Ablation of placental REST deregulates fetal brain metabolism and impacts gene expression of the offspring brain at the postnatal and adult stages.

In this study, the transcriptional repressor REST (Repressor Element 1 Silencing Transcription factor) was ablated in the mouse placenta to investigate molecular and cellular impacts on the offspring brain at different life stages. Ablation of placental REST deregulated several brain metabolites, including glucose and lactate that fuel brain energy, vitamin C (ascorbic acid) that functions in the epigenetic programming of the brain during postnatal development, and glutamate and creatine that help the brain to respond to stress conditions during adult life. Bulk RNA-seq analysis showed that a lack of placental REST persistently altered multiple transport genes, including those related to oxygen transportation in the offspring brain. While metabolic genes were impacted in the postnatal brain, different stress response genes were activated in the adult brain. DNA methylation was also impacted in the adult brain due to the loss of placental REST, but in a sex-biased manner. Single-nuclei RNA-seq analysis showed that specific cell types of the brain, particularly those of the choroid plexus and ependyma, which play critical roles in producing cerebrospinal fluid and maintaining metabolic homeostasis, were significantly impacted due to the loss of placental REST. These cells showed significant differential expression of genes associated with the metabotropic (G coupled protein) and ionotropic (ligand-gated ion channel) glutamate receptors, suggesting an impact of ablation of placental REST on the glutamatergic signaling of the offspring brain. The study expands our understanding of placental influences on the offspring brain.

The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

CRISPR Advancements for Human Health.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.

Prss29 Cre recombinase mice are useful to study adult uterine gland function.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

Optimal Load Magnitude and Placement for Peak Power Production in a Vertical Jump: A Segmental Contribution Analysis.

ABSTRACT: Bordelon, NM, Jones, DH, Sweeney, KM, Davis, DJ, Critchley, ML, Rochelle, LE, George, AC, and Dai, B. Optimal load magnitude and placement for peak power production in a vertical jump: A segmental contribution analysis. J Strength Cond Res 36(4): 911-919, 2022-Weighted jumps are widely used in power training, however, there are discrepancies regarding which loading optimizes peak jump power. The purpose was to quantify the effects of load magnitudes and placements on the force, velocity, and power production in a countermovement vertical jump. Sixteen male and 15 female subjects performed vertical jumps in 7 conditions: no external load, 10 and 20% dumbbell loads, 10 and 20% vest loads, and 10 and 20% barbell loads with load percentages relative to body weight. Arm swing was encouraged for all, but the barbell load conditions. Kinematics were collected to quantify the whole-body (the person and external loads) forces, velocities, and power as well as segments' contributions to the whole-body forces and velocities. Repeated-measure analyses of variance were performed followed by paired comparisons. Jump heights were the greatest for the no external load and 10% dumbbell conditions. The 10 and 20% dumbbell conditions demonstrated the greatest peak whole-body power, while the 2 barbell conditions showed the lowest peak whole-body power. At the time of peak whole-body power, the 2 dumbbell and 2 vest conditions resulted in greater whole-body forces. Whole-body velocities were the greatest for the no external load and 10% dumbbell conditions. Holding the dumbbells in the hands magnified the effects of external loads in producing forces and velocities. The constraint of arm movements in the barbell conditions limited power production. These findings highlight the importance of load placement and arm swing in identifying the optimal configuration for power production in weighted jumps.

Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

Vertical Ground Reaction Force Estimation From Benchmark Nonstationary Kinematic Data.

Time-differentiating kinematic signals from optical motion capture amplifies the inherent noise content of those signals. Commonly, biomechanists address this problem by applying a Butterworth filter with the same cutoff frequency to all noisy displacement signals prior to differentiation. Nonstationary signals, those with time-varying frequency content, are widespread in biomechanics (eg, those containing an impact) and may necessitate a different filtering approach. A recently introduced signal filtering approach wherein signals are divided into sections based on their energy content and then Butterworth filtered with section-specific cutoff frequencies improved second derivative estimates in a nonstationary kinematic signal. Utilizing this signal-section filtering approach for estimating running vertical ground reaction forces saw more of the signal's high-frequency content surrounding heel strike maintained without allowing inappropriate amounts of noise contamination in the remainder of the signal. Thus, this signal-section filtering approach resulted in superior estimates of vertical ground reaction forces compared with approaches that either used the same filter cutoff frequency across the entirety of each signal or across the entirety of all signals. Filtering kinematic signals using this signal-section filtering approach is useful in processing data from tasks containing an impact when accurate signal second derivative estimation is of interest.

Mid-flight lateral trunk bending increased ipsilateral leg loading during landing: a center of mass analysis.

Increased lateral trunk bending to the injured side has been observed when ACL injuries occur. The purpose was to quantify the effect of mid-flight lateral trunk bending on center of mass (COM) positions and subsequent landing mechanics during a jump-landing task. Forty-one recreational athletes performed a jump-landing task with or without mid-flight lateral trunk bending. When the left and right trunk bending conditions were compared with the no trunk bending condition, participants moved the COM of the upper body to the bending direction, while the COM of the pelvis, ipsilateral leg, and contralateral leg moved away from the bending direction relative to the whole body COM. Participants demonstrated increased peak vertical ground reaction forces (VGRF) and knee valgus and internal rotation angles at peak VGRF for the ipsilateral leg, but decreased peak VGRF and knee internal rotation angles at peak VGRF and increased knee varus angles at peak VGRF for the contralateral leg. Mid-flight lateral trunk resulted in an asymmetric landing pattern associated with increased ACL loading for the ipsilateral leg. The findings may help to understand altered trunk motion during ACL injury events and the discrepancy in ACL injuries related to limb dominance in badminton and volleyball.

Influence of preoperative septic peritonitis and anastomotic technique on the dehiscence of enterectomy sites in dogs: A retrospective review of 210 anastomoses.

OBJECTIVE: To determine the influence of preoperative septic peritonitis (PSP) and stapled versus hand-sewn anastomoses on the dehiscence of intestinal resection and anastomosis (IRA). We hypothesized that the incidence of IRA dehiscence would be greater (1) when performed with PSP and (2) for hand-sewn anastomoses. STUDY DESIGN: Retrospective. ANIMAL POPULATIONS: Client-owned dogs at Michigan State University Veterinary Teaching Hospital. METHODS: Records of dogs surviving 72 hours after IRAs between 2003 and 2013 were reviewed for age, gender, neuter status, weight, presence of PSP, preoperative albumin, IRA indication and location, anastomotic technique, suture type, postoperative dehiscence and timing, duration of hospitalization, last follow-up, and other complications. Univariate logistic regression and chi-square analysis were used to screen prognostic factors; factors with P < .3 were included in a multivariate analysis. RESULTS: Two hundred and ten IRAs in 198 dogs fulfilled the inclusion criteria. Dehiscence was diagnosed in 11.4% cases, 6.6% without PSP, and 21.1% with PSP (P = .01). Indication for IRA did not influence the risk of dehiscence. No association was detected between anastomotic technique and IRA dehiscence in dogs without PSP (stapled 4.2%, hand-sewn 8.1%); however, stapled anastomoses were less likely to dehisce in dogs with PSP (stapled 9.7%, hand-sewn 28.9%). Risk factors for dehiscence included PSP (P = .005) and hand-sewn technique (P = .02). CONCLUSION: Our results confirmed that PSP is a risk factor for dehiscence of IRA and suggest that patients with PSP may be a unique surgical population, in which stapling may be preferred over hand-sewn anastomoses after enterectomies.

Sex-specific effects of docosahexaenoic acid (DHA) on the microbiome and behavior of socially-isolated mice.

Dietary supplementation with the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has been shown to have a beneficial effect on reducing the symptoms associated with several neuropsychiatric conditions including anxiety and depression. However, the mechanisms underlying this effect remain largely unknown. Increasing evidence suggests that the vast repertoire of commensal bacteria within the gut plays a critical role in regulating various biological processes in the brain and may contribute to neuropsychiatric disease risk. The present study determined the contribution of DHA on anxiety and depressive-like behaviors through modulation of the gut microbiota in a paradigm of social isolation. Adult male and female mice were subjected to social isolation for 28days and then placed either on a control diet or a diet supplemented with 0.1% or 1.0% DHA. Fecal pellets were collected both 24h and 7days following the introduction of the new diets. Behavioral testing revealed that male mice fed a DHA diet, regardless of dose, exhibited reduced anxiety and depressive-like behaviors compared to control fed mice while no differences were observed in female mice. As the microbiota-brain-axis has been recently implicated in behavior, composition of microbial communities were analyzed to examine if these sex-specific effects of DHA may be associated with changes in the gut microbiota (GM). Clear sex differences were observed with males and females showing distinct microbial compositions prior to DHA supplementation. The introduction of DHA into the diet also induced sex-specific interactions on the GM with the fatty acid producing a significant effect on the microbial profiles in males but not in females. Interestingly, levels of Allobaculum and Ruminococcus were found to significantly correlate with the behavioral changes observed in the male mice. Predictive metagenome analysis using PICRUSt was performed on the fecal samples collected from males and identified enrichment in functional KEGG pathway terms relevant to processes such as the biosynthesis of unsaturated fatty acids and antioxidant metabolism. These results indicate that DHA alters commensal community composition and produces beneficial effects on anxiety and depressive-like behaviors in a sex-specific manner. The present study provides insight into the mechanistic role that gut microbes may play in the regulation of anxiety and depressive-like behaviors and how dietary intervention can modulate these effects.

Associations between cytokines, endocrine stress response, and gastrointestinal symptoms in autism spectrum disorder.

Many children and adolescents with autism spectrum disorder (ASD) have significant gastrointestinal (GI) symptoms, but the etiology is currently unknown. Some individuals with ASD show altered reactivity to stress and altered immune markers relative to typically-developing individuals, particularly stress-responsive cytokines including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Acute and chronic stress is associated with the onset and exacerbation of GI symptoms in those without ASD. The present study examined whether GI symptoms in ASD were associated with increases in cortisol, a stress-associated endocrine marker, and TNF-α and IL-6 in response to stress. As hypothesized, a greater amount of lower GI tract symptoms were significantly associated with post-stress cortisol concentration. The relationship between cortisol response to stress and GI functioning was greater for children who had a history of regressive autism. Exploratory analyses revealed significant correlations between cortisol response, intelligence, and inappropriate speech. In contrast, symptoms of the lower GI tract were not associated with levels of TNF-α or IL-6. Significant correlations were found, however, between TNF-α and IL-6 and irritability, socialization, and intelligence. These findings suggest that individuals with ASD and symptoms of the lower GI tract may have an increased response to stress, but this effect is not associated with concomitant changes in TNF-α and IL-6. The relationship between cortisol stress response and lower GI tract symptoms in children with regressive autism, as well as the relationships between cortisol, IL-6, and intelligence in ASD, warrant further investigation.

Displacement in new economy labor markets: Post-displacement wage loss in high tech versus low tech cities.

While scholars and politicians tout education as the salve to employment disruptions, we argue that the geography of the new economy, and the social closure mechanisms that geography creates, may be just as important as individuals' characteristics for predicting post-displacement wage loss (or gain). We use data from the 2012 Displaced Workers ement of the Current Population Survey and from the 2010 United States Census to test hypotheses linking local labor markets in different industrial contexts to post-displacement wage loss. Our results point to age as a closure mechanism, and to the partially protective effect of education in high-tech versus low-tech economic sectors. This study is the first to use national level data to examine how employment in high-tech cities influences post-displacement wages. These findings are relevant both for theorizing about the new economy and for public policy.

Explosive radiation or uninformative genes? Origin and early diversification of tachinid flies (Diptera: Tachinidae).

Molecular phylogenetic studies at all taxonomic levels often infer rapid radiation events based on short, poorly resolved internodes. While such rapid episodes of diversification are an important and widespread evolutionary phenomenon, much of this poor phylogenetic resolution may be attributed to the continuing widespread use of "traditional" markers (mitochondrial, ribosomal, and some nuclear protein-coding genes) that are often poorly suited to resolve difficult, higher-level phylogenetic problems. Here we reconstruct phylogenetic relationships among a representative set of taxa of the parasitoid fly family Tachinidae and related outgroups of the superfamily Oestroidea. The Tachinidae are one of the most species rich, yet evolutionarily recent families of Diptera, providing an ideal case study for examining the differential performance of loci in resolving phylogenetic relationships and the benefits of adding more loci to phylogenetic analyses. We assess the phylogenetic utility of nine genes including both traditional genes (e.g., CO1 mtDNA, 28S rDNA) and nuclear protein-coding genes newly developed for phylogenetic analysis. Our phylogenetic findings, based on a limited set of taxa, include: a close relationship between Tachinidae and the calliphorid subfamily Polleninae, monophyly of Tachinidae and the subfamilies Exoristinae and Dexiinae, subfamily groupings of Dexiinae+Phasiinae and Tachininae+Exoristinae, and robust phylogenetic placement of the somewhat enigmatic genera Strongygaster, Euthera, and Ceracia. In contrast to poor resolution and phylogenetic incongruence of "traditional genes," we find that a more selective set of highly informative genes is able to more precisely identify regions of the phylogeny that experienced rapid radiation of lineages, while more accurately depicting their phylogenetic context. Although much expanded taxon sampling is necessary to effectively assess the monophyly of and relationships among major tachinid lineages and their relatives, we show that a small number of well-chosen nuclear protein-coding genes can successfully resolve even difficult phylogenetic problems.

Increasing midtarsal joint stiffness reduces triceps surae metabolic costs in walking simulations but has little effect on total stance limb metabolic cost.

The human foot's arch is thought to be beneficial for efficient gait. This study addresses the extent to which arch stiffness changes alter the metabolic energy requirements of human gait. Computational musculoskeletal simulations of steady state walking using direct collocation were performed. Across a range of foot arch stiffnesses, the metabolic cost of transport decreased by less than 1% with increasing foot arch stiffness. Increasing arch stiffness increased the metabolic efficiency of the triceps surae during push-off, but these changes were almost entirely offset by other muscle groups consuming more energy with increasing foot arch stiffness.

Comparison of Thermal and Mechanical Pain Testing Modalities in Sprague Dawley and Fischer 344 Rats (Rattus norvegicus).

While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344). Male and female, 10- to 14-wk-old F344 and SD rats were used to assess withdrawal thresholds in 3 different modalities: the Randall-Selitto test (RST), Hargreaves test (HT), and tail flick test (TFT). The RST was performed by using an operator-controlled handheld instrument to generate a noxious pressure stimulus to the left hind paw. The HT and the TFT used an electronically controlled light source to deliver a noxious thermal stimulus to the left hind paw or tail tip, respectively. Rats of each sex and genetic background underwent one type of test on day 0 and day 7. Withdrawal thresholds and thermal latencies were compared among tests. No significant differences were observed. Our findings can serve as a guide for researchers considering these nociceptive tests for their experiments.

Behavioral characterization of G-protein-coupled receptor 160 knockout mice.

ABSTRACT: Neuropathic pain is a devastating condition where current therapeutics offer little to no pain relief. Novel nonnarcotic therapeutic targets are needed to address this growing medical problem. Our work identified the G-protein-coupled receptor 160 (GPR160) as a potential target for therapeutic intervention. However, the lack of small-molecule ligands for GPR160 hampers our understanding of its role in health and disease. To address this void, we generated a global Gpr160 knockout (KO) mouse using CRISPR-Cas9 genome editing technology to validate the contributions of GPR160 in nociceptive behaviors in mice. Gpr160 KO mice are healthy and fertile, with no observable physical abnormalities. Gpr160 KO mice fail to develop behavioral hypersensitivities in a model of neuropathic pain caused by constriction of the sciatic nerve. On the other hand, responses of Gpr160 KO mice in the hot-plate and tail-flick assays are not affected. We recently deorphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a potential ligand. Using Gpr160 KO mice, we now report that the development of behavioral hypersensitivities after intrathecal or intraplantar injections of CARTp are dependent on GPR160. Cocaine- and amphetamine-regulated transcript peptide plays a role in various affective behaviors, such as anxiety, depression, and cognition. There are no differences in learning, memory, and anxiety between Gpr160 KO mice and their age-matched and sex-matched control floxed mice. Results from these studies support the pronociceptive roles of CARTp/GPR160 and GPR160 as a potential therapeutic target for treatment of neuropathic pain.

Characterizing the mechanical function of the foot's arch across steady-state gait modes.

The arch of the human foot has historically been likened to either a truss, a rigid lever, or a spring. Growing evidence indicates that energy is stored, generated, and dissipated actively by structures crossing the arch, suggesting that the arch can further function in a motor- or spring-like manner. In the present study, participants walked, ran with a rearfoot strike pattern, and ran with a non-rearfoot strike pattern overground while foot segment motions and ground reaction forces were recorded. To quantify the midtarsal joint's (i.e., arch's) mechanical behavior, a brake-spring-motor index was defined as the ratio between midtarsal joint net work and the total magnitude of joint work. This index was statistically significantly different between each gait condition. Index values decreased from walking to rearfoot strike running to non-rearfoot strike running, indicating that the midtarsal joint was most motor-like when walking and most spring-like in non-rearfoot running. The mean magnitude of elastic strain energy stored in the plantar aponeurosis mirrored the increase in spring-like arch function from walking to non-rearfoot strike running. However, the behavior of the plantar aponeurosis could not account for a more motor-like arch in walking and rearfoot strike running, given the lack of main effect of gait condition on the ratio between net work and total work performed by force in the plantar aponeurosis about the midtarsal joint. Instead, the muscles of the foot are likely altering the motor-like mechanical function of the foot's arch, the operation of these muscles between gait conditions warrants further investigation.

Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.

Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.

Gene Targeting in Rat Embryonic Stem Cells.

Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.

Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.

Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.