associated with burn wound infection:Staphylococcus aureus and Pseudomonas aeruginosa. The following dressing materials were sutured in place: gauze, nanocrystalline silver, silver-plated nylon, and polyethylene/polyester coated with high-oxidation silver salts. After 1 and 3 days, the wounds were assessed for erythema, swelling, and re-epithelialization, tissue was biopsied to determine the recovery of the challenge microorganisms, and histology was performed. We also examined the number of microorganisms present on the dressings themselves. RESULTS: Histology indicated that 30 s was sufficient to produce burns extending into the deep dermal layer. After 3 days, nanocrystalline silver and silver-plated nylon led to slightly reduced swelling relative to simple gauze, although none of the dressings significantly affected erythema or wound re-epithelialization. All the dressings led to decreased recovery of the challenge organisms from the burn tissue, relative to simple gauze. However, the magnitude of the reduction was greatest for nanocrystalline silver (log10 reduction = 4-5); additionally, only nanocrystalline silver gave a statistically significant decrease (P = 0.02). Notably, the antimicrobial effect for all dressings was reduced by Day 3 relative to Day 1. Similar trends were observed for microbial retention on the dressings themselves. CONCLUSION: Nanocrystalline silver-based wound dressings generally outperformed silver-plated nylon and high-oxidation silver salts in thisin vivo model of burn wounds. Relative to prophylactic use, it may be advisable to change the dressings more frequently when treating an infected wound.

Cellular roles of the human Obg-like ATPase 1 (hOLA1) and its YchF homologs.

P-loop NTPases comprise one of the major superfamilies of nucleotide binding proteins, which mediate a variety of cellular processes, such as mRNA translation, signal transduction, cell motility, and growth regulation. In this review, we discuss the structure and function of two members of the ancient Obg-related family of P-loop GTPases: human Obg-like ATPase 1 (hOLA1), and its bacterial/plant homolog, YchF. After a brief discussion of nucleotide binding proteins in general and the classification of the Obg-related family in particular, we discuss the sequence and structural features of YchF and hOLA1. We then explore the various functional roles of hOLA1 in mammalian cells during stress response and cancer progression, and of YchF in bacterial cells. Finally, we directly compare and contrast the structure and function of hOLA1 with YchF before summarizing the future perspectives of hOLA1 research. This review is timely, given the variety of recent studies aimed at understanding the roles of hOLA1 and YchF in such critical processes as cellular-stress response, oncogenesis, and protein synthesis.

Eukaryotic initiation factor 5B (eIF5B) regulates temozolomide-mediated apoptosis in brain tumour stem cells (BTSCs).

Glioblastoma multiforme (GBM) is among the deadliest cancers, owing in part to complex inter- and intra-tumor heterogeneity and the presence of a population of stem-like cells called brain tumour stem cells (BTSCs/BTICs). These cancer stem cells survive treatment and confer resistance to the current therapies - namely, radiation and the chemotherapeutic, temozolomide (TMZ). TMZ induces cell death by alkylating DNA, and BTSCs resist this mechanism via a robust DNA damage response. Hence, recent studies aimed to sensitize BTSCs to TMZ using combination therapy, such as inhibition of DNA repair machinery. We have previously demonstrated in established GBM cell lines that eukaryotic initiation factor 5B (eIF5B) promotes the translation of pro-survival and anti-apoptotic proteins. Consequently, silencing eIF5B sensitizes these cells to TRAIL-induced apoptosis. However, established cell lines do not always recapitulate the features of human glioma. Therefore, we investigated this mechanism in patient-derived BTSCs. We show that silencing eIF5B leads to increased TMZ sensitivity in two BTSC lines: BT25 and BT48. Depletion of eIF5B decreases the levels of anti-apoptotic proteins in BT48 and sensitizes these cells to TMZ-induced activation of caspase-3, cleavage of PARP, and apoptosis. We suggest that eIF5B represents a rational target to sensitize GBM tumors to the current standard-of-care.

The LhrC sRNAs control expression of T cell-stimulating antigen TcsA in Listeria monocytogenes by decreasing tcsA mRNA stability.

The bacterial pathogen Listeria monocytogenes encodes seven homologous small regulatory RNAs, named the LhrC family of sRNAs. The LhrCs are highly induced under infection-relevant conditions and are known to inhibit the expression of multiple target mRNAs encoding virulence-associated surface proteins. In all cases studied so far, the LhrCs use their CU-rich regions for base pairing to complementary AG-rich sequences of the ribosomal binding site (RBS) of specific target mRNAs. Consequently, LhrC-mRNA interaction results in inhibition of translation followed by mRNA degradation, corresponding to the canonical model for sRNA-mediated gene regulation in bacteria. Here, we demonstrate that the LhrC sRNAs employ a different regulatory mechanism when acting to down-regulate the expression of tcsA, encoding a T cell-stimulating antigen. In this case, LhrC base pairs to an AG-rich site located well upstream of the RBS in tcsA mRNA. Using an in vitro translation assay, we found that LhrC could not prevent the ribosome from translating the tcsA messenger. Rather, the LhrC sRNAs act to decrease the half-life of tcsA mRNA in vivo. Importantly, LhrC-mediated destabilization of tcsA mRNA relies on an intact LhrC binding site near the 5´-end of the tcsA mRNA and occurs independently of translation. Based on these findings, we propose an alternative mechanism for LhrC-mediated control in L. monocytogenes that relies solely on sRNA-induced degradation of a target mRNA.

Eukaryotic initiation factor 5B (eIF5B) provides a critical cell survival switch to glioblastoma cells via regulation of apoptosis.

Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, -9, and -7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIPS. We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.

Eukaryotic Initiation Factor 5B (eIF5B) Cooperates with eIF1A and eIF5 to Facilitate uORF2-Mediated Repression of ATF4 Translation.

A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B-which can substitute for eIF2 in delivering Met-tRNAi-leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.

The Genetic Architecture of Intra-Species Hybrid Mito-Nuclear Epistasis.

Genetic variants that are neutral within, but deleterious between, populations (Dobzhansky-Muller Incompatibilities) are thought to initiate hybrid dysfunction and then to accumulate and complete the speciation process. To identify the types of genetic differences that might initiate speciation, it is useful to study inter-population (intra-species) hybrids that exhibit reduced fitness. In Caenorhabditis briggsae, a close relative of the nematode C. elegans, such minor genetic incompatibilities have been identified. One incompatibility between the mitochondrial and nuclear genomes reduces the fitness of some hybrids. To understand the nuclear genetic architecture of this epistatic interaction, we constructed two sets of recombinant inbred lines by hybridizing two genetically diverse wild populations. In such lines, selection is able to eliminate deleterious combinations of alleles derived from the two parental populations. The genotypes of surviving hybrid lines thus reveal favorable allele combinations at loci experiencing selection. Our genotype data from the resulting lines are consistent with the interpretation that the X alleles participate in epistatic interactions with autosomes and the mitochondrial genome. We evaluate this possibility given predictions that mitochondria-X epistasis should be more prevalent than between mitochondria and autosomes. Our empirical identification of inter-genomic linkage disequilibrium supports the body of literature indicating that the accumulation of mito-nuclear genetic incompatibilities might initiate the speciation process through the generation of less-fit inter-population hybrids.

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding-a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Paternal Mitochondrial Transmission in Intra-Species Caenorhabditis briggsae Hybrids.

To study mitochondrial-nuclear genetic interactions in the nematode Caenorhabditis briggsae, our three laboratories independently created 38 intra-species cytoplasmic-nuclear hybrid (cybrid) lines. Although the cross design combines maternal mitotypes with paternal nuclear genotypes, eight lines (21%) unexpectedly contained paternal mitotypes. All eight share in common ancestry of one of two genetically related strains. This unexpected parallel observation of paternal mitochondrial transmission, undesirable given our intent of creating cybrids, provides a serendipitous experimental model and framework to study the molecular and evolutionary basis of uniparental mitochondrial inheritance.