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Abstract Olfactory dysfunction is associated with aging and the earliest stages of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases; it is thought to be an early biomarker of cognitive decline. In marmosets, a small non-human primate model used in brain research, olfactory pathway activity during olfactory stimulation has not been well studied because of the difficulty in clearly switching olfactory stimuli inside a narrow MRI. Here, we developed an olfactory-stimulated fMRI system using a small-aperture MRI machine. The olfactory presentation system consisted of two tubes, one for supply and one for suction of olfactory stimulants and a balloon valve. A balloon valve installed in the air supply tube controlled the presentation of the olfactory stimulant, which enabled sharp olfactory stimulation within MRI, such as 30 s of stimulation repeated five times at five-minute intervals. The olfactory stimulation system was validated in vivo and in a simulated system. fMRI analysis showed a rapid increase in signal values within 30 s of olfactory stimulation in eight regions related to the sense of smell. As these regions include those associated with Alzheimer’s and Parkinson’s diseases, olfactory stimulation fMRI may be useful in clarifying the relationship between olfactory dysfunction and dementia in non-human primates.

Abstract Assessment of social interactions and behavioral changes in nonhuman primates is useful for understanding brain function changes during life events and pathogenesis of neurological diseases. The common marmoset (Callithrix jacchus), which lives in a nuclear family like humans, is a useful model, but longitudinal automated behavioral observation of multiple animals has not been achieved. Here, we developed a Full Monitoring and Animal Identification (FulMAI) system for longitudinal detection of three-dimensional (3D) trajectories of each individual in multiple marmosets under free-moving conditions by combining video tracking, Light Detection and Ranging, and deep learning. Using this system, identification of each animal was more than 97% accurate. Location preferences and inter-individual distance could be calculated, and deep learning could detect grooming behavior. The FulMAI system allows us to analyze the natural behavior of individuals in a family over their lifetime and understand how behavior changes due to life events together with other data.

Abstract Adverse early life experiences are well-established risk factors for neurological disorders later in life. However, the molecular mechanisms underlying the impact of adverse experiences on neurophysiological systems throughout life remain incompletely understood. Previous studies suggest that social attachment to parents in early development are indispensable for infants to grow into healthy adults. In situations where multiple offspring are born in a single birth in common marmosets, human hand-rearing is employed to ensure the survival of the offspring in captivity. However, hand-reared marmosets often exhibit behavioral abnormalities, including abnormal vocalizations, excessive attachment to the caretaker, and aggressive behavior. In this study, comprehensive transcriptome analyses were conducted on hippocampus tissues, a neuroanatomical region sensitive to social attachment, obtained from human hand-reared (N = 6) and parent-reared male marmosets (N = 5) at distinct developmental stages. Our analyses revealed consistent alterations in a subset of genes, including those related to neurodevelopmental diseases, across different developmental stages, indicating their continuous susceptibility to the effects of early parental deprivation. These findings highlight the dynamic nature of gene expression in response to early life experiences and suggest that the impact of early parental deprivation on gene expression may vary across different stages of development.

Abstract Reconstitution of the germ cell lineage using pluripotent stem cells provides a unique platform to deepen our understanding of the mechanisms underlying germ cell development and to produce functional gametes for reproduction. This study aimed to establish a culture system that induces a robust number of primordial germ cell-like cells (PGCLCs) from common marmoset (Callithrix jacchus) embryonic stem cells. The robust induction was achieved by not only activation of the conserved PGC-inducing signals, WNT and BMP4, but also temporal inhibitions of WNT and retinoic acid signals, which prevent mesodermal and neural differentiation, respectively, during PGCLC differentiation. Many of the gene expression and differentiation properties of common marmoset PGCLCs were similar to those of human PGCLCs, making this culture system a reliable and useful primate model. Finally, we identified PDPN and KIT as surface marker proteins by which PGCLCs can be isolated from embryonic stem cells without genetic manipulation. This study will expand the opportunities for research on germ cell development and production of functional gametes to the common marmoset.

Primary intraosseous adenoid cystic carcinoma (PIACC) of the jaw is rare. To our knowledge, only 51 cases have been reported in the English literature. We present a rare case of PIACC arising in the mandible with multiple bone metastases and review the previous articles. A 70-year-old woman presented with paresthesia of the right chin and lower gingiva for 4 months. Radiography revealed an irregular radiolucent region on the right side of the ramus, infiltrating to the mandibular canal. Biopsy revealed a pathological diagnosis of adenoid cystic carcinoma. Multiple bone metastases were present in the sternum, scapula, and thighs. The treatment effect was progressive disease for chemotherapy; therefore, best supportive care was provided for 3 years.

ABSTRACT The intestinal microbiome is closely related to host health, and metatranscriptomic analysis can be used to assess the functional activity of microbiomes by quantifying microbial gene expression levels, helping elucidate the interactions between the microbiome and the environment. However, the functional changes in the microbiome along the host intestinal tract remain unknown, and previous analytical methods have limitations, such as potentially overlooking unknown genes due to dependence on existing databases. The objective of this study is to develop a computational pipeline combined with next-generation sequencing for spatial covariation analysis of the functional activity of microbiomes at multiple intestinal sites (biogeographic locations) within the same individual. This method reconstructs a reference metagenomic sequence across multiple intestinal sites and integrates the metagenome and metatranscriptome, allowing the gene expression levels of the microbiome, including unknown bacterial genes, to be compared among multiple sites. When this method was applied to metatranscriptomic analysis in the intestinal tract of common marmosets, a New World monkey, the reconstructed metagenome covered most of the expressed genes and revealed that the differences in microbial gene expression among the cecum, transverse colon, and feces were more dynamic and sensitive to environmental shifts than the abundances of the genes. In addition, metatranscriptomic profiling at three intestinal sites of the same individual enabled covariation analysis incorporating spatial relevance, accurately predicting the function of a total of 10,856 unknown genes. Our findings demonstrate that our proposed analytical method captures functional changes in microbiomes at the gene resolution level. IMPORTANCE We developed an analysis method that integrates metagenomes and metatranscriptomes from multiple intestinal sites to elucidate how microbial function varies along the intestinal tract. This method enables spatial covariation analysis of the functional activity of microbiomes and accurate identification of gene expression changes among intestinal sites, including changes in the expression of unknown bacterial genes. Moreover, we applied this method to the investigation of the common marmoset intestine, which is anatomically and pharmacologically similar to that of humans. Our findings indicate the expression pattern of the microbiome varies in response to changes in the internal environment along the intestinal tract, and this microbial change may affect the intestinal environment.

Measurement(s) sequence_assembly • genome • DNA Technology Type(s) sequence assembly process • DNA sequencing Sample Characteristic - Organism Macaca fascicularis • Callithrix jacchus Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14665314

Abstract GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer’s disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed (1st generation models). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL–G–F and AppNL–F mice) were developed (2nd generation models). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL–F and Psen1P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL–G–F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative AppG–F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-KATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

Abstract Background The common marmoset (Callithrix jacchus) is one of the most studied primate model organisms. However, the marmoset genomes available in the public databases are highly fragmented and filled with sequence gaps, hindering research advances related to marmoset genomics and transcriptomics. Results Here we utilize single-molecule, long-read sequence data to improve and update the existing genome assembly and report a near-complete genome of the common marmoset. The assembly is of 2.79 Gb size, with a contig N50 length of 6.37 Mb and a chromosomal scaffold N50 length of 143.91 Mb, representing the most contiguous and high-quality marmoset genome up to date. Approximately 90% of the assembled genome was represented in contigs longer than 1 Mb, with approximately 104-fold improvement in contiguity over the previously published marmoset genome. More than 98% of the gaps from the previously published genomes were filled successfully, which improved the mapping rates of genomic and transcriptomic data on to the assembled genome. Conclusions Altogether the updated, high-quality common marmoset genome assembly provide improvements at various levels over the previous versions of the marmoset genome assemblies. This will allow researchers working on primate genomics to apply the genome more efficiently for their genomic and transcriptomic sequence data.

Lycopene is a lipophilic unsaturated carotenoid exhibiting a strong singlet oxygen-quenching ability. Herein, we investigated the effect of lycopene intake on the fasting blood glucose (FBG) level by conducting a systematic review and meta-analyses. We searched 15 databases (from the earliest date to June 2022 for PubMed or to August or September 2018 for the other databases) and included human interventional studies that assessed the effects of oral lycopene intake on FBG levels of participants ≥ 18 years of age. Three authors independently selected applicable studies and then assessed the study quality. Data were pooled as standardized mean difference (SMD) and analyzed by the random-effects model. Heterogeneity was assessed by I2 statistics. A meta-analysis including 11 trial arms (n = 750) revealed a tendency towards a significant decrease in FBG level with not-important heterogeneity [SMD = −0.15 (95% CI: −0.31, 0.00), p = 0.05, I2 = 9%]. Subgroup meta-analysis including two studies (n = 152) in type 2 diabetes patients revealed significantly decreased FBG levels with not-important heterogeneity [SMD = −0.37 (95% CI: −0.69, −0.05), p = 0.02, I2 = 0%]. Most studies meeting the eligibility criteria had a moderate risk of bias. The funnel plot for FBG suggested an absence of publication bias. In conclusion, this systematic review and meta-analyses suggested that lycopene intake exerted an FBG-decreasing effect.

Mutations in C8orf37 cause Bardet-Biedl syndrome (BBS), retinitis pigmentosa (RP), and cone–rod dystrophy (CRD), all manifest in photoreceptor degeneration. Little is known about which proteins C8orf37 interacts with to contribute to photoreceptor survival. To determine the proteins that potentially interact with C8orf37, we carried out a yeast two-hybrid (Y2H) screen using C8orf37 as a bait. FAM161A, a microtubule-binding protein localized at the photoreceptor cilium required for photoreceptor survival, was identified as one of the preys. Double immunofluorescence staining and proximity ligation assay (PLA) of marmoset retinal sections showed that C8orf37 was enriched and was co-localized with FAM161A at the ciliary base of photoreceptors. Epitope-tagged C8orf37 and FAM161A, expressed in HEK293 cells, were also found to be co-localized by double immunofluorescence staining and PLA. Furthermore, interaction domain mapping assays identified that the N-terminal region of C8orf37 and amino acid residues 341-517 within the PFAM UPF0564 domain of FAM161A were critical for C8orf37–FAM161A interaction. These data suggest that the two photoreceptor survival proteins, C8orf37 and FAM161A, interact with each other which may contribute to photoreceptor health.

Pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and induced PSCs (iPSCs) are excellent tools for studying embryonic development in organisms and classified into naïve and primed states. ESC-derived germline chimera individuals can be produced by injecting naïve ESCs/iPSCs into preimplantation embryos, and conversion of primed human ESCs/iPSCs into a naïve state provides insights into epiblast cell features. Non-human ESCs/iPSCs are alternatives to human naïve ESCs/iPSCs, which elicit ethical issues. In this study, we used the common marmoset (Callithrix jacchus) as an animal model. Since 1996, 16 marmoset ESC lines have been established. Because most of these ESC lines are female and were derived >10 years ago, new ESCs, particularly male marmoset ESC lines, are needed. Here, we successfully established 17 novel marmoset ESC lines, including six male ESC lines from in vitro-fertilized (IVF) embryos and 12 ESC lines under feeder-free conditions. This report is the first to establish ESC lines using feeder-free conditions and IVF preimplantation blastocysts in marmosets, and these novel ESC lines could potentially facilitate future non-human primate ESC studies.

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA).

We previously reported the efficient targeted introduction of transgenes into the genomic DNA of the common marmoset (Callithrix jacchus) using CRISPR-Cas9. In this study, we generated a marmoset embryonic stem cell (ESC) line that ubiquitously expresses the tamoxifen-inducible Cre-driver ERT2CreERT2. We validated the pluripotency of the ESC line and also successfully demonstrated the temporal control of the Cre-driver in a tamoxifen-dependent manner in the ESCs. This ESC line, named ActiCre-B1, will be a valuable resource for in vitro investigation of phenotypes related to embryonic lethality by targeted knockout of functionally important genes.

BLIMP1 (PRDM1) and VASA (DDX4) play pivotal roles in the development of the germ cell linage. Importantly, these genes are specifically expressed in germ cells; BLIMP1 in primordial germ cells (PGCs) to early-stage gonocytes, and VASA in migration-stage PGCs to mature gametes. The high reproductive efficiency of common marmosets (marmosets; Callithrix jacchus) makes them advantageous for use in germ cell research. We herein report the generation of a male marmoset embryonic stem cell (ESC) line harboring BLIMP1 and DDX4 double reporters. This ESC line will be a useful tool for investigating male gametogenesis in non-human primates.

Marmosets are an important model organism in neuroscience but there has only been limited success in training them on behavioral tasks. Here the authors report their ability to train marmosets in various motor tasks and simultaneously image neural dynamics in motor cortex with 2-photon imaging.

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

Understanding the connectivity architecture of entire vertebrate brains is a fundamental but difficult task. Here we present an integrated neuro-histological pipeline as well as a grid-based tracer injection strategy for systematic mesoscale connectivity mapping in the common marmoset (Callithrix jacchus). Individual brains are sectioned into ~1700 20 µm sections using the tape transfer technique, permitting high quality 3D reconstruction of a series of histochemical stains (Nissl, myelin) interleaved with tracer labeled sections. Systematic in-vivo MRI of the individual animals facilitates injection placement into reference-atlas defined anatomical compartments. Further, by combining the resulting 3D volumes, containing informative cytoarchitectonic markers, with in-vivo and ex-vivo MRI, and using an integrated computational pipeline, we are able to accurately map individual brains into a common reference atlas despite the significant individual variation. This approach will facilitate the systematic assembly of a mesoscale connectivity matrix together with unprecedented 3D reconstructions of brain-wide projection patterns in a primate brain.

Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.

To control infectious diseases in humans, it is important to understand the pathogenicity of the infecting organism(s). Although non-human primates, such as cynomolgus and rhesus macaques, have been used for influenza virus infection models, their size can limit their use in confined animal facilities. In this study, we investigated the susceptibility of marmosets to influenza viruses to assess the possibility of using these animals as a non-human primate model for influenza research. We first used an influenza A (H1N1)pdm09 virus to compare two inoculation routes: the conventional route, via a combination of the intratracheal, intranasal, ocular, and oral routes; and the tracheal spray route. In marmosets inoculated via the tracheal spray route, we found inflammation throughout the lungs and trachea. In contrast, in marmosets inoculated via the conventional route, the inflammation was confined to roughly the center of the lung. These data suggest that the tracheal spray route may be more suitable than the conventional route to inoculate marmosets with influenza viruses. We also tested an influenza A(H5N1) highly pathogenic avian influenza (HPAI) virus and found that some marmosets inoculated with this virus via the tracheal spray route showed weight loss, decreased body temperature, and loss of appetite and activity. The replication of this H5N1 virus in respiratory organs was confirmed. These results indicate the potential of marmosets as an animal model for infection with seasonal or HPAI viruses.

Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1-2 h, 2-4 h, 4-6 h, 6-8 h, and 8-10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1-2 h was lowest among groups at 2-4 h, 4-6 h, 6-8 h, and 8-10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p>0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets.

The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals for drug development in the future.

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins-an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a-were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

Passa-se de relance por menção dos complexos fatores de azares e riscos dominantes naturais erráticos menos previsíveis do que os dos maciços construídos sob controle: relembra-se o princípio básico dos atendimentos mediante a seleção do "universo físico" menos vulnerável, sob domínio da estatística de médias. Exemplifica-se na barragem de terra (de Mello, 1977; Viotti, 1989). No tocante as análises convencionais de instabilizações de taludes, demonstra-se que coincidências históricas e regionais (Bishop, 1952) introduziram um "gene poluidor'' em todos os métodos e programas de computação. Gerados por uma hipótese simplificadora de Terzaghi (1936), prevalecem ao longo de 67 anos com um erro significativo conceitual e numérico, obviamente a favor da segurança, anti-económico. Ressalta-se a sedução preponderante da computação, que desviou os civis-geotécnicos dos entusiasmos pela geotecnia apaixonante de aprimoramento dos fatores razoavelmente controláveis. Incorporam-se correcções conceituais e práticas bem fundadas nos avanços pesquisadores, e no questionamento convencido e convincente dos primórdios, inescapavelmente mais grosseiros, de parâmetros e hipóteses que nos serviram por longo tempo, com grato reconhecimento. Exemplificam-se alguns erros a corrigir de imediato.