Precise sensorimotor control impacts reproductive fitness of C. elegans in 3D environments.

The ability of animals to sense and navigate towards relevant cues in complex and elaborate habitats is paramount for their survival and reproductive success. The nematode Caenorhabditis elegans uses a simple and elegant sensorimotor program to track odors in its environments. Whether this allows the worm to effectively navigate a complex environment and increase its evolutionary success has not been tested yet. We designed an assay to test whether C. elegans can track odors in a complex 3D environment. We then used a previously established 3D cultivation system to test whether defect in tracking odors to find food in a complex environment affected their brood size. We found that wild-type worms can accurately migrate toward a variety of odors in 3D. However, mutants of the muscarinic acetylcholine receptor GAR-3 which have a sensorimotor integration defect that results in a subtle navigational defect steering towards attractive odors, display decreased chemotaxis to the odor butanone not seen in the traditional 2D assay. We also show that the decreased ability to locate appetitive stimuli in 3D leads to reduced brood size not observed in the standard 2D culture conditions. Our study shows that mutations in genes previously overlooked in 2D conditions can have a significant impact in the natural habitat, and highlights the importance of considering the evolutionary selective pressures that have shaped the behavior, as well as the underlying genes and neural circuits.

A siRNA targets and inhibits a broad range of SARS-CoV-2 infections including Delta variant.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants has altered the trajectory of the COVID-19 pandemic and raised some uncertainty on the long-term efficiency of vaccine strategy. The development of new therapeutics against a wide range of SARS-CoV-2 variants is imperative. We, here, have designed an inhalable siRNA, C6G25S, which covers 99.8% of current SARS-CoV-2 variants and is capable of inhibiting dominant strains, including Alpha, Delta, Gamma, and Epsilon, at picomolar ranges of IC50 in vitro. Moreover, C6G25S could completely inhibit the production of infectious virions in lungs by prophylactic treatment, and decrease 96.2% of virions by cotreatment in K18-hACE2-transgenic mice, accompanied by a significant prevention of virus-associated extensive pulmonary alveolar damage, vascular thrombi, and immune cell infiltrations. Our data suggest that C6G25S provides an alternative and effective approach to combating the COVID-19 pandemic.

Facile Transformation of Murine and Human Primary Dendritic Cells into Robust and Modular Artificial Antigen-Presenting Systems by Intracellular Hydrogelation.

The growing enthusiasm for cancer immunotherapies and adoptive cell therapies has prompted increasing interest in biomaterials development mimicking natural antigen-presenting cells (APCs) for T-cell expansion. In contrast to conventional bottom-up approaches aimed at layering synthetic substrates with T-cell activation cues, transformation of live dendritic cells (DCs) into artificial APCs (aAPCs) is demonstrated herein using a facile and minimally disruptive hydrogelation technique. Through direct intracellular permeation of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel monomer and UV-activated radical polymerization, intracellular hydrogelation is rapidly accomplished on DCs with minimal influence on cellular morphology and surface antigen display, yielding highly robust and modular cell-gel hybrid constructs amenable to peptide antigen exchange, storable by freezing and lyophilization, and functionalizable with cytokine-releasing carriers for T-cell modulation. The DC-derived aAPCs are shown to induce prolonged T-cell expansion and improve anticancer efficacy of adoptive T-cell therapy in mice compared to nonexpanded control T cells, and the gelation technique is further demonstrated to stabilize primary DCs derived from human donors. The work presents a versatile approach for generating a new class of cell-mimicking biomaterials and opens new venues for immunological interrogation and immunoengineering.

Consumption of Oleic Acid During Matriphagy in Free-Living Nematodes Alleviates the Toxic Effects of the Bacterial Metabolite Violacein.

Maternal behaviors benefit the survival of young, contributing directly to the mother's reproductive fitness. An extreme form of this is seen in matriphagy, when a mother performs the ultimate sacrifice and offers her body as a meal for her young. Whether matriphagy offers only a single energy-rich meal or another possible benefit to the young is unknown. Here, we characterized the toxicity of a bacterial secondary metabolite, namely, violacein, in Caenorhabditis elegans and found it is not only toxic towards adults, but also arrests growth and development of C. elegans larvae. To counteract this, C. elegans resorted to matriphagy, with the mothers holding their eggs within their bodies and hatching the young larvae internally, which eventually led to the mothers' death. This violacein-induced matriphagy alleviated some of the toxic effects of violacein, allowing a portion of the internally-hatched young to bypass developmental arrest. Using genetic and pharmacological experiments, we found the consumption of oleate, a monounsaturated fatty acid produced by the mother, during matriphagy is partially responsible. As such, our study provides experimental evidence of why such a drastic and peculiar maternal behavior may have arisen in nematode natural habitats.

Odor-dependent temporal dynamics in Caenorhabitis elegans adaptation and aversive learning behavior.

Animals sense an enormous number of cues in their environments, and, over time, can form learned associations and memories with some of these. The nervous system remarkably maintains the specificity of learning and memory to each of the cues. Here we asked whether the nematode Caenorhabditis elegans adjusts the temporal dynamics of adaptation and aversive learning depending on the specific odor sensed. C. elegans senses a multitude of odors, and adaptation and learned associations to many of these odors requires activity of the cGMP-dependent protein kinase EGL-4 in the AWC sensory neuron. We identified a panel of 17 attractive odors, some of which have not been tested before, and determined that the majority of these odors require the AWC primary sensory neuron for sensation. We then devised a novel assay to assess odor behavior over time for a single population of animals. We used this assay to evaluate the temporal dynamics of adaptation and aversive learning to 13 odors and find that behavior change occurs early in some odors and later in others. We then examined EGL-4 localization in early-trending and late-trending odors over time. We found that the timing of these behavior changes correlated with the timing of nuclear accumulation of EGL-4 in the AWC neuron suggesting that temporal changes in behavior may be mediated by aversive learning mechanisms. We demonstrate that temporal dynamics of adaptation and aversive learning in C. elegans can be used as a model to study the timing of memory formation to different sensory cues.

Hypergravity hinders axonal development of motor neurons in Caenorhabditis elegans.

As space flight becomes more accessible in the future, humans will be exposed to gravity conditions other than our 1G environment on Earth. Our bodies and physiology, however, are adapted for life at 1G gravity. Altering gravity can have profound effects on the body, particularly the development of muscles, but the reasons and biology behind gravity's effect are not fully known. We asked whether increasing gravity had effects on the development of motor neurons that innervate and control muscle, a relatively unexplored area of gravity biology. Using the nematode model organism Caenorhabditis elegans, we examined changes in response to hypergravity in the development of the 19 GABAergic DD/VD motor neurons that innervate body muscle. We found that a high gravity force above 10G significantly increases the number of animals with defects in the development of axonal projections from the DD/VD neurons. We showed that a critical period of hypergravity exposure during the embryonic/early larval stage was sufficient to induce defects. While characterizing the nature of the axonal defects, we found that in normal 1G gravity conditions, DD/VD axonal defects occasionally occurred, with the majority of defects occurring on the dorsal side of the animal and in the mid-body region, and a significantly higher rate of error in the 13 VD axons than the 6 DD axons. Hypergravity exposure increased the rate of DD/VD axonal defects, but did not change the distribution or the characteristics of the defects. Our study demonstrates that altering gravity can impact motor neuron development.

NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.

The nematodeCaenorhabditiselegansis one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a two dimensional habitat. In nature, however,C. elegansis found in three dimensional environments such as rotting fruit. To investigate the biology ofC. elegansin a 3D controlled environment we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. NGT-3D allows for the growth of both nematodes and the bacteria they consume. Worms show comparable rates of growth, reproduction and lifespan when bacterial colonies in the 3D matrix are abundant. However, when bacteria are sparse, growth and brood size fail to reach levels observed in standard 2D plates. Using NGT-3D we observe drastic deficits in fertility in a sensory mutant in 3D compared to 2D, and this defect was likely due to an inability to locate bacteria. Overall, NGT-3D will sharpen our understanding of nematode biology and allow scientists to investigate questions of nematode ecology and evolutionary fitness in the laboratory.

Two Endogenous Antiangiogenic Inhibitors, Endostatin and Angiostatin, Demonstrate Biphasic Curves in their Antitumor Profiles.

Angiogenesis refers to growth of blood vessels from pre-existing ones. In 1971, Folkman proposed that by choking off the blood supply to tumors, they are starved, leading to their demise. A few years ago, the monoclonal antibody Avastin became the first antiangiogenic biological approved by FDA, for treatment of cancer patients. Two other antiangiogenic endogenous protein fragments were isolated in Folkman's laboratory more than a decade ago. Here, we present a short review of data demonstrating that angiostatin and endostatin display a biphasic antitumor dose-response. This behavior is common among a large number of antiangiogenic agents and the reduced effectiveness of antiangiogenic agents at high dose rates may be due to suppression of growth of new vessels carrying the agent into the critical region around the tumor.

HSPG-binding peptide corresponding to the exon 6a-encoded domain of VEGF inhibits tumor growth by blocking angiogenesis in murine model.

Vascular endothelial growth factor VEGF(165) is a critical element for development of the vascular system in physiological and pathological angiogenesis. VEGF isoforms have different affinities for heparan sulphate proteoglycan (HSPG) as well as for VEGF receptors; HSPGs are important regulators in vascular development. Therefore, inhibition of interactions between VEGF and HSPGs may prevent angiogenesis. Here, we demonstrate that an HSPG-binding synthetic peptide, corresponding to exon 6a-encoded domain of VEGF gene, has anti-angiogenic property. This 20 amino acids synthetic peptide prevents VEGF(165) binding to several different cell types, mouse embryonic sections and inhibits endothelial cell migration, despite its absence in VEGF(165) sequence. Our in vivo anti-tumor studies show that the peptide inhibits tumor growth in both mouse Lewis-Lung Carcinoma and human Liposarcoma tumor-bearing animal models. This is the first evidence that a synthetic VEGF fragment corresponding to exon 6a has functional antagonism both in vitro and in vivo. We conclude that the above HPSG binding peptide (6a-P) is a potent inhibitor of angiogenesis-dependent diseases.

Angiostatin regulates the expression of antiangiogenic and proapoptotic pathways via targeted inhibition of mitochondrial proteins.

Angiostatin, a proteolytic fragment of plasminogen, is a potent endogenous antiangiogenic agent. The molecular mechanisms governing angiostatin's antiangiogenic and antitumor effects are not well understood. Here, we report the identification of mitochondrial compartment as the ultimate target of angiostatin. After internalization of angiostatin into the cell, at least 2 proteins within the mitochondria bind this molecule: malate dehydrogenase, a member of Krebs cycle, and adenosine triphosphate synthase. In vitro and in vivo studies revealed differential regulation of key prosurvival and angiogenesis-related proteins in angiostatin-treated tumors and tumor-endothelium. Angiostatin induced apoptosis via down-regulation of mitochondrial BCL-2. Angiostatin treatment led to down-regulation of c-Myc and elevated levels of another key antiangiogenic protein, thrombospondin-1, reinforcing its antitumor and antiangiogenic effects. Further evidence is provided for reduced recruitment and infiltration of bone marrow-derived macrophages in angiostatin-treated tumors. The observed effects of angiostatin were restricted to the tumor site and were not observed in other major organs of the mice, indicating unique tumor specific bioavailability. Together, our data suggest mitochondria as a novel target for antiangiogenic therapy and provide mechanistic insights to the antiangiogenic and antitumor effects of angiostatin.

Linking antibody Fc domain to endostatin significantly improves endostatin half-life and efficacy.

PURPOSE: The half-life of the antiangiogenic molecule endostatin that has been used in clinical trial is short ( approximately 2 h). In addition, approximately 50% of the clinical grade endostatin molecules lack four amino acids at their NH(2) termini. Lack of these amino acids gives rise to a molecule that is devoid of zinc, resulting in no antitumor activity. Our goal was to develop a new version of endostatin that does not show such deficiency. EXPERIMENTAL DESIGN: A recombinant human endostatin conjugated to the Fc domain of IgG was constructed and expressed in mammalian cell culture. The presence of Fc has been shown by previous investigators to play a major role in increasing the half-life of the molecule. Fc-endostatin was tested in tumor-bearing mice, and its half-life was compared with the clinical grade endostatin. RESULTS: The antitumor dose of Fc-endostatin was found to be approximately 100 times less than the clinical grade endostatin. The half-life of Fc-endostatin in the circulation was found to be weeks rather than hours, as observed for endostatin alone. In addition, a U-shaped curve was observed for antitumor activity of endostatin as a function of endostatin concentration delivered to the animals. CONCLUSION: Fc-endostatin is a superior molecule to the original clinical endostatin. Due to its long half-life, the amount of protein required is substantially reduced compared with the clinically tested endostatin. Furthermore, in view of the U-shaped curve of efficacy observed for endostatin, we estimate that the requirement for Fc-endostatin is approximately 700-fold less than endostatin alone. The half-life of endostatin is similar to that of vascular endothelial growth factor-Trap and Avastin, two other antiangiogenic reagents. We conclude that a new clinical trial of endostatin, incorporating Fc, may benefit cancer patients.

Peptide-mediated targeting to tumor blood vessels of lung cancer for drug delivery.

Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5-52 peptides also bound vascular endothelial growth factor-stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Furthermore, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer-bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors.

A novel peptide specifically binding to nasopharyngeal carcinoma for targeted drug delivery.

Nasopharyngeal carcinoma (NPC) is a common cancer among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate in the early stage of NPC has been reported as high as 90 to 95% with the use of radiotherapy, but in the advanced cases, even with the use of both chemotherapy and radiotherapy, the survival rate is still <50%. To improve the survival rate, we identify a 12-mer peptide (L-peptide) specifically binding to NPC cells with a phage displayed random peptide library. The L-phage and synthetic L-peptide bound to the tumor cell surfaces of most NPC cell lines and biopsy specimens, but not normal nasal mucosal cells, and the L-peptide-linked liposomes containing fluorescent substance (L-peptide-Lipo-HPTS) were capable of binding to and translocating across plasma membranes. L-Peptide-linked liposomes that carried doxorubicin (L-peptide-Lipo-Dox) caused marked cytotoxicity in NPC cells. In SCID mice bearing NPC xenografts, the L-phages specifically bound to the tumor mass, an effect that was inhibited by competition with synthetic L-peptide. In addition, the L-peptide-Lipo-Dox suppressed tumor growth better than Lipo-Dox. These results indicate that the novel L-peptide specifically binds NPC cells and is a good candidate for targeted drug delivery to NPC solid tumors.