Population genetics of marmosets in Asian primate research centers and loci associated with epileptic risk revealed by whole-genome sequencing

The common marmoset(Callithrix jacchus)has emerged as a valuable nonhuman primate model in biomedical research with the recent release of high-quality reference genome assemblies.Epileptic marmosets have been independently reported in two Asian primate research centers.Nevertheless,the population genetics within these primate centers and the specific genetic variants associated with epilepsy in marmosets have not yet been elucidated.Here,we characterized the genetic relationships and risk variants for epilepsy in 41 samples from two epileptic marmoset pedigrees using whole-genome sequencing.We identified 14558184 single nucleotide polymorphisms(SNPs)from the 41 samples and found higher chimerism levels in blood samples than in fingernail samples.Genetic analysis showed fourth-degree of relatedness among marmosets at the primate centers.In addition,SNP and copy number variation(CNV)analyses suggested that the WW domain-containing oxidoreductase(WWOX)and Tyrosine-protein phosphatase nonreceptor type 21(PTPN21)genes may be associated with epilepsy in marmosets.Notably,KCTD18-like gene deletion was more common in epileptic marmosets than control marmosets.This study provides valuable population genomic resources for marmosets in two Asian primate centers.Genetic analyses identified a reasonable breeding strategy for genetic diversity maintenance in the two centers,while the case-control study revealed potential risk genes/variants associated with epilepsy in marmosets.

Generation of chimeric monkeys using embryonic stem cells

Embryonic stem cells(ESCs)represent a subtype of pluripotent stem cells(PSCs)derived from the inner cell mass of blastocysts.These cells exhibit three principal features:the capacity for unlimited self-replication,the ability to differentiate into diverse somatic cell types in vitro,and the potential to contribute to chimera animals in vivo upon reintroduction into the host blastocyst.Thus,ESCs are widely used in basic and biomedical research,as well as in applications such as cell replacement therapy and the creation of genetically modified animal models.

Ultrasensitive proteomics depicted an in-depth landscape for the very early stage of mouse maternal-to-zygotic transition

Single-cell or low-input multi-omics techniques have revolutionized the study of pre-implantation embryo development.However,the single-cell or low-input proteomic research in this field is relatively underdeveloped because of the higher threshold of the starting material for mammalian embryo samples and the lack of hypersensitive proteome technology.In this study,a comprehensive solution of ultrasensitive proteome technology(CS-UPT)was developed for single-cell or low-input mouse oocyte/embryo samples.The deep coverage and high-throughput routes significantly reduced the starting material and were selected by investigators based on their demands.Using the deep coverage route,we provided the first large-scale snapshot of the very early stage of mouse maternal-to-zygotic transition,including almost 5,500 protein groups from 20 mouse oocytes or zygotes for each sample.Moreover,significant protein regulatory networks centered on transcription factors and kinases between the MII oocyte and 1-cell embryo provided rich insights into minor zygotic genome activation.

Mitophagy in neurodegenerative disease pathogenesis

Mitochondria are critical cellular energy resources and are central to the life of the neuron.Mitophagy selectively clears damaged or dysfunctional mitochondria through autophagic machinery to maintain mitochondrial quality control and homeostasis.Mature neurons are postmitotic and consume substantial energy,thus require highly efficient mitophagy pathways to turn over damaged or dysfunctional mitochondria.Recent evidence indicates that mitophagy is pivotal to the pathogenesis of neurological diseases.However,more work is needed to study mitophagy pathway components as potential therapeutic targets.In this review,we briefly discuss the characteristics of nonselective autophagy and selective autophagy,including ERphagy,aggrephagy,and mitophagy.We then introduce the mechanisms of Parkin-dependent and Parkin-independent mitophagy pathways under physiological conditions.Next,we summarize the diverse repertoire of mitochondrial membrane receptors and phospholipids that mediate mitophagy.Importantly,we review the critical role of mitophagy in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease,Parkinson’s disease,and amyotrophic lateral sclerosis.Last,we discuss recent studies considering mitophagy as a potential therapeutic target for treating neurodegenerative diseases.Together,our review may provide novel views to better understand the roles of mitophagy in neurodegenerative disease pathogenesis.

Developmental Patterns and Gender Differences of Vocal Production in Marmoset Monkeys

Dear Editor,The common marmoset(Callithrix jacchus),a small New World monkey species,is considered an ideal non-human primate model for studying the evolution and neural mechanism of vocal behavior due to their rich call repertoire and vocal communication[1-3].Especially,the early vocal development of marmoset monkeys has attracted much attention,because newborn marmosets have been shown to exhibit a vocal development trajectory.Within two months after birth,marmosets'vocalizations undergo dramatic changes from cries to adult-like mature calls,a process that is influenced not only by inner physical maturation but also by parental care and feedback[4].

Enhanced Beta2-band Oscillations Denote Auditory Hallucination in Schizophrenia Patients and a Monkey Model of Psychosis

An electroencephalographic(EEG)signature of auditory hallucinations(AHs)is important for facilitating the diagnosis and treatment of AHs in schizophrenia.We recorded EEG from 25 schizophrenia patients with recurrent AHs.During the period of AHs,EEG recordings exhibited significantly elevated beta2-band power in the temporal region,as compared to those recorded in the absence of AHs or during stimulation with verbal sounds.We further generated methamphetamine-treated rhesus monkeys exhibiting psychosis-like behaviors,including repetitive sudden searching actions in the absence of external intrusion,suggesting the occurrence of AHs.Epidural EEG beta2-band power in the temporal region of these monkeys was enhanced immediately after methamphetamine treatment and positively correlated with the frequency of sudden searching actions.Thus,the enhancement of temporal beta2-band oscillations represents a signature for AHs in both patients and a monkey model of psychosis,and this monkey model can be used for developing closed-loop neuromodulation approaches for the treatment of refractory AHs in schizophrenia.

Characterization of gut microbial alterations in cynomolgus macaques during growth and maturation

Aging is closely related to physiology and disease development in animals.Gut microbiota varies with lifecycle and exerts profound influences on the host.To investigate gut microbial alterations during growth and maturation,41 female cynomolgus monkeys(Macaca fascicularis)ranging in age from 1 month to 15 years were divided into four groups(infant,young,adult,and middle-aged).

Adaptation of Ocular Opponency Neurons Mediates Attention-Induced Ocular Dominance Plasticity

Previous research has shown that ocular dominance can be biased by prolonged attention to one eye.The ocular-opponency-neuron model of binocular rivalry has been proposed as a candidate account for this phenomenon.Yet direct neural evidence is still lacking.By manipulating the contrast of dichoptic testing gratings,here we measured the steady-state visually evoked potentials(SSVEPs)at the intermodulation frequencies to selectively track the activities of ocular-opponency-neurons before and after the“dichoptic-backward-movie”adaptation.One hour of adaptation caused a shift of perceptual and neural ocular dominance towards the unattended eye.More importantly,we found a decrease in the intermodulation SSVEP response after adaptation,which was significantly greater when high-contrast gratings were presented to the attended eye than when they were presented to the unattended eye.These results strongly support the view that the adaptation of ocular-opponency-neurons contributes to the ocular dominance plasticity induced by prolonged eye-based attention.

Distributions of Visual Receptive Fields from Retinotopic to Craniotopic Coordinates in the Lateral Intraparietal Area and Frontal Eye Fields of the Macaque

Even though retinal images of objects change their locations following each eye movement,we perceive a stable and continuous world.One possible mechanism by which the brain achieves such visual stability is to construct a craniotopic coordinate by integrating retinal and extraretinal information.There have been several proposals on how this may be done,including eye-position modulation(gain fields)of retinotopic receptive fields(RFs)and craniotopic RFs.In the present study,we investigated coordinate systems used by RFs in the lateral intraparietal(LIP)cortex and frontal eye fields(FEF)and compared the two areas.We mapped the two-dimensional RFs of neurons in detail under two eye fixations and analyzed how the RF of a given neuron changes with eye position to determine its coordinate representation.The same recording and analysis procedures were applied to the two brain areas.We found that,in both areas,RFs were distributed from retinotopic to craniotopic representations.There was no significant difference between the distributions in the LIP and FEF.Only a small fraction of neurons was fully craniotopic,whereas most neurons were between the retinotopic and craniotopic representations.The distributions were strongly biased toward the retinotopic side but with significant craniotopic shifts.These results suggest that there is only weak evidence for craniotopic RFs in the LIP and FEF,and that transformation from retinotopic to craniotopic coordinates in these areas must rely on other factors such as gain fields.

Nanotechnology-based combination therapy for overcoming multidrug-resistant cancer

Multidrug resistance(MDR) is a major obstacle to successful cancer treatment and is crucial to cancer metastasis and relapse.Combination therapy is an effective strategy for overcoming MDR. However, the different pharmacokinetic(PK) profiles of combined drugs often undermine the combination effect in vivo, especially when greatly different physicochemical properties(e.g.,those of macromolecules and small drugs) combine. To address this issue, nanotechnology-based codelivery techniques have been actively explored. They possess great advantages for tumor targeting, controlled drug release, and identical drug PK profiles. Thus,a powerful tool for combination therapy is provided, and the translation from in vitro to in vivo is facilitated. In this review, we present a summary of various combination strategies for overcoming MDR and the nanotechnology-based combination therapy.

Flexible multichannel electrodes for acute recording in nonhuman primates

Flexible electrodes have demonstrated better biocompatibility than rigid electrodes in relieving tissue encapsulation and long-term recording.Nonhuman primates are closer to humans in their brains’structural and functional properties,thus making them more suitable than rodents as animal models for potential clinical usage.However,the application of flexible electrodes on nonhuman primates has rarely been reported.In the present study,a flexible multichannel electrode array for nonhuman primates was developed and implemented for extracellular recording in behaving monkeys.To minimize the window of durotomy for reducing possible risks,a guide-tube-compatible implantation solution was designed to deliver the flexible electrodes through the dura into the cortex.The proposed structure for inserting flexible electrodes was characterized ex vivo and validated in vivo.Furthermore,acute recording of multichannel flexible electrodes for the primates was performed.The results showed that the flexible electrodes and implantation method used in this study meet the needs of extracellular recording in nonhuman primates.Task-related neuronal activities with a high signal-to-noise ratio of spikes demonstrated that our whole device is currently a minimally invasive and clinically viable approach for extracellular recording.

Neural Mechanism Underlying Task-Specific Enhancement of Motor Learning by Concurrent Transcranial Direct Current Stimulation

The optimal protocol for neuromodulation by transcranial direct current stimulation(tDCS)remains unclear.Using the rotarod paradigm,we found that mouse motor learning was enhanced by anodal tDCS(3.2 mA/cm^(2))during but not before or after the performance of a task.Dual-task experiments showed that motor learning enhancement was specific to the task accompanied by anodal tDCS.Studies using a mouse model of stroke induced by middle cerebral artery occlusion showed that concurrent anodal tDCS restored motor learning capability in a task-specific manner.Transcranial in vivo Ca^(2+)imaging further showed that anodal tDCS elevated and cathodal tDCS suppressed neuronal activity in the primary motor cortex(M1).Anodal tDCS specifically promoted the activity of task-related M1 neurons during task performance,suggesting that elevated Hebbian synaptic potentiation in task-activated circuits accounts for the motor learning enhancement.Thus,application of tDCS concurrent with the targeted behavioral dysfunction could be an effective approach to treating brain disorders.

The monkey microbial biobank brings previously uncultivated bioresources for nonhuman primate and human gut microbiomes

Impact statement Nonhuman primates(NHPs)such as monkeys are the closest living relatives to humans and are the best available models for causative studies of human health and diseases.Gut microbiomes are intensively involved in host health.In this study,by large-scale cultivation of microbes from fecal samples of monkeys,we obtained previously uncultured bacterial species and constructed a Macaca fascicularis Gut Microbial Biobank(MfGMB).The MfGMB consisted of 250 strains that represent 97 species of 63 genera,25 families,and 4 phyla.The information of the 250 strains and the genomes of 97 cultured species are publicly accessible.The MfGMB represented nearly 50% of core gut microbial compositions at the genus level and covered over 80% of the KO-based known gut microbiome functions of M.fascicularis.Data mining showed that the bacterial species in the MfGMB were prevalent not only in NHPs gut microbiomes but also in human gut microbiomes.This study will help the understanding and future investigations on how gut microbiomes interact with their mammalian hosts.

Genetic Approaches for Neural Circuits Dissection in Non-human Primates

Genetic tools,which can be used for the morphology study of specific neurons,pathway-selective connectome mapping,neuronal activity monitoring,and manipulation with a spatiotemporal resolution,have been widely applied to the understanding of complex neural circuit formation,interactions,and functions in rodents.Recently,similar genetic approaches have been tried in non-human primates(NHPs)in neuroscience studies for dissecting the neural circuits involved in sophisticated behaviors and clinical brain disorders,although they are still very preliminary.In this review,we introduce the progress made in the development and application of genetic tools for brain studies on NHPs.We also discuss the advantages and limitations of each approach and provide a perspective for using genetic tools to study the neural circuits of NHPs.

Extension of the Lifespan of a Mouse Model of Rett Syndrome by Intracerebroventricular Delivery of MECP2

DearEditors,Rett Syndrome(RTT)is a severe neurodevelopmental disorder characterized by neural dysfunctions and a reduced lifespan,mainly in female patients,involving loss-of-function mutations in the methyl-CpG binding protein 2(MECP2)gene[1-4].

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.

enOsCas12f1-mediated exon skipping for Duchenne muscular dystrophy therapy in humanized mouse model

Duchenne muscular dystrophy(DMD)is a severe neuromuscular disorder caused by mutations in the dystrophin gene,which encodes the essential protein dystrophin.This genetic condition,affecting approximately 1 in 5000 male births worldwide(Birnkrant et al.,2018),is characterized by progressive muscle weakness and wasting,leading to premature death in the second or third decade of life(Farini et al.,2016).The absence of dystrophin protein causes instability in the sarcolemma,which predisposes individuals to myonecrosis and activation of inflammatory signaling cascades.Unfortunately,there is currently no effective cure for DMD,and glucocorticoid steroids are commonly used in clinical settings to delay symptom development(Merlini et al.,2003).

lahysiological links of circadian clock and biological clock of aging

Circadian rhythms orchestrate biochemical and physi-ological processes in living organisms to respond the day/night cycle. In mammals, nearly all cells hold self- sustained circadian clocks meanwhile couple the intrinsic rhythms to systemic changes in a hierarchical manner. The suprachiasmatic nucleus （SCN） of the hypothalamus functions as the master pacemaker to initiate daily synchronization according to the photope-riod, in turn determines the phase of peripheral cellular clocks through a variety of signaling relays, including endocrine rhythms and metabolic cycles. With aging, circadian desynchrony occurs at the expense of peripheral metabolic pathologies and central neurode- generative disorders with sleep symptoms, and genetic ablation of circadian genes in model organisms resem- bled the aging-related features. Notably, a number of studies have linked longevity nutrient sensing pathways in modulating circadian clocks. Therapeutic strategies that bridge the nutrient sensing pathways and circadian clock might be rational designs to defy aging.

Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.

Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

Dear Editor,RNA knockdown in vivo carries significant potential for dis-ease modeling and therapies.Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes,strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of genome DNA is not appropriate,and RNA virus infection diseases when pathogenic DNA is not available(such as SARS-Cov-2 and MERS infections).