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Background: The coronavirus (COVID-19) pandemic and associated public health restrictions created unprecedented challenges for parents and their young dependent children. While psycho-social impacts of natural disasters on families are well studied, a typography of parent specific concerns in the COVID-19 context was yet to be articulated. Objective: Using a bioecological systems framework, we adopted a mixed-methods research design to examine parents' core concerns about the impacts of the pandemic on themselves and their children, testing for differences in concern foci of mothers compared with fathers. Method: Data were drawn from the Australian Temperament Project Generation 3 (ATPG3) study, a prospective study of children born to a 40-year population-based cohort. During enforced COVID-19 lockdown restrictions between May to September 2020, ATPG3 parents (n = 516) were surveyed about their own and their children's functioning in the context of the pandemic. Subject of qualitative content analysis, parents (n = 192) experiencing wellbeing concerns offered additional free-text responses about the nature of stress impacting themselves and their child/ren. Results: Parents reported far-reaching impacts for themselves and their children across multiple bioecological systems. Core concerns were for emotional rather than physical health, specifically, for parents this was represented by increased levels of anxiety and stress, and for children, these impacts were notable from a developmental perspective. Greater frequency of parenting related concern was expressed by mothers in comparison to fathers. Conclusions: Findings demonstrate the complex and interrelated nature of multi-systemic and gendered stressors impacting parents during the pandemic, and importantly point to modifiable risk factors which may inform early risk detection efforts.

Abstract Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.

Abstract Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Abstract Promoter-proximal pausing by RNA Pol II is a rate-determining step in gene transcription that is hypothesized to be a prominent point at which regulatory factors act. The pausing factor NELF is known to induce and stabilize pausing, but not all kinds of pausing are NELF-mediated. Here, we find that NELF-depleted Drosophila melanogaster cells functionally recapitulate the NELF-independent pausing we previously observed in fission yeast (which lack NELF). Critically, only NELF-mediated pausing establishes a strict requirement for Cdk9 kinase activity for the release of paused Pol II into productive elongation. Upon inhibition of Cdk9, cells with NELF efficiently shutdown gene transcription, while in NELF-depleted cells, defective, non-productive transcription continues unabated. By introducing a strict checkpoint for Cdk9, the evolution of NELF was likely critical to enable increased regulation of Cdk9 in higher eukaryotes, as Cdk9 availability can be restricted to limit gene transcription without inducing wasteful, non-productive transcription.

Abstract Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3–5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.

Sea turtle nesting beaches are experiencing increased sand temperatures as climate change progresses. In one major green turtle (Chelonia mydas) nesting beach in the northern Great Barrier Reef, over 99 percent of hatchlings are female. The effects of contaminants on sea turtle hatchling sex determination are not often explored. Liver samples were collected from green turtle hatchlings that were sacrificed for histological sex determination in a parallel study on the effects of sand cooling on sex ratios, and analysed for trace elements via acid digestion and organic contaminants via in vitro cytotoxicity bioassays. Chromium, antimony, barium, and cadmium have previously been demonstrated to be estrogenic, and concentrations of these elements were used to calculate three estrogenic indexes for each clutch: predicted relative estrogenic potency (PEEQA), the sum of percent trace elements above the median of all samples (TEOM), and the sum of percent estrogenic elements above the median of all samples (EstroEOM). Excluding an outlier clutch, cadmium, antimony, and EstroEOM had significant positive relationships with sex ratio deviation. Mean clutch cobalt, lead, antimony and barium, also had a significant positive relationship with clutch sex ratio. There was no relationship between in vitro cytotoxicity of liver extracts and sex ratio, however, 9% of hatchlings had organic contaminants high enough to suggest potential cellular damage. Contaminant effects on sex determination are likely to be caused by a mixture of contaminant interactions as well as temperature. Many trace elements detected in this study have also been linked to negative health effects on hatchlings in previous studies. Considering the risks of feminization due to climate change and potential contaminant effects on hatchling health and sex determination, future studies exploring contaminant effects on sea turtle hatchling sex determination are recommended.

Abstract Introduction Aimed to develop a simple and robust volumetric modulated arc radiotherapy (VMAT) solution for comprehensive lymph node (CLN) breast cancer without increase in low‐dose wash. Methods Forty CLN‐breast patient data sets were utilised to develop a knowledge‐based planning (KBP) VMAT model, which limits low‐dose wash using iterative learning and base‐tangential methods as benchmark. Another twenty data sets were employed to validate the model comparing KBP‐generated ipsilateral VMAT (ipsi‐VMAT) plans against the benchmarked hybrid (h)‐VMAT (departmental standard) and bowtie‐VMAT (published best practice) methods. Planning target volume (PTV), conformity/homogeneity index (CI/HI), organ‐at‐risk (OAR), remaining‐volume‐at‐risk (RVR) and blinded radiation oncologist (RO) plan preference were evaluated. Results Ipsi‐ and bowtie‐VMAT plans were dosimetrically equivalent, achieving greater nodal target coverage (P

Background and purpose: Deep inspiration breath-hold (DIBH) is a technique that is widely utilised to spare the heart and lungs during breast radiotherapy. In this study, a method was developed to validate directly the intrafraction accuracy of DIBH during breast volumetric modulated arc therapy (VMAT) via internal chest wall (CW) monitoring. Materials and methods: In-house software was developed to automatically extract and compare the treatment position of the CW in cine-mode electronic portal image device (EPID) images with the planned CW position in digitally reconstructed radiographs (DRR) for breast VMAT treatments. Feasibility of this method was established by evaluating the percentage of total dose delivered to the target volume when the CW was sufficiently visible for monitoring. Geometric accuracy of the approach was quantified by applying known displacements to an anthropomorphic thorax phantom. The software was used to evaluate (offline) the geometric treatment accuracy for ten patients treated using real-time position management (RPM)-guided DIBH. Results: The CW could be monitored within the tangential sub-arcs which delivered a median 89% (range 73% to 97%) of the dose to target volume. The phantom measurements showed a geometric accuracy within 1 mm, with visual inspection showing good agreement between the software-derived and user-determined CW positions. For the RPM-guided DIBH treatments, the CW was found to be within ±5 mm of the planned position in 97% of EPID frames in which the CW was visible. Conclusion: An intrafraction monitoring method with sub-millimetre accuracy was successfully developed to validate target positioning during breast VMAT DIBH.

Abstract Understanding how climate change impacts species and ecosystems is integral to conservation. When studying impacts of climate change, warming temperatures are a research focus, with much less attention given to extreme weather events and their impacts. Here, we show how localized, extreme rainfall events can have a major impact on a species that is endangered in many parts of its range. We report incubation temperatures from the world's largest green sea turtle rookery, during a breeding season when two extreme rainfall events occurred. Rainfall caused nest temperatures to drop suddenly and the maximum drop in temperature for each rain‐induced cooling averaged 3.6°C (n = 79 nests, min = 1.0°C, max = 7.4°C). Since green sea turtles have temperature‐dependent sex determination, with low incubation temperatures producing males, such major rainfall events may have a masculinization effect on primary sex ratios. Therefore, in some cases, extreme rainfall events may provide a “get‐out‐of‐jail‐free card” to avoid complete feminization of turtle populations as climate warming continues.

Radiological water equivalence of solid phantoms used for radiotherapy is often desired, but is non-trivial to achieve across the range of therapeutic energies. This study evaluated the water equivalence of a new solid phantom material in beam qualities relevant to radiotherapy applications. In-phantom measured depth distributions were compared to that in water to assess the relative attenuation and scatter characteristics of the material. The phantom material was found to be dosimetrically equivalent to water within (1.0 ± 1.0)% for megavoltage photon beam qualities, (1.5 ± 1.3)% for megavoltage electron beam qualities, (1.5 ± 1.5)% for medium-energy kilovoltage X-rays and (3.0 ± 1.5)% for low-energy kilovoltage X-rays.

Hearing loss is a genetically and phenotypically heterogeneous disorder. The purpose of this study was to determine the genetic cause underlying hearing loss in four Ashkenazi Jewish families. We screened probands from each family using a combination of targeted mutation screening and exome sequencing to identifiy the genetic cause of hearing loss in each family. We identified four variants in MYO15A, two novel variants never previously linked to deafness (c.7212+5G>A and p.Leu2532ArgfsTer37) and two recurrent variants (p.Tyr2684His and p.Gly3287Gly). One family showed locus heterogeneity, segregrating two genetic forms of hearing loss. Mini-gene assays revealed the c.7212+5G>A variant results in abnormal splicing and is most likely a null allele. We show that families segregrating the p.Gly3287Gly variant show both inter and intra-familial phenotypic differences. These results add to the list of MYO15A deafness-causing variants, further confirm the pathogenicity of the p.Gly3287Gly variant and shed further light on the genetic etiology of hearing loss in the Ashkenazi Jewish population.

AbstractControl of gene expression is fundamental at every level of cell function. Promoter-proximal pausing and divergent transcription at promoters and enhancers, which are prominent features in animals, have only been studied in a handful of research experiments in plants. PRO-Seq analysis in cassava (Manihot esculenta

Summary: Mutations in the gene encoding the type II transmembrane protease 3 (TMPRSS3) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3-related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3-KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders. : The role of TMPRSS3 in auditory hair cell (HC) remains unclear. Nelson and colleagues generated stem cell-derived inner ear organoids with Tmprss3 mutations, which demonstrate that (1) inner ear organoids exhibit comparative effects of the genetic abnormality to its in vivo counterparts, (2) Tmprss3 mutations lead to HC apoptosis and reduced BK channels, and (3) TMPRSS3 localizes to the cell membrane. Keywords: deafness, degeneration, disease modeling, embryonic stem cell, hair cell, inner ear, organoid, TMPRSS3, vestibular system

Abstract Background Liver tumors are subject to motion with respiration, which is typically accounted for by increasing the target volume. The prescription dose is often reduced to keep the mean liver dose under a threshold level to limit the probability of radiation induced liver toxicity. A retrospective planning study was performed to determine the potential clinical gains of removal of respiratory motion from liver SABR treatment volumes, which may be achieved with gating or tumor tracking. Methods Twenty consecutive liver SABR patients were analysed. The treated PTV included the GTV in all phases of respiration (ITV) with a 5 mm margin. The goal prescription was 50Gy/5# (BED 100 Gy10) but was reduced by 2.5 Gy increments to meet liver dose constraints. Elimination of motion was modelled by contouring the GTV in the expiration phase only, with a 5 mm PTV margin. All patients were replanned using the no-motion PTV and tumor dose was escalated to higher prescription levels where feasible given organ-at-risk constraints. For the cohort of patients with metastatic disease, BED gains were correlated to increases in tumour control probability (TCP). The effect of the gradient of the TCP curve on the magnitude of TCP increase was evaluated by repeating the study for an additional prescription structure, 54Gy/3# (BED 151 Gy10). Results Correlation between PTV size and prescribed dose exists; PTVs encompassing

While post-translational modifications of the RNA polymerase II complex regulate gene expression, the exact mechanism remains unknown. Here, the authors use altered-specificity kinase mutations to examine the role of Cdk9, Mcs6/Cdk7, and Lsk1/Cdk12 on transcription at base-pair resolution in yeast.

Abstract Defects of CIB2, calcium‐ and integrin‐binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type‐IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2−/− mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2−/− mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair‐cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2−/− mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.

Abstract Introduction Accurate delivery of radiation while reducing dose to organs at risk is essential in prostate treatment. The Calypso motion management system detects and corrects both inter‐ and intra‐fraction motion which offers potential benefits over standard alignment to fiducial markers. The aims of this study were to implement Calypso with Dynamic Edge™ gating and to assess both the motion seen, and interventions required. Methods An implementation group was formed which assessed changes needed to standard workflows. Three patients had Calypso beacons inserted into their prostate. All patients were treated using volumetric modulated arc therapy to a dose of 80 Gy in 40 fractions. Standard inter‐fraction motion correction using either kilovoltage (kV) orthogonal paired imaging or cone beam computed tomography (CBCT) image‐guided radiotherapy techniques, were used along with the Calypso system to compare accuracy. A gating threshold of >0.5 cm was used during treatment. Workflow variations along with inter‐ and intra‐fraction motion and interventions required were assessed. Results A total of 116 fractions were treated using Calypso with Dynamic Edge™ gating. There was a strong concordance between aligning beacons using kV orthogonal imaging or CBCT and Calypso (mean variation ≤0.06 cm). The mean intra‐fraction motion detected was ≤0.2 cm in all directions with the largest motion recorded being 2.2 cm in the left direction while the treatment beam was off. Prostate rotation was largest in the pitch direction and 28 fractions exceeded the 10° tolerance. A total of 78 couch shift corrections of ≥0.3 cm were required, usually following standard imaging, and before treatment starting. Three gating events due to intra‐fraction motion occurred during treatment. Conclusions Intra‐fraction motion monitoring with Calypso was successfully implemented. Greatest movement was seen between time of standard imaging and treatment starting with more than half the treatments requiring a ≥0.3 cm adjustment. This would not have been detected without intra‐fraction monitoring.

Bordetella bacteria are respiratory pathogens of humans, birds, and livestock. Bordetella pertussis the causative agent of whopping cough remains a significant health issue. The transcriptional regulator, BpsR, represses a number of Bordetella genes relating to virulence, cell adhesion, cell motility, and nicotinic acid metabolism. DNA binding of BpsR is allosterically regulated by interaction with 6-hydroxynicotinic acid (6HNA), the first product in the nicotinic acid degradation pathway. To understand the mechanism of this regulation, we have determined the crystal structures of BpsR and BpsR in complex with 6HNA. The structures reveal that BpsR binding of 6HNA induces a conformational change in the protein to prevent DNA binding. We have also identified homologs of BpsR in other Gram negative bacteria in which the amino acids involved in recognition of 6HNA are conserved, suggesting a similar mechanism for regulating nicotinic acid degradation.

The mammalian hearing organ is a regular array of two types of hair cells (HCs) surrounded by six types of supporting cells. Along the tonotopic axis, this conserved radial array of cell types shows longitudinal variations to enhance the tuning properties of basilar membrane. We present the current evidence supporting the hypothesis that quantitative local variations in gene expression profiles are responsible for local cell responses to global gene manipulations. With the advent of next generation sequencing and the unprecedented array of technologies offering high throughput analyses at the single cell level, transcriptomics will become a common tool to enhance our understanding of the inner ear. We provide an overview of the approaches and landmark studies undertaken to date to analyze single cell variations in the organ of Corti and discuss the current limitations. We next provide an overview of the complexity of known regulatory mechanisms in the inner ear. These mechanisms are tightly regulated temporally and spatially at the transcription, RNA-splicing, mRNA-regulation, and translation levels. Understanding the intricacies of regulatory mechanisms at play in the inner ear will require the use of complementary approaches, and most probably, a combinatorial strategy coupling transcriptomics, proteomics, and epigenomics technologies. We highlight how these data, in conjunction with recent insights into molecular cell transformation, can advance attempts to restore lost hair cells.

Marine turtles are vulnerable to climate change because their life history and reproduction are tied to environmental temperatures. The egg incubation stage is arguably the most vulnerable stage, because marine turtle eggs require a narrow range of temperatures for successful incubation. Additionally, incubation temperature affects sex, emergence success, morphology and locomotor performance of hatchlings. Hatchlings often experience high rates of predation in the first few hours of their life, and increased size or locomotor ability may improve their chances of survival. Between 2010 and 2013 we monitored the temperature of loggerhead (Caretta caretta; Linnaeus 1758) turtle nests at Mon Repos Rookery, and used these data to calculate a mean three day maximum temperature (T3dm) for each nest. We calculated the hatching and emergence success for each nest, then measured the mass, size and locomotor performance of hatchlings that emerged from those nests. Nests with a T3dm greater than 34°C experienced a lower emergence success and produced smaller hatchlings than nests with a T3dm lower than 34°C. Hatchlings from nests with a T3dm below 34°C performed better in crawling and swimming trials than hatchlings from nests with a T3dm above 34°C. Thus even non-lethal increases in global temperatures have the potential to detrimentally affect fitness and survival of marine turtle hatchlings.

Excessive sea turtle nest predation is a problem for conservation management of sea turtle populations. This study assessed predation on nests of the endangered loggerhead sea turtle (Caretta caretta) at Wreck Rock beach adjacent to Deepwater National Park in Southeast Queensland, Australia after a control program for feral foxes was instigated. The presence of predators on the nesting dune was evaluated by tracking plots (2 × 1 m) every 100 m along the dune front. There were 21 (2014–2015) and 41 (2015–2016) plots established along the dune, and these were monitored for predator tracks daily over three consecutive months in both nesting seasons. Predator activities at nests were also recorded by the presence of tracks on top of nests until hatchlings emerged. In addition, camera traps were set to record the predator activity around selected nests. The tracks of the fox (Vulpes vulpes) and goanna (Varanus spp) were found on tracking plots. Tracking plots, nest tracks and camera traps indicated goanna abundance varied strongly between years. Goannas were widely distributed along the beach and had a Passive Activity Index (PAI) (0.31 in 2014–2015 and 0.16 in 2015–2016) approximately seven times higher than that of foxes (PAI 0.04 in 2014–2015 and 0.02 in 2015–2016). Five hundred and twenty goanna nest visitation events were recorded by tracks but no fox tracks were found at turtle nests. Camera trap data indicated that yellow-spotted goannas (Varanus panoptes) appeared at loggerhead turtle nests more frequently than lace monitors (V. varius) did, and further that lace monitors only predated nests previously opened by yellow-spotted goannas. No foxes were recorded at nests with camera traps. This study suggests that large male yellow-spotted goannas are the major predator of sea turtle nests at the Wreck Rock beach nesting aggregation and that goanna activity varies between years.

Observations of federally endangered Southern Steelhead Trout (Oncorhynchus mykiss) were compiled for the southern California Distinct Population Segment (DPS) that extends approximately 500 kilometers from the Santa Maria River (Santa Barbara County) south to the Mexican border. Existing monitoring programs and anecdotal observations documented 177 adult Steelhead in the past 25 years. The level of monitoring effort and technology used varied by watershed and year. Thus, the low numbers of adult Steelhead observed during the past 25 years provides relative abundance and distribution documentation of occurrences but likely underestimates the abundance of adult Steelhead throughout the DPS. The patchy distribution observed suggests that fish are opportunistically entering creeks and river mouths based on available hydrologic passage opportunities associated with higher rainfall years. Given the extremely low numbers of anadromous adults, long recovery timeline, future uncertainty with respect to climate change, and a multitude of anthropogenic factors, additional conservation actions including state and federal protection of resident O. mykiss are recommended to increase the likelihood of the continued existence of the species within the DPS. Additional focused monitoring, research, and implementation of recovery actions are needed to expand our knowledge of southern California Steelhead distribution and abundance.

Hatchling sea turtles emerge from nests, crawl down the beach and enter the sea where they typically enter a stereotypical hyperactive swimming frenzy. During this swim the front flippers are moved up and down in a flapping motion and are the primary source of thrust production. I used high-speed video linked with simultaneous measurement of thrust production in tethered hatchlings, along with high-speed video of free swimming hatchlings swimming at different water speeds in a swim flume to investigate the links between kinematics of front flipper movement, thrust production and swimming speed. In particular I tested the hypotheses that (1) increased swimming speed is achieved through an increased stroke rate; (2) force produced per stroke is proportional to stroke amplitude, (3) that forward thrust is produced during both the down and up phases of stroking; and (4) that peak thrust is produced towards the end of the downstroke cycle. Front flipper stroke rate was independent of water speed refuting the hypothesis that swimming speed is increased by increasing stroke rate. Instead differences in swimming speed were caused by a combination of varying flipper amplitude and the proportion of time spent powerstroking. Peak thrust produced per stroke varied within and between bouts of powerstroking, and these peaks in thrust were correlated with both flipper amplitude and flipper angular momentum during the downstroke supporting the hypothesis that stroke force is a function of stroke amplitude. Two distinct thrust production patterns were identified, monophasic in which a single peak in thrust was recorded during the later stages of the downstroke, and biphasic in which a small peak in thrust was recorded at the very end of the upstroke and this followed by a large peak in thrust during the later stages of the downstroke. The biphasic cycle occurs in ∼20% of hatchlings when they first started swimming, but disappeared after one to two hours of swimming. The hypothesis that forward thrust is produced during both the up and down stroke was only supported relatively rarely in hatchlings that exhibited the diphasic cycle, the majority of time forward thrust was only produced during the downstroke phase. The hypothesis that peak forward thrust is produced during the end of the downstroke was supported in both the monophasic and biphasic thrust producing stroke cycles.

Cannabinoid receptor interacting protein 1a (CRIP1a) is an important CB1 cannabinoid receptor-associated protein, first identified from a yeast two-hybrid screen to modulate CB1-mediated N-type Ca2+ currents. In this paper we review studies of CRIP1a function and structure based upon in vitro experiments and computational chemistry, which elucidate the specific mechanisms for the interaction of CRIP1a with CB1 receptors. N18TG2 neuronal cells overexpressing or silencing CRIP1a highlighted the ability of CRIP1 to regulate cyclic adenosine 3′,5′monophosphate (cAMP) production and extracellular signal-regulated kinase (ERK1/2) phosphorylation. These studies indicated that CRIP1a attenuates the G protein signaling cascade through modulating which Gi/o subtypes interact with the CB1 receptor. CRIP1a also attenuates CB1 receptor internalization via β-arrestin, suggesting that CRIP1a competes for β-arrestin binding to the CB1 receptor. Predictions of CRIP1a secondary structure suggest that residues 34-110 are minimally necessary for association with key amino acids within the distal C-terminus of the CB1 receptor, as well as the mGlu8a metabotropic glutamate receptor. These interactions are disrupted through phosphorylation of serines and threonines in these regions. Through investigations of the function and structure of CRIP1a, new pharmacotherapies based upon the CRIP-CB1 receptor interaction can be designed to treat diseases such as epilepsy, motor dysfunctions and schizophrenia.