Circuit-specific gene therapy reverses core symptoms in a primate Parkinson's disease model.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.

Pathogen protein modularity enables elaborate mimicry of a host phosphatase.

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Controlling Visually Guided Behavior by Holographic Recalling of Cortical Ensembles.

Neurons in cortical circuits are often coactivated as ensembles, yet it is unclear whether ensembles play a functional role in behavior. Some ensemble neurons have pattern completion properties, triggering the entire ensemble when activated. Using two-photon holographic optogenetics in mouse primary visual cortex, we tested whether recalling ensembles by activating pattern completion neurons alters behavioral performance in a visual task. Disruption of behaviorally relevant ensembles by activation of non-selective neurons decreased performance, whereas activation of only two pattern completion neurons from behaviorally relevant ensembles improved performance, by reliably recalling the whole ensemble. Also, inappropriate behavioral choices were evoked by the mistaken activation of behaviorally relevant ensembles. Finally, in absence of visual stimuli, optogenetic activation of two pattern completion neurons could trigger behaviorally relevant ensembles and correct behavioral responses. Our results demonstrate a causal role of neuronal ensembles in a visually guided behavior and suggest that ensembles implement internal representations of perceptual states.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

Maternal manipulation of offspring size can trigger the evolution of eusociality in promiscuous species.

Eusocial organisms typically live in colonies with one reproductive queen supported by thousands of sterile workers. It is widely believed that monogamous mating is a precondition for the evolution of eusociality. Here, we present a theoretical model that simulates a realistic scenario for the evolution of eusociality. In the model, mothers can evolve control over resource allocation to offspring, affecting offspring's body size. The offspring can evolve body-size-dependent dispersal, by which they disperse to breed or stay at the nest as helpers. We demonstrate that eusociality can evolve even if mothers are not strictly monogamous, provided that they can constrain their offspring's reproduction through manipulation. We also observe the evolution of social polymorphism with small individuals that help and larger individuals that disperse to breed. Our model unifies the traditional kin selection and maternal manipulation explanations for the evolution of eusociality and demonstrates that-contrary to current consensus belief-eusociality can evolve despite highly promiscuous mating.

Tools for editing the mammalian mitochondrial genome.

The manipulation of animal mitochondrial genomes has long been a challenge due to the lack of an effective transformation method. With the discovery of specific gene editing enzymes, designed to target pathogenic mitochondrial DNA mutations (often heteroplasmic), the selective removal or modification of mutant variants has become a reality. Because mitochondria cannot efficiently import RNAs, CRISPR has not been the first choice for editing mitochondrial genes. However, the last few years witnessed an explosion in novel and optimized non-CRISPR approaches to promote double-strand breaks or base-edit of mtDNA in vivo. Engineered forms of specific nucleases and cytidine/adenine deaminases form the basis for these techniques. I will review the newest developments that constitute the current toolbox for animal mtDNA gene editing in vivo, bringing these approaches not only to the exploration of mitochondrial function, but also closer to clinical use.

Tools to analyze the organization and formation of the germline cyst in zebrafish oogenesis.

Oocytes develop in the germline cyst, a cellular organization in which germ cells are tightly interconnected and surrounded by somatic cells. The cyst produces oocytes for follicle formation and is a hub for essential processes in meiosis and oocyte differentiation. However, the formation and organization of the cyst, and their contribution to oocyte production in vertebrates remain unclear. Here, we provide tools for three-dimensional and functional in vivo analyses of the germline cyst in the zebrafish ovary. We describe the use of serial block-face scanning electron microscopy (SBF-SEM) to resolve the three-dimensional architecture of cells and organelles in the cyst at ultrastructural resolution. We present a deep learning-based pipeline for high-throughput quantitative analysis of three-dimensional confocal datasets of cysts in vivo. We provide a method for laser ablation of cellular components for manipulating cyst cells in ovaries. These methods will facilitate the investigation of the cyst cellular organization, expand the toolkit for the study of the zebrafish ovary, and advance our understanding of female developmental reproduction. They could also be further applied to the investigation of other developmental systems.

Mouse embryos, chimeras, and embryonal carcinoma stem cells-Reflections on the winding road to gene manipulation.

The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell- or embryo-derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny, they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal.

Open millifluidics based on powder-encased channels.

Millifluidics, the manipulation of liquid flow in millimeter-sized channels, has been a revolutionary concept in chemical processing and engineering. The solid channels that contain the liquids, though, are not flexible in their design and modification, and prevent contact with the external environment. All-liquid constructs, on the other hand, while flexible and open, are imbedded in a liquid environment. Here, we provide a route to circumvent these limitations by encasing the liquids in a hydrophobic powder in air that jams on the surface, containing and isolating flowing fluids, offering flexibility and adaptability in design, as manifest in the ability to reconfigure, graft, and segment the constructs. Along with the open nature of these powder-contained channels that allow arbitrary connections/disconnections and substance addition/extraction, numerous applications can be opened in the biological, chemical, and material arenas.

Male-killing virus in a noctuid moth Spodoptera litura.

A female-biased sex ratio is considered advantageous for the cytoplasmic elements that inhabit sexually reproducing organisms. There are numerous examples of bacterial symbionts in the arthropod cytoplasm that bias the host sex ratio toward females through various means, including feminization and male killing. Recently, maternally inherited RNA viruses belonging to the family Partitiviridae were found to cause male killing in moths and flies, but it was unknown whether male-killing viruses were restricted to Partitiviridae or could be found in other taxa. Here, we provide compelling evidence that a maternally inherited RNA virus, Spodoptera litura male-killing virus (SlMKV), selectively kills male embryos of the tobacco caterpillar Spodoptera litura, resulting in all-female broods. SlMKV injected into uninfected S. litura can also be inherited maternally and causes male killing. SlMKV has five genomic segments encoding seven open reading frames, has no homolog of known male-killing genes, and belongs to an unclassified group of arthropod-specific viruses closely related to Tolivirales. When transinfected into larvae, both male and female recipients allow SlMKV to proliferate, but only males die at the pupal stage. The viral RNA levels in embryonic and pupal male killing suggest that the mechanism of male killing involves the constitutive expression of viral products that are specifically lethal to males, rather than the male-specific expression of viral products. Our results, together with recent findings on male-killing partiti-like viruses, suggest that diverse viruses in arthropods tend to acquire male killing independently and that such viruses may be important components of intragenomic conflict in arthropods.

Propulsive cell entry diverts pathogens from immune degradation by remodeling the phagocytic synapse.

Phagocytosis is a critical immune function for infection control and tissue homeostasis. During phagocytosis, pathogens are internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors are required to disrupt the biogenesis of phagolysosomes. In contrast, we present here that physical forces from motile pathogens during cell entry divert them away from the canonical degradative pathway. This altered fate begins with the force-induced remodeling of the phagocytic synapse formation. We used the parasite Toxoplasma gondii as a model because live Toxoplasma actively invades host cells using gliding motility. To differentiate the effects of physical forces from virulence factors in phagocytosis, we employed magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophages. Experiments and computer simulations show that large propulsive forces hinder productive activation of receptors by preventing their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites are engulfed into vacuoles that fail to mature into degradative units, similar to the live motile parasite's intracellular pathway. Using yeast cells and opsonized beads, we confirmed that this mechanism is general, not specific to the parasite used. These results reveal new aspects of immune evasion by demonstrating how physical forces during active cell entry, independent of virulence factors, enable pathogens to circumvent phagolysosomal degradation.

Engineered exosomes for studies in tumor immunology.

Exosomes are a type of extracellular vesicle (EV) with diameters of 30-150 nm secreted by most of the cells into the extracellular spaces and can alter the microenvironment through cell-to-cell interactions by fusion with the plasma membrane and subsequent endocytosis and release of the cargo. Because of their biocompatibility, low toxicity and immunogenicity, permeability (even through the blood-brain barrier (BBB)), stability in biological fluids, and ability to accumulate in the lesions with higher specificity, investigators have started making designer's exosomes or engineered exosomes to carry biologically active protein on the surface or inside the exosomes as well as using exosomes to carry drugs, micro RNA, and other products to the site of interest. In this review, we have discussed biogenesis, markers, and contents of various exosomes including exosomes of immune cells. We have also discussed the current methods of making engineered and designer's exosomes as well as the use of engineered exosomes targeting different immune cells in the tumors, stroke, as well as at peripheral blood. Genetic engineering and customizing exosomes create an unlimited opportunity to use in diagnosis and treatment. Very little use has been discovered, and we are far away to reach its limits.

Host plant physiological transformation and microbial population heterogeneity as important determinants of the Soft Rot Pectobacteriaceae-plant interactions.

Pectobacterium and Dickeya species belonging to the Soft Rot Pectobacteriaceae (SRP) are one of the most devastating phytopathogens. They degrade plant tissues by producing an arsenal of plant cell wall degrading enzymes. However, SRP-plant interactions are not restricted to the production of these "brute force" weapons. Additionally, these bacteria apply stealth behavior related to (1) manipulation of the host plant via induction of susceptible responses and (2) formation of heterogeneous populations with functionally specialized cells. Our review aims to summarize current knowledge on SRP-induced plant susceptible responses and on the heterogeneity of SRP populations. The review shows that SRP are capable of adjusting the host's hormonal balance, inducing host-mediated plant cell wall modification, promoting iron assimilation by the host, stimulating the accumulation of reactive oxygen species and host cell death, and activating the synthesis of secondary metabolites that are ineffective in limiting disease progression. By this means, SRP facilitate host plant susceptibility. During host colonization, SRP populations produce various functionally specialized cells adapted for enhanced virulence, increased resistance, motility, vegetative growth, or colonization of the vascular system. This enables SRP to perform self-contradictory tasks, which benefits a population's overall fitness in various environments, including host plants. Such stealthy tactical actions facilitate plant-SRP interactions and disease progression.

In vivo manufacture and manipulation of CAR-T cells for better druggability.

The current CAR-T cell therapy products have been hampered in their druggability due to the personalized preparation required, unclear pharmacokinetic characteristics, and unpredictable adverse reactions. Enabling standardized manufacturing and having clear efficacy and pharmacokinetic characteristics are prerequisites for ensuring the effective practicality of CAR-T cell therapy drugs. This review provides a broad overview of the different approaches for controlling behaviors of CAR-T cells in vivo. The utilization of genetically modified vectors enables in vivo production of CAR-T cells, thereby abbreviating or skipping the lengthy in vitro expansion process. By equipping CAR-T cells with intricately designed control elements, using molecule switches or small-molecule inhibitors, the control of CAR-T cell activity can be achieved. Moreover, the on-off control of CAR-T cell activity would yield potential gains in phenotypic remodeling. These methods provide beneficial references for the future development of safe, controllable, convenient, and suitable for standardized production of CAR-T cell therapy products.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

A versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems.

Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs) and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan, and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra ) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in future.

30†years of nanobodies - an ongoing success story of small binders in biological research.

A milestone in the field of recombinant binding molecules was achieved 30 years ago with the discovery of single-domain antibodies from which antigen-binding variable domains, better known as nanobodies (Nbs), can be derived. Being only one tenth the size of conventional antibodies, Nbs feature high affinity and specificity, while being highly stable and soluble. In addition, they display accessibility to cryptic sites, low off-target accumulation and deep tissue penetration. Efficient selection methods, such as (semi-)synthetic/naïve or immunized cDNA libraries and display technologies, have facilitated the isolation of Nbs against diverse targets, and their single-gene format enables easy functionalization and high-yield production. This Review highlights recent advances in Nb applications in various areas of biological research, including structural biology, proteomics and high-resolution and in vivo imaging. In addition, we provide insights into intracellular applications of Nbs, such as live-cell imaging, biosensors and targeted protein degradation.

Use of Elasticsearch-based business intelligence tools for integration and visualization of biological data.

The emergence of massive datasets exploring the multiple levels of molecular biology has made their analysis and knowledge transfer more complex. Flexible tools to manage big biological datasets could be of great help for standardizing the usage of developed data visualizations and integration methods. Business intelligence (BI) tools have been used in many fields as exploratory tools. They have numerous connectors to link numerous data repositories with a unified graphic interface, offering an overview of data and facilitating interpretation for decision makers. BI tools could be a flexible and user-friendly way of handling molecular biological data with interactive visualizations. However, it is rather uncommon to see such tools used for the exploration of massive and complex datasets in biological fields. We believe that two main obstacles could be the reason. Firstly, we posit that the way to import data into BI tools are not compatible with biological databases. Secondly, BI tools may not be adapted to certain particularities of complex biological data, namely, the size, the variability of datasets and the availability of specialized visualizations. This paper highlights the use of five BI tools (Elastic Kibana, Siren Investigate, Microsoft Power BI, Salesforce Tableau and Apache Superset) onto which the massive data management repository engine called Elasticsearch is compatible. Four case studies will be discussed in which these BI tools were applied on biological datasets with different characteristics. We conclude that the performance of the tools depends on the complexity of the biological questions and the size of the datasets.