Establishment of porcine fecal-derived ex vivo microbial communities to evaluate the impact of livestock feed on gut microbiome.

Sustainable livestock production requires reducing competition for food and feed resources and increasing the utilization of food by-products in livestock feed. This study describes the establishment of an anaerobic batch culture model to simulate pig microbiota and evaluate the effects of a food by-product, wakame seaweed stalks, on ex vivo microbial communities. We selected one of the nine media to support the growth of a bacterial community most similar in composition and diversity to that observed in pig donor feces. Supplementation with wakame altered the microbial profile and short-chain fatty acid composition in the ex vivo model, and a similar trajectory was observed in the in vivo pig experimental validation. Notably, the presence of wakame increased the abundance of Lactobacillus species, which may have been due to cross-feeding with Bacteroides. These results suggest the potential of wakame as a livestock feed capable of modulating the pig microbiome. Collectively, this study highlights the ability to estimate the microbiome changes that occur when pigs are fed a specific feed using an ex vivo culture model.

A systematic review and meta-analysis of how social memory is studied.

Social recognition is crucial for survival in social species, and necessary for group living, selective reproduction, pair bonding, and dominance hierarchies. Mice and rats are the most commonly used animal models in social memory research, however current paradigms do not account for the complex social dynamics they exhibit in the wild. To assess the range of social memories being studied, we conducted a systematic analysis of neuroscience articles testing the social memory of mice and rats published within the past two decades and analyzed their methods. Our results show that despite these rodent's rich social memory capabilities, the majority of social recognition papers explore short-term memories and short-term familiarity levels with minimal exposure between subject and familiar stimuli-a narrow type of social memory. We have identified several key areas currently understudied or underrepresented: kin relationships, mates, social ranks, sex variabilities, and the effects of aging. Additionally, reporting on social stimulus variables such as housing history, strain, and age, is limited, which may impede reproducibility. Overall, our data highlight large gaps in the diversity of social memories studied and the effects social variables have on social memory mechanisms.

Phase 3 Clinical Trial Comparing the Safety and Efficacy of Netarsudil to Ripasudil in Patients with Primary Open-Angle Glaucoma or Ocular Hypertension: Japan Rho Kinase Elevated Intraocular Pressure Treatment Trial (J-ROCKET).

INTRODUCTION: A clinical trial evaluated ocular hypotensive efficacy and safety of netarsudil 0.02% once daily (QD) relative to ripasudil 0.4% twice daily (BID). METHODS: This was a single-masked, randomized, phase 3, superiority study. Japanese patients were randomized to either the netarsudil 0.02% group or the ripasudil 0.4% group in a 1:1 ratio and treated for 4 weeks. The primary efficacy variable was mean diurnal intraocular pressure (IOP) (average of diurnal time points at 09:00, 11:00, and 16:00) at Week 4. RESULTS: A total of 245 patients were included in the primary analysis. At Week 4, least squares (LS) mean of diurnal IOP adjusted for baseline was 15.96 and 17.71 mmHg in the netarsudil 0.02% and ripasudil 0.4% groups, respectively, demonstrating the superiority of netarsudil 0.02% QD over ripasudil 0.4% BID by a margin of - 1.74 mmHg (p < 0.0001). Mean reduction from baseline in mean diurnal IOP at Week 4 was 4.65 and 2.98 mmHg, respectively. Adverse events (AEs) occurred less frequently in netarsudil 0.02% than in ripasudil 0.4%, with the incidence of ocular AEs being 59.8% and 66.7%, respectively. The most frequently reported AE was conjunctival hyperemia in both groups, with an incidence of 54.9% and 62.6%, respectively. No serious eye-related AEs were reported. CONCLUSION: Netarsudil ophthalmic solution 0.02% dosed QD (p.m.) was well tolerated and more effective in reducing IOP than ripasudil ophthalmic solution 0.4% dosed BID. Netarsudil 0.02% QD may become an important option for the treatment of Japanese patients with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT04620135.

RNA virus discoveries in the electric ant, Wasmannia auropunctata.

Despite being one of the most destructive invasive species of ants, only two natural enemies are known currently for Wasmannia auropunctata, commonly known as the electric ant or little fire ant. Because viruses can be effective biological control agents against many insect pests, including ants, a metagenomics/next-generation sequencing approach was used to facilitate discovery of virus sequences from the transcriptomes of W. auropunctata. Five new and complete positive sense, single-stranded RNA virus genomes, and one new negative sense, single-stranded RNA virus genome were identified, sequenced, and characterized from W. auropunctata collected in Argentina by this approach, including a dicistrovirus (Electric ant dicistrovirus), two polycipiviruses (Electric ant polycipivirus 1; Electric ant polycipivirus 2), a solinvivirus (Electric ant solinvivirus), a divergent genome with similarity to an unclassified group in the Picornavirales (Electric ant virus 1), and a rhabdovirus (Electric ant rhabdovirus). An additional virus genome was detected that is likely Solenopsis invicta virus 10 (MH727527). The virus genome sequences were absent from the transcriptomes of W. auropunctata collected in the USA (Hawaii and Florida). Additional limited field surveys corroborated the absence of these viruses in regions where the electric ant is invasive (the USA and Australia). The replicative genome strand of four of the viruses (Electric ant polycipivirus 2, Electric ant solinvivirus, Electric ant virus 1, and Solenopsis invicta virus 10 (in the electric ant) was detected in Argentinean-collected W. auropunctata indicating that the ant is a host for these viruses. These are the first virus discoveries to be made from W. auropunctata.

Mind the gap: A systematic review and meta-analysis of how social memory is studied.

Social recognition is crucial for survival in social species, and necessary for group living, selective reproduction, pair bonding, and dominance hierarchies. Mice and rats are the most commonly used animal models in social memory research, however current paradigms do not account for the complex social dynamics they exhibit in the wild. To assess the range of social memories being studied, we conducted a systematic analysis of neuroscience articles testing the social memory of mice and rats published within the past two decades and analyzed their methods. Our results show that despite these rodent's rich social memory capabilities, the majority of social recognition papers explore short-term memories and short-term familiarity levels with minimal exposure between subject and familiar stimuli - a narrow type of social memory. We have identified several key areas currently understudied or underrepresented: kin relationships, mates, social ranks, sex variabilities, and the effects of aging. Additionally, reporting on social stimulus variables such as housing history, strain, and age, is limited, which may impede reproducibility. Overall, our data highlight large gaps in the diversity of social memories studied and the effects social variables have on social memory mechanisms.

In vivo Optical Access to Olfactory Sensory Neuronsin the Mouse Olfactory Epithelium.

In neuroscience, it is fundamental to understand how sensory stimuli are translated into neural activity at the entry point of sensory systems. In the olfactory system, odorants inhaled into the nasal cavity are detected by ~1,000 types of odorant receptors (ORs) that are expressed by olfactory sensory neurons (OSNs). Since each OSN expresses only one type of odorant receptor, the odor-evoked responses reflect the interaction between odorants and the expressed OR. The responses of OSN somata are often measured by calcium imaging and electrophysiological techniques; however, previous techniques require tissue dissection or cell dissociation, rendering it difficult to investigate physiological responses. Here, we describe a protocol that allows us to observe odor-evoked responses of individual OSN somata in the mouse olfactory epithelium in vivo. Two-photon excitation through the thinned skull enables highly-sensitive calcium imaging using a genetically encoded calcium indicator, GCaMP. Recording of odor-evoked responses in OSN somata in freely breathing mice will be fundamental to understanding how odor information is processed at the periphery and higher circuits in the brain.

Widespread Inhibition, Antagonism, and Synergy in Mouse Olfactory Sensory Neurons In Vivo.

Sensory information is selectively or non-selectively enhanced and inhibited in the brain, but it remains unclear whether and how this occurs at the most peripheral level. Using in vivo calcium imaging of mouse olfactory bulb and olfactory epithelium in wild-type and mutant animals, we show that odors produce not only excitatory but also inhibitory responses in olfactory sensory neurons (OSNs). Heterologous assays indicate that odorants can act as agonists to some but inverse agonists to other odorant receptors. We also demonstrate that responses to odor mixtures are extensively suppressed or enhanced in OSNs. When high concentrations of odors are mixed, widespread antagonism suppresses the overall response amplitudes and density. In contrast, a mixture of low concentrations of odors often produces synergistic effects and boosts the faint odor inputs. Thus, odor responses are extensively tuned by inhibition, antagonism, and synergy at the most peripheral level, contributing to robust sensory representations.

Cognitive burden and polypharmacy in elderly Japanese patients treated with anticholinergics for an overactive bladder.

This study aimed to investigate the cognitive burden and polypharmacy in elderly patients treated with anticholinergics for an overactive bladder. We conducted a retrospective study of patients with an overactive bladder receiving treatment at two hospitals in Japan (Nara Medical University Hospital and Saiseikai Nara Hospital). Prescription data were collected from the medical records of the patients registered between January 2013 and April 2014. The Anticholinergic Cognitive Burden Scale was used to estimate the severity of the anticholinergic effects on the cognition of each patient. We collected the prescription data of 584 and 246 patients from the Nara Medical University Hospital and Saiseikai Nara Hospital, respectively. The mean daily total Anticholinergic Cognitive Burden score ranged between 3 and 4 (3.59 ± 1.16 at Nara Medical University Hospital vs 3.32 ± 0.78 at Saiseikai Nara Hospital, P < 0.01). At both hospitals, the mean number of prescriptions was >5 in patients ≥75 years (5.95 ± 4.43 and 5.64 ± 3.90 at Nara Medical University Hospital and Saiseikai Nara Hospitals, respectively). Our findings suggest that 10%-20% of elderly patients (≥65 years) receiving treatment with anticholinergics for an overactive bladder are in a state of polypharmacy. The total anticholinergic cognitive burden of each patient mainly depends on the anticholinergics being used for treating the overactive bladder. Especially for elderly patients with a high risk of adverse effects, including cognitive impairment, careful attention needs to be paid during selection of drugs for treating patients with an overactive bladder.

Matrin 3 Is a Component of Neuronal Cytoplasmic Inclusions of Motor Neurons in Sporadic Amyotrophic Lateral Sclerosis.

Mutations in the MATR3 gene have been identified as a cause of familial amyotrophic lateral sclerosis, but involvement of the matrin 3 (MATR3) protein in sporadic amyotrophic lateral sclerosis (SALS) pathology has not been fully assessed. We immunohistochemically analyzed MATR3 pathology in the spinal cords of SALS and control autopsy specimens. MATR3 immunostaining of the motor neuron nuclei revealed two distinct patterns: mild and strong staining. There were no differences in the ratio of mild versus strong nuclear staining between the SALS and control cases. MATR3-containing neuronal cytoplasmic inclusions (NCIs) were observed in 60% of SALS cases. Most motor neurons with MATR3-positive NCIs exhibited a mild nuclear staining pattern. Although 16.8% of NCIs positive for transactivating response region DNA-binding protein 43 (TDP-43) were estimated as double-labeled by MATR3, no MATR3-positive or TDP-43-negative NCIs were observed. Although a previous study found that MATR3-positive NCIs are present only in cases with C9orf72 hexanucleotide repeat expansion, ubiquitin-positive granular NCIs were not observed in the cerebellum, which have been reported as specific to C9orf72-related ALS. Six ALS cases were confirmed to be negative for the GGGGCC hexanucleotide. Our results reveal that MATR3 is a component of TDP-43-positive NCIs in motor neurons, even in SALS, and indicate the broader involvement of MATR3 in ALS pathology and the heterogeneity of TDP-43-positive NCIs.

Mechanosensory-Based Phase Coding of Odor Identity in the Olfactory Bulb.

Mitral and tufted (M/T) cells in the olfactory bulb produce rich temporal patterns of activity in response to different odors. However, it remains unknown how these temporal patterns are generated and how they are utilized in olfaction. Here we show that temporal patterning effectively discriminates between the two sensory modalities detected by olfactory sensory neurons (OSNs): odor and airflow-driven mechanical signals. Sniff-induced mechanosensation generates glomerulus-specific oscillatory activity in M/T cells, whose phase was invariant across airflow speed. In contrast, odor stimulation caused phase shifts (phase coding). We also found that odor-evoked phase shifts are concentration invariant and stable across multiple sniff cycles, contrary to the labile nature of rate coding. The loss of oscillatory mechanosensation impaired the precision and stability of phase coding, demonstrating its role in olfaction. We propose that phase, not rate, coding is a robust encoding strategy of odor identity and is ensured by airflow-induced mechanosensation in OSNs.

Distorted Coarse Axon Targeting and Reduced Dendrite Connectivity Underlie Dysosmia after Olfactory Axon Injury.

The glomerular map in the olfactory bulb (OB) is the basis for odor recognition. Once established during development, the glomerular map is stably maintained throughout the life of an animal despite the continuous turnover of olfactory sensory neurons (OSNs). However, traumatic damage to OSN axons in the adult often leads to dysosmia, a qualitative and quantitative change in olfaction in humans. A mouse model of dysosmia has previously indicated that there is an altered glomerular map in the OB after the OSN axon injury; however, the underlying mechanisms that cause the map distortion remain unknown. In this study, we examined how the glomerular map is disturbed and how the odor information processing in the OB is affected in the dysosmia model mice. We found that the anterior-posterior coarse targeting of OSN axons is disrupted after OSN axon injury, while the local axon sorting mechanisms remained. We also found that the connectivity of mitral/tufted cell dendrites is reduced after injury, leading to attenuated odor responses in mitral/tufted cells. These results suggest that existing OSN axons are an essential scaffold for maintaining the integrity of the olfactory circuit, both OSN axons and mitral/tufted cell dendrites, in the adult.

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide.

PURPOSE: Oxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1. METHODS: The sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken ß actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection. RESULTS: Expression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1ß secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1ß and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model. CONCLUSIONS: The intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.

A novel factor Iss10 regulates Mmi1-mediated selective elimination of meiotic transcripts.

A number of meiosis-specific transcripts are selectively eliminated during the mitotic cell cycle in fission yeast. Mmi1, an RNA-binding protein, plays a crucial role in this selective elimination. Mmi1 recognizes a specific region, namely, the determinant of selective removal (DSR) on meiotic transcripts and induces nuclear exosome-mediated elimination. During meiosis, Mmi1 is sequestered by a chromosome-associated dot structure, Mei2 dot, allowing meiosis-specific transcripts to be stably expressed. Red1, a zinc-finger protein, is also known to participate in the Mmi1/DSR elimination system, although its molecular function has remained elusive. To uncover the detailed molecular mechanisms underlying the Mmi1/DSR elimination system, we sought to identify factors that interact genetically with Mmi1. Here, we show that one of the identified factors, Iss10, is involved in the Mmi1/DSR system by regulating the interaction between Mmi1 and Red1. In cells lacking Iss10, association of Red1 with Mmi1 is severely impaired, and target transcripts of Mmi1 are ectopically expressed in the mitotic cycle. During meiosis, Iss10 is downregulated, resulting in dissociation of Red1 from Mmi1 and subsequent suppression of Mmi1 activity.

A sexually conditioned switch of chemosensory behavior in C. elegans.

In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.

Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt concentration chemotaxis in Caenorhabditis elegans.

It is poorly understood how sensory systems memorize the intensity of sensory stimulus, compare it with a newly sensed stimulus, and regulate the orientation behaviour based on the memory. Here we report that Caenorhabditis elegans memorizes the environmental salt concentration during cultivation and exhibits a strong behavioural preference for this concentration. The right-sided amphid gustatory neuron known as ASER, senses decreases in salt concentration, and this information is transmitted to the postsynaptic AIB interneurons only in the salt concentration range lower than the cultivation concentration. In this range, animals migrate towards higher concentration by promoting turning behaviour upon decreases in salt concentration. These observations provide a mechanism for adjusting the orientation behaviour based on the memory of sensory stimulus using a simple neural circuit.

Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioral plasticity at the sensory neuron synapses of Caenorhabditis elegans.

Growing evidence suggests that sensory neuron synapses not merely pass, but actively encode sensory information and convey it to the central nervous system. The chemosensory preferences of Caenorhabditis elegans, as manifested in the direction of chemotaxis, are reversibly regulated by prior experience at the level of sensory neurons; the attractive drive is promoted by diacylglycerol (DAG) signaling, whereas the counteracting repulsive drive requires PtdIns(3,4,5)P(3) signaling. Here we report that the two opposing drives require a class IIA phosphatidylinositol transfer protein (PITP), PITP-1, which localizes to the sensory neuron synapses. In pitp-1 mutants, attraction behavior to salt is reduced, whereas conditioned repulsion from salt is eliminated: the mutants inflexibly show weak attraction behavior to salt, irrespective of prior experience. To generate flexible behavioral outputs, attraction and repulsion, PITP-1 acts in the gustatory neuron ASER and likely regulates neurotransmission from ASER, as pitp-1 mutations do not affect the ASER Ca(2+) response to sensory stimulus. Furthermore, full attraction to salt is restored in pitp-1 mutants by expression of the phosphatidylinositol transfer domain alone, and also by mutations of a DGK gene that cause accumulation of DAG, suggesting that PITP-1 serves for DAG production via phosphatidylinositol transport and, hence, regulates synaptic transmission. In addition to gustatory behavior, olfactory behaviors and osmotic avoidance are also regulated by PITP-1 in the sensory neurons that detect each sensory stimulus. Thus, PITP-1-dependent phosphatidylinositol transport is essential for sensory neuron synapses to couple sensory inputs to effective behavioral responses.

Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells.

A number of meiosis-specific mRNAs are initially weakly transcribed, but then selectively removed during fission yeast mitotic growth. These mRNAs harbour a region termed DSR (determinant of selective removal), which is recognized by the YTH family RNA-binding protein Mmi1p. Mmi1p directs the destruction of these mRNAs in collaboration with nuclear exosomes. However, detailed molecular mechanisms underlying this process of selective mRNA elimination have remained elusive. In this study, we demonstrate the critical role of polyadenylation in this process. Two-hybrid and genetic screens revealed potential interactions between Mmi1p and proteins involved in polyadenylation. Additional investigations showed that destruction of DSR-containing mRNAs by exosomes required polyadenylation by a canonical poly(A) polymerase. The recruitment of Pab2p, a poly(A)-binding protein, to the poly(A) tail was also necessary for mRNA destruction. In cells undergoing vegetative growth, Mmi1p localized with exosomes, Pab2p, and components of the polyadenylation complex in several patchy structures in the nucleoplasm. These patches may represent the sites for degradation of meiosis-specific mRNAs with untimely expression.

A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans.

The molecular mechanism by which neurites are selected for elimination or incorporation into the mature circuit during developmental pruning remains unknown. The trophic theory postulates that local cues provided by target or surrounding cells act to inhibit neurite elimination. However, no widely conserved factor mediating this trophic function has been identified. We found that the developmental survival of specific neurites in Caenorhabditis elegans largely depends on detection of the morphogen Wnt by the Ror kinase CAM-1, which is a transmembrane tyrosine kinase with a Frizzled domain. Mutations in Wnt genes or in cam-1 enhanced neurite elimination, whereas overexpression of cam-1 inhibited neurite elimination in a Wnt-dependent manner. Moreover, mutations in these genes counteracted the effect of a mutation in mbr-1, which encodes a transcription factor that promotes neurite elimination. These results reveal the trophic role of an atypical Wnt pathway and reinforce the classical model of developmental pruning.