Selective Expansion Research Articles

A multimodal continuum robot constructed using three stable state characteristics of Kresling origami

Abstract Inspired by the Kresling origami structure and its three stable state characteristics, a novel multimodal continuum robot (MCR) has been proposed in this paper, which achieve its own movement through gas-driven and actively elastic deformed Kresling origami. Air bags are utilized to auxiliary drive in this paper, which make the specific plane of origami unit transform from first stable state to third stable state. The high stiffness of third stable state endows the origami unit better carrying capacity and more motion characteristics, the driving force of the central chamber and selective expansion of air bags enable the multimodal motion of MCR. The theoretical model of MCR and three stable state characteristics of Kresling origami are established to predict and control the behavior of actuator. The experimental results of MCR prototype demonstrate the high carrying capacity of proposed origami unit, the feasibility and high accuracy of multimodal motion. The designing concept of this paper improves the ability of continuum robots to perform complex tasks and promotes the development of origami structures in the field of robotics.

Therapeutic liver cell transplantation to treat murine PKU.

For gene therapy of the liver, in vivo applications based on adeno-associated virus are the most advanced vectors despite limitations, including low efficacy and episomal loss, potential integration and safety issues, and high production costs. Alternative vectors and/or delivery routes are of high interest. The regenerative ability of the liver bears the potential for ex vivo therapy using liver cell transplantation for disease correction if provided with a selective advantage to expand and replace the existing cell mass. Here we present such treatment of a mouse model of human phenylketonuria (PKU). Primary hepatocytes from wild-type mice were gene modified in vitro (with a lentiviral vector) that carries a gene editing system (CRISPR) to inhibit Cypor. Cypor inactivation confers paracetamol (or acetaminophen) resistance to hepatocytes and thus a growth advantage to eliminate the pre-existing liver cells upon grafting (via the spleen) and exposure to repeated treatment with paracetamol. Grafting Cypor-inactivated wild-type hepatocytes into inbred young adult enu2 (PKU) mice, followed by selective expansion by paracetamol dosing, resulted in replacing up to 5% of cell mass, normalization of blood phenylalanine, and permanent correction of PKU. Hepatocyte transplantation offers thus an armamentarium of novel therapy options for genetic liver defects.

Tracking the progeny of bacterial persisters using a CRISPR-based genomic recorder

The rise of antimicrobial failure is a global emergency, and causes beyond typical genetic resistance must be determined. One probable factor is the existence of subpopulations of transiently growth-arrested bacteria, persisters, that endure antibiotic treatment despite genetic susceptibility to the drug. The presence of persisters in infected hosts has been successfully established, notably through the development of fluorescent reporters. It is proposed that infection relapse is caused by persisters resuming growth after cessation of the antibiotic treatment, but to date, there is no direct evidence for this. This is because no tool or reporter currently exists to track the extent to which infection relapse is initiated by regrowth of persisters in the host. Indeed, once they have transitioned out of the persister state, the progeny of persisters are genetically and phenotypically identical to susceptible bacteria in the population, making it virtually impossible to ascertain the source of relapse. We designed pSCRATCH (plasmid for Selective CRISPR Array expansion To Check Heritage), a molecular tool that functions to record the state of antibiotic persistence in the genome of Salmonella persisters. We show that pSCRATCH successfully marks persisters by adding spacers in their CRISPR arrays and the genomic label is stable in persister progeny after exit from persistence. We further show that in a Salmonella infection model the system enables the discrimination of treatment failure originating from persistence versus resistance. Thus, pSCRATCH provides proof of principle for stable marking of persisters and a prototype for applications to more complex infection models and other pathogens.

An integrative strategy used by the aphid Uroleucon formosanum to counter host sesquiterpene lactone defense: Insights from combined genomic and transcriptomic analysis.

Insect herbivores adapt and develop strategies to counteract plant chemical defenses. The aphid Uroleucon formosanum is a serious sap-sucking pest that infests lettuces containing toxic sesquiterpene lactones (STLs). Herein, we employed a combination of genome sequencing and RNA-seq transcriptome profiling to understand the mechanisms underlying phytotoxin tolerance in U.formosanum. We generated the first chromosome-level genome assembly for U.formosanum, with a total size of 453.26Mb and a scaffold N50 of 33.22Mb. Comparative genomic analyses revealed an enrichment of signals for positive selection and gene family expansion in immune-related pathways. Specifically, the expanded set of heat shock protein70 (HSP70) genes showed upregulation after treatment with lactucin, suggesting that they may play a role in the immune response against STLs. The expression of takeout-like genes and cuticle-associated genes was also significantly increased in the lactucin-treated samples. Additionally, 53 cytochrome P450 monooxygenase, 30 carboxylesterase, 19 glutathione S-transferase, 32 uridine diphosphate glycosyltransferase and 63 ATP-binding cassette (ABC) transporter genes were identified in the U.formosanum genome. CYP4C1, CYP6A13 and 7 ABC genes were strongly upregulated in response to lactucin treatment, indicating the involvement of detoxifying enzymes in the tolerance of U.formosanum to STLs. Our findings suggest that the cuticle barrier, immune response and enzyme-mediated metabolic detoxification jointly enhance the tolerance of U.formosanum to phytotoxins and promote its adaptation to host plants. This study presents a valuable genomic resource and provides insights into insect adaptation to plant chemical challenges and future technological developments for pest management.

MicroRNA-146a deficiency enhances host protection against murine cytomegalovirus.

Natural killer (NK) cells are innate lymphoid cells that protect a host from viral infections and malignancies. MicroRNA-146a (miR-146a) is an important regulator of immune function that is highly expressed in NK cells and is further upregulated during murine cytomegalovirus (MCMV) infection. Here we utilized mice with a global targeted deletion of miR-146a to understand its impact on the innate immune responses to MCMV infection. MiR-146a-/- mice were protected from lethal MCMV infection, which was intrinsic to the hematopoietic compartment based on bone marrow chimera experiments. NK cell depletion abrogated this protection, implicating NK cells as critical for the miR-146a-/- protection from MCMV. Surprisingly, NK cells from miR-146a-deficient mice were largely similar to control NK cells with respect to development, maturation, trafficking, and effector functions. However, miR-146a-/- mice had increased NK cell numbers and frequency of the most mature Stage IV (CD27-CD11b+) NK cells in the liver at baseline, enhanced STAT1 phosphorylation, and increased selective expansion of Ly49H+ NK cells and T cells during MCMV infection. This study demonstrates a critical role for miR-146a in the host response to MCMV, arising from mechanisms that include increased NK cell numbers and early T-cell expansion.

Selective Weighting and Prediction Error Expansion for High-Fidelity Images

Selective Weighting and Prediction Error Expansion for High-Fidelity Images

406.7: Inducing long-term heart allograft tolerance through selective Treg expansion by IL-2 complex treatment.

406.7: Inducing long-term heart allograft tolerance through selective Treg expansion by IL-2 complex treatment.

Comparative Genomics Supports Ecologically Induced Selection as a Putative Driver of Banded Penguin Diversification.

The relative importance of genetic drift and local adaptation in facilitating speciation remains unclear. This is particularly true for seabirds, which can disperse over large geographic distances, providing opportunities for intermittent gene flow among distant colonies that span the temperature and salinity gradients of the oceans. Here, we delve into the genomic basis of adaptation and speciation of banded penguins, Galápagos (Spheniscus mendiculus), Humboldt (Spheniscus humboldti), Magellanic (Spheniscus magellanicus), and African penguins (Spheniscus demersus), by analyzing 114 genomes from the main 16 breeding colonies. We aim to identify the molecular mechanism and genomic adaptive traits that have facilitated their diversifications. Through positive selection and gene family expansion analyses, we identified candidate genes that may be related to reproductive isolation processes mediated by ecological thermal niche divergence. We recover signals of positive selection on key loci associated with spermatogenesis, especially during the recent peripatric divergence of the Galápagos penguin from the Humboldt penguin. High temperatures in tropical habitats may have favored selection on loci associated with spermatogenesis to maintain sperm viability, leading to reproductive isolation among young species. Our results suggest that genome-wide selection on loci associated with molecular pathways that underpin thermoregulation, osmoregulation, hypoxia, and social behavior appears to have been crucial in local adaptation of banded penguins. Overall, these results contribute to our understanding of how the complexity of biotic, but especially abiotic, factors, along with the high dispersal capabilities of these marine species, may promote both neutral and adaptive lineage divergence even in the presence of gene flow.

LIM-domain-only 4 (LMO4) enhances CD8+ T-cell stemness and tumor rejection by boosting IL-21-STAT3 signaling

High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8+ T cells. Using IFN-γ production as a proxy for CD8+ T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8+ T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8+ T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8+ memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8+ T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8+ T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies.

Activation of Antiviral Host Responses against Avian Influenza Virus and Remodeling of Gut Microbiota by rLAB Vector Expressing rIL-17A in Chickens.

Low-pathogenic avian influenza virus (LPAIV) remains the most common subtype of type-A influenza virus that causes moderate to severe infection in poultry with significant zoonotic and pandemic potential. Due to high mutability, increasing drug resistance, and limited vaccine availability, the conventional means to prevent intra- or interspecies transmission of AIV is highly challenging. As an alternative to control AIV infections, cytokine-based approaches to augment antiviral host defense have gained significant attention. However, the selective application of cytokines is critical since unregulated expression of cytokines, particularly proinflammatory ones, can cause substantial tissue damage during acute phases of immune responses. Moreover, depending on the type of cytokine and its impact on intestinal microbiota, outcomes of cytokine-gut microflora interaction can have a critical effect on overall host defense against AIV infections. Our recent study demonstrated some prominent roles of chicken IL-17A (ChIL-17A) in regulating antiviral host responses against AIV infection, however, in an in vitro model. For more detailed insights into ChIL-17A function, in the present study, we investigated whether ChIL-17A-meditated elevated antiviral host responses can translate into effective immune protection against AIV infection in an in vivo system. Moreover, considering the role of gut health in fostering innate or local host responses, we further studied the contributory relationships between gut microbiota and host immunity against AIV infection in chickens. For this, we employed a recombinant lactic acid-producing bacterial (LAB) vector, Lactococcus lactis, expressing ChIL-17A and analyzed the in vivo functionality in chickens against an LPAIV (A/H9N2) infection. Our study delineates that mucosal delivery of rL. lactis expressing ChIL-17A triggers proinflammatory signaling cascades and can drive a positive shift in phylum Firmicutes, along with a marked decline in phylum Actinobacteriota and Proteobacteria, favoring effective antiviral host responses against AIV infection in chickens. We propose that ChIL-17A-mediated selective expansion of beneficial gut microbiota might form a healthy microbial community that augments the effective immune protection against AIV infections in chickens.

PD-1 controls differentiation, survival, and TCR affinity evolution of stem-like CD8+ T cells.

Stem-like progenitors are a critical subset of cytotoxic T cells that self-renew and give rise to expanded populations of effector cells critical for successful checkpoint blockade immunotherapy. Emerging evidence suggests that the tumor-draining lymph nodes can support the continuous generation of these stem-like cells that replenish the tumor sites and act as a critical source of expanded effector populations, underlining the importance of understanding what factors promote and maintain activated T cells in the stem-like state. Using advanced 3D multiplex immunofluorescence imaging, here we identified antigen-presentation niches in tumor-draining lymph nodes that support the expansion, maintenance, and affinity evolution of a unique population of TCF-1+PD-1+SLAMF6 hi stem-like CD8+ T cells. Our results show that contrary to the prevailing view that persistent TCR signaling drives terminal effector differentiation, prolonged antigen engagement well beyond the initial priming phase sustained the proliferation and self-renewal of these stem-like T cells in vivo . The inhibitory PD-1 pathway plays a central role in this process by mediating the fine-tuning of TCR and co-stimulatory signal input that enables selective expansion of high affinity TCR stem-like clones, enabling them to act as a renewable source of high affinity effector cells. PD-1 checkpoint blockade disrupts this fine tuning of input signaling, leading to terminal differentiation to the effector state or death of the most avid anti-tumor stem-like cells. Our results thus reveal an unexpected relationship between TCR ligand affinity recognition, a key negative feedback regulatory loop, and T cell stemness programming. Furthermore, these findings raise questions about whether anti-PD-1 checkpoint blockade during cancer immunotherapy provides a short-term anti-tumor effect that comes at the cost of diminishing efficacy due to progressive loss of these critical high affinity stem-like precursors.

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

CHIP: a clonal odyssey of the bone marrow niche.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.

Sepsis shapes the human γδ TCR repertoire in an age- and pathogen-dependent manner.

Sepsis affects 25 million children per year globally, leading to 2.9 million deaths and substantial disability in survivors. Extensive characterization of interactions between the host and bacteria in children is required to design novel preventive and therapeutic strategies tailored to this age group. Vγ9Vδ2 T cells are the first T cells generated in humans. These cells are defined by the expression of Vγ9Vδ2 T-cell receptors (TCRs, using the TRGV9 and TRDV2 gene segments), which react strongly against the prototypical bacterial phosphoantigen HMBPP. We investigated this reactivity by analyzing the TCR δ (TRD) repertoire in the blood of 76 children (0-16 years) with blood culture-proven bacterial sepsis caused by HMBPP-positive Escherichia coli or by HMBPP-negative Staphylococcus aureus or by HMBPP-negative Streptococcus pneumoniae. Strikingly, we found that S. aureus, and to a lesser extent E. coli but not S. pneumoniae, shaped the TRDV2 repertoire in young children (<2 years) but not in older children or adults. This dichotomy was due to the selective expansion of a fetal TRDV2 repertoire. Thus, young children possess fetal-derived Vγ9Vδ2 T cells that are highly responsive toward specific bacterial pathogens.

Reconciling Britain’s agricultural trade policy initiatives with world trade rules: 1960–1975

ABSTRACT Between 1960 and 1972 successive British governments explored, and sometimes pursued, a series of agricultural trade policy initiatives designed to shift the burden of supporting UK farmers from taxpayers to consumers, and to reduce the UK’s agri-food imports by expanding home production. These included a minimum import price scheme for cereals; a Selective Expansion Programme; debate about Agriculture’s Import Saving Role; and 1970 proposals for a fundamental change to farm policy that would apply whether or not the UK joined the European Communities (EC). Prior to EC membership the rules of the General Agreement on Tariffs and Trade (GATT), bilateral agreements with a number of countries, and pressure from the UK’s trading partners (particularly the United States) constrained its room for manoeuvre. Consequently, this dimension of farm policy could not safely be left to agriculture ministers and the Treasury, but was one that involved other departments, and prime ministers, as it necessitated detailed international negotiations. Following EC entry, GATT Article XXIV deliberations and the UK’s renegotiation of its terms of entry failed to secure meaningful reform of the common agricultural policy (CAP). The 1975 referendum confirming EC membership meant the UK no longer had an independent agri-food trade policy.

Reconfiguration of the visual code and retinal cell type complement in closely related diurnal and nocturnal mice.

How does evolution act on neuronal populations to match computational characteristics to functional demands? We address this problem by comparing visual code and retinal cell composition in closely related murid species with different behaviours. Rhabdomys pumilio are diurnal and have substantially thicker inner retina and larger visual thalamus than nocturnal Mus musculus. High-density electrophysiological recordings of visual response features in the dorsal lateral geniculate nucleus (dLGN) reveals that Rhabdomys attains higher spatiotemporal acuity both by denser coverage of the visual scene and a selective expansion of elements of the code characterised by non-linear spatiotemporal summation. Comparative analysis of single cell transcriptomic cell atlases reveals that realignment of the visual code is associated with increased relative abundance of bipolar and ganglion cell types supporting OFF and ON-OFF responses. These findings demonstrate how changes in retinal cell complement can reconfigure the coding of visual information to match changes in visual needs.

Regulatory T Cell Dysfunction in Autoimmune Diseases.

Regulatory T cells (Tregs), a suppressive subpopulation of T cells, are potent mediators of peripheral tolerance, responsible for immune homeostasis. Many autoimmune diseases exhibit disruptions in Treg function or quantity, resulting in an imbalance between protective and pathogenic immune cells. Selective expansion or manipulation of Tregs is a promising therapeutic approach for autoimmune diseases. However, the extensive diversity of Treg subpopulations and the multiple approaches used for Treg identification leads to high complexity, making it difficult to develop a successful treatment capable of modulating Tregs. In this review, we describe the suppressive mechanisms, subpopulations, classification, and identification methodology for Tregs, and their role in the pathogenesis of autoimmune diseases.

Multiscale formulation for materials composed by a saturated porous matrix and solid inclusions

Despite all the progress achieved in the characterization of heterogeneous materials by using multiscale paradigms based on the Representative Volume Element concept (RVE), there are still many aspects that demand ongoing development. We mention, for instance, in-homogeneous media with internal micro-structure comprising a mixture of components that require a dissimilar number/character of primary fields to describe their physical behavior. Micro-structures constituted by a saturated porous matrix (described through the pair displacement/pore pressure fields) endowed with impermeable non-porous solid inclusions (described only by the displacement field) are a typical example among many other practical applications. This new level of heterogeneity, between the set of primal descriptors for each micro-scale constituent, claims detailed revisions and novel adjustments in multiscale RVE-based theories.In this work, we present a first-order RVE-based formulation to address with the aforementioned phenomenology at a smaller length scale. At the macro-scale, the well-known poromechanics theory is preserved, where the constitutive response is provided by homogenization of the corresponding micro-scale problem. Two important attributes feature the model: (i) it is encompassed within the general framework posited by the “Method of Multiscale Virtual Power (MMVP)”, this leads to a variationally consistent methodology; (ii) our formulation preserves the objectivity with respect to the RVE-size (an indispensable but uncommon characteristic in RVE-based multiscale approaches for saturated porous media), due to it is based in a recent contribution of the authors (the so-called Selective Order Expansion technique “SOE”).The formulation is implemented following the squared Finite Element (FE2) numerical scheme. The potentialities of the model are demonstrated through a detailed series of numerical examples, including rigorous comparisons against the Direct Numerical Simulation (DNS) procedure.

Neutral selection and clonal expansion during the development of colon cancer metastasis.

Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours.

CarcSeq detection of lorcaserin-induced clonal expansion of Pik3ca H1047R mutants in rat mammary tissue.

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a nongenotoxic rat carcinogen, which induced mammary tumors in male and female rats in a 2-yr bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 wk. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary, and liver samples using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as nongenotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations, namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error-corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, P < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 wk as compared with 12 wk. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a nongenotoxic carcinogen using a treatment duration as short as 3 months.