A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory.

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

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.

Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans.

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy.

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay.

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually leave their food source and prefer to remain outside the bacterial lawn. The assay is an easy way to test the worms' ability to sense external or internal cues to properly respond to harmful conditions. Though a simple assay, counting is time consuming, particularly with multiple samples, and assay durations that span overnight are inconvenient for researchers. An imaging system that can image many plates over a long period is useful but costly. Here, we describe a smartphone-based imaging method to record lawn avoidance in C. elegans. The method requires only a smartphone and a light emitting diode (LED) light box, to serve as a transmitted light source. Using free time-lapse camera applications, each phone can image up to six plates, with sufficient sharpness and contrast to manually count worms outside the lawn. The resulting movies are processed into 10 s audio video interleave (AVI) files for every hourly time point, then cropped to show each single plate to make them more amenable for counting. This method is a cost-effective way for those looking to examine avoidance defects and can potentially be extended to other C. elegans assays.

Green Tea Consumption and the COVID-19 Omicron Pandemic Era: Pharmacology and Epidemiology.

In spite of the development of numerous vaccines for the prevention of COVID-19 and the approval of several drugs for its treatment, there is still a great need for effective and inexpensive therapies against this disease. Previously, we showed that green tea and tea catechins interfere with coronavirus replication as well as coronavirus 3CL protease activity, and also showed lower COVID-19 morbidity and mortality in countries with higher green tea consumption. However, it is not clear whether green tea is still effective against the newer SARS-CoV-2 variants including omicron. It is also not known whether higher green tea consumption continues to contribute to lower COVID-19 morbidity and mortality now that vaccination rates in many countries are high. Here, we attempted to update the information regarding green tea in relation to COVID-19. Using pharmacological and ecological approaches, we found that EGCG as well as green tea inhibit the activity of the omicron variant 3CL protease efficiently, and there continues to be pronounced differences in COVID-19 morbidity and mortality between groups of countries with high and low green tea consumption as of December 6, 2022. These results collectively suggest that green tea continues to be effective against COVID-19 despite the new omicron variants and increased vaccination.

Making "Sense" of Ecology from a Genetic Perspective: Caenorhabditis elegans, Microbes and Behavior.

Our knowledge of animal and behavior in the natural ecology is based on over a century's worth of valuable field studies. In this post-genome era, however, we recognize that genes are the underpinning of ecological interactions between two organisms. Understanding how genes contribute to animal ecology, which is essentially the intersection of two genomes, is a tremendous challenge. The bacterivorous nematode Caenorhabditis elegans, one of the most well-known genetic animal model experimental systems, experiences a complex microbial world in its natural habitat, providing us with a window into the interplay of genes and molecules that result in an animal-microbial ecology. In this review, we will discuss C. elegans natural ecology, how the worm uses its sensory system to detect the microbes and metabolites that it encounters, and then discuss some of the fascinating ecological dances, including behaviors, that have evolved between the nematode and the microbes in its environment.

Comparison of Life Traits in Two Bacterivorous Nematodes Suggest Different Ecological Strategies to Exploit Similar Habitats.

Environments can be in states of dynamic change as well as persistent stability. These different states are a result of outside external conditions, but also the constant flux of living organisms in that ecological fauna. Nematodes are tremendously diverse, and many types can reside in the same soil microenvironments at the same time. To examine how so many nematodes can thrive and exploit a single environment, we identified two bacterivorous nematodes, Caenorhabditis elegans and Acrobeloides tricornis, that can inhabit rotting apple and soil environments. We cultured both nematodes in the laboratory and compared their life traits. We found that whereas C. elegans develops and reproduces extremely quickly, A. tricornis reaches sexual maturity much later and lays eggs at a slower rate but remains fertile for a longer time. In addition, A. tricornis displays a slower feeding behavior than C. elegans. Finally, A. tricornis has a significantly longer lifespan than C. elegans. These differences in development, physiology and behavior between the two nematodes hint at different ecological strategies to exploit the same habitat over different time periods, C. elegans as a colonizer-type nematode, and A. tricornis as more of a persister.

LNX1 Contributes to Cell Cycle Progression and Cisplatin Resistance.

The ligand of numb-protein X1 (LNX1) acts as a proto-oncogene by inhibiting p53 stability; however, the regulation of LNX1 expression has not been investigated. In this study, we screened chemicals to identify factors that potentially regulate LNX1 expression. We found that LNX1 expression levels were decreased by DNA damage, including that by cisplatin. Upon treatment with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), LNX1 expression levels increased. In addition, cell-cycle progression increased upon LNX1 expression; the levels of S and G2/M populations were correlated with LNX1 expression. Moreover, in CRISPR-Cas9-mediated LNX1 knockout cells, we observed a delay in cell-cycle progression and a downregulation of genes encoding the cell-cycle markers cyclin D1 and cyclin E1. Finally, the upregulation of LNX1-activated cell-cycle progression and increased resistance to cisplatin-mediated cell death. Taken together, these results suggest that LNX1 contributes to cell-cycle progression and cisplatin resistance.

Elevated Levels of CTRP1 in Obesity Contribute to Tumor Progression in a p53-Dependent Manner.

Mounting evidence supports the relationship between obesity and cancer. However, the molecular mechanisms linking obesity with cancer remain largely uninvestigated. In this study, we demonstrate that the expression of C1q/TNF-related protein 1 (CTRP1), an adiponectin paralogue, contributes to tumor growth by regulating the tumor suppressor p53. In our study, obese mice on a high-fat diet showed higher serum CTRP1 levels. Through in vitro experiments, we showed that the secreted form of CTRP1 in the culture medium decreased p53 expression and p53-dependent transcription in the cells. Moreover, CTRP1 treatment enhanced colony formation and cell migration. These results collectively suggest that elevated levels of CTRP1 in obesity significantly contribute to tumor progression.

Portal modulation effects of terlipressin on liver regeneration and survival in a porcine model subjected to 90% hepatectomy.

BACKGROUND: Excessive postoperative portal pressure is associated with post-hepatectomy liver failure and small-for-size syndrome after partial liver transplantation. This study aimed to identify the portal modulation effects of terlipressin on liver regeneration and survival in a porcine model subjected to 90% hepatectomy. METHODS: Twenty pigs undergoing 90% hepatectomy were divided into control (n = 10) and terlipressin (n = 10) groups. Terlipressin 0.5 mg was injected subcutaneously three times a day, from immediately before hepatectomy to 7 days after surgery, for surviving pigs in the terlipressin group. Portal pressure measurement, biochemical analysis, assessment of molecular markers for liver regeneration, and immunohistochemistry were performed in both groups. RESULTS: The 7-day survival rate was significantly higher in the terlipressin group than that in the control group. Portal pressure in the terlipressin group was lower than that in the control group at 30 min and 1 h after hepatectomy. Total bilirubin level was lower in the terlipressin group than that in the control group at 1 h and 6 h after hepatectomy. Proliferating cell nuclear antigen expression was higher in the control group than that in the terlipressin group at 6 h after hepatectomy, while the proportion of Ki-67-positive cells was higher in the terlipressin group than that in the control group at 7 days after hepatectomy. Endothelin-1 level reflecting liver injury was lower in the terlipressin group than that in the control group at 1 h and 6 h after hepatectomy. CONCLUSION: Terlipressin could optimize liver regeneration and improve survival through rapid and effective portal modulation after extensive hepatectomy.

The beneficial impacts of splanchnic vasoactive agents on hepatic functional recovery in massive hepatectomy porcine model.

BACKGROUND: Excessive portal pressure after massive hepatectomy can cause hepatic sinusoidal injury and have deleterious impacts on hepatic functional recovery, contributing to developing post-hepatectomy liver failure. This study aimed to assess the effects of splanchnic vasoactive agents on hepatic functional recovery and regeneration while clarifying the underlying mechanism, using a 70% hepatectomy porcine model. METHODS: Eighteen pigs undergoing 70% hepatectomy were involved in this study and divided into three groups: control (n=6), terlipressin (n=6), and octreotide (n=6). Terlipressin (0.5 mg) and octreotide (0.2 mg) were administered 3 times a day for each group with the first dose starting just before surgery until the 7th postoperative day, at which time the surviving pigs were sacrificed. During the period, portal pressure, liver weight, biochemical analysis, histological injury score, and molecular markers were evaluated and compared between groups. RESULTS: The 7-day survival rates in the octreotide, terlipressin, and control groups were 100%, 83.3%, and 66.7%, respectively. The portal pressures decreased in both terlipressin and octreotide groups than the control group at 30 minutes, 1 hour and 6 hours after hepatectomy. The amount of regeneration measured by liver weight to body weight ratio at the time of sacrifice in the terlipressin group was smaller than that in the control group (117% vs. 129%, P=0.03). Serum aspartate aminotransferase (AST) and total bilirubin levels at 1 and 6 hours after hepatectomy and prothrombin time/international normalized ratio (PT/INR) at 6 hours after hepatectomy were significantly improved in the terlipressin and octreotide groups compared to the control group. Serum endothelin-1 (ET-1) was significantly lower in the terlipressin group than that in the control group 6 hours after hepatectomy (P<0.01). The histological injury score in the control group was significantly higher than that in the terlipressin group on the 7th postoperative day (P<0.01). CONCLUSIONS: Splanchnic vasoactive agents, such as terlipressin and octreotide, could effectively decrease portal pressure and attenuate liver injury after massive hepatectomy.

Biotechnological Activities and Applications of Bacterial Pigments Violacein and Prodigiosin.

In this review, we discuss violacein and prodigiosin, two chromogenic bacterial secondary metabolites that have diverse biological activities. Although both compounds were "discovered" more than seven decades ago, interest into their biological applications has grown in the last two decades, particularly driven by their antimicrobial and anticancer properties. These topics will be discussed in the first half of this review. The latter half delves into the current efforts of groups to produce these two compounds. This includes in both their native bacterial hosts and heterogeneously in other bacterial hosts, including discussing some of the caveats related to the yields reported in the literature, and some of the synthetic biology techniques employed in this pursuit.

EGCG, a green tea polyphenol, inhibits human coronavirus replication in vitro.

COVID-19 pandemic results in record high deaths in many countries. Although a vaccine for SARS-CoV-2 is now available, effective antiviral drugs to treat coronavirus diseases are not available yet. Recently, EGCG, a green tea polyphenol, was reported to inhibit SARS-CoV-2 3CL-protease, however the effect of EGCG on coronavirus replication is unknown. In this report, human coronavirus HCoV-OC43 (beta coronavirus) and HCoV-229E (alpha coronavirus) were used to examine the effect of EGCG on coronavirus. EGCG treatment decreases 3CL-protease activity of HCoV-OC43 and HCoV-229E. Moreover, EGCG treatment decreased HCoV-OC43-induced cytotoxicity. Finally, we found that EGCG treatment decreased the levels of coronavirus RNA and protein in infected cell media. These results indicate that EGCG inhibits coronavirus replication.

Diacetyl odor shortens longevity conferred by food deprivation in C. elegans via downregulation of DAF-16/FOXO.

Dietary restriction extends lifespan in various organisms by reducing the levels of both nutrients and non-nutritional food-derived cues. However, the identity of specific food-derived chemical cues that alter lifespan remains unclear. Here, we identified several volatile attractants that decreased the longevity on food deprivation, a dietary restriction regimen in Caenorhabditis elegans. In particular, we found that the odor of diacetyl decreased the activity of DAF-16/FOXO, a life-extending transcription factor acting downstream of insulin/IGF-1 signaling. We then demonstrated that the odor of lactic acid bacteria, which produce diacetyl, reduced the nuclear accumulation of DAF-16/FOXO. Unexpectedly, we showed that the odor of diacetyl decreased longevity independently of two established diacetyl receptors, ODR-10 and SRI-14, in sensory neurons. Thus, diacetyl, a food-derived odorant, may shorten food deprivation-induced longevity via decreasing the activity of DAF-16/FOXO through binding to unidentified receptors.

Muscle and epidermal contributions of the structural protein ß-spectrin promote hypergravity-induced motor neuron axon defects in C. elegans.

Biology is adapted to Earth's gravity force, and the long-term effects of varying gravity on the development of animals is unclear. Previously, we reported that high gravity, called hypergravity, increases defects in the development of motor neuron axons in the nematode Caenorhabditis elegans. Here, we show that a mutation in the unc-70 gene that encodes the cytoskeletal ß-spectrin protein suppresses hypergravity-induced axon defects. UNC-70 expression is required in both muscle and epidermis to promote the axon defects in high gravity. We reveal that the location of axon defects is correlated to the size of the muscle cell that the axon traverses. We also show that mutations that compromise key proteins of hemidesmosomal structures suppress hypergravity-induced axon defects. These hemidesmosomal structures play a crucial role in coupling mechanical force between the muscle, epidermis and the external cuticle. We speculate a model in which the rigid organization of muscle, epidermal and cuticular layers under high gravity pressure compresses the narrow axon migration pathways in the extracellular matrix hindering proper axon pathfinding of motor neurons.

Lipidomic signatures of post-hepatectomy liver failure using porcine hepatectomy models.

BACKGROUND: Clinical diagnosis of post-hepatectomy liver failure (PHLF) can only be made on or after the 5th postoperative day. Biomarker for early diagnosis is considered as a critical unmet need. METHODS: Twenty domestic female crossbreed (Yorkshire-landrace and duroc) pigs underwent sham operation (n=6), 70% (n=7) and 90% (n=7) partial hepatectomy (PH). A comprehensive lipidomic analysis was conducted using sera collected at pre-operation (PO), 14, 30, and 48 h after PH using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: Of the 184 quantified lipids, 14 lipids showed significant differences between the two resection groups starting at 30 h after surgery. Four phosphatidylcholine (PC) plasmalogen species (P-16:0/16:0, P-18:0/18:2, P-18:0/20:4, and P-18:0/22:6) and PC 32:2 significantly increased in the 90% PH group while these returned to PO level after 30 h in the 70% PH group, presumably implying the failure markers. In contrast, eight triacylglycerol (TG) species (40:0, 42:1, 42:0, 44:1, 44:2, 46:1, 46:2, and 48:3) and sphingomyelin d18:1/20:0 showed an opposite trend, wherein they significantly decreased in the 90% PH group while these in the 70% PH group were abruptly increased until 30 h but returned to near PO levels at 48 h, implying the recovery markers. Same trends could also be observed in the level of whole lipid classes of PC plasmalogens and TGs, in addition to selected individual lipid species. CONCLUSIONS: Characteristic lipidomic signatures of PHLF could be identified using large animal models. These candidates have a potential to serve as a tool for early diagnosis and may open new paths to the study to overcome PHLF.

Tea Polyphenols EGCG and Theaflavin Inhibit the Activity of SARS-CoV-2 3CL-Protease In Vitro.

COVID-19, a global pandemic, has caused over 750,000 deaths worldwide as of August 2020. A vaccine or remedy for SARS-CoV-2, the virus responsible for COVID-19, is necessary to slow down the spread and lethality of COVID-19. However, there is currently no effective treatment available against SARS-CoV-2. In this report, we demonstrated that EGCG and theaflavin, the main active ingredients of green tea and black tea, respectively, are potentially effective to inhibit SARS-CoV-2 activity. Coronaviruses require the 3CL-protease for the cleavage of its polyprotein to make individual proteins functional. EGCG and theaflavin showed inhibitory activity against the SARS-CoV-2 3CL-protease in a dose-dependent manner, and the half inhibitory concentration (IC50) was 7.58 µg/ml for EGCG and 8.44 µg/ml for theaflavin. In addition, we did not observe any cytotoxicity for either EGCG or theaflavin at the concentrations tested up to 40 µg/ml in HEK293T cells. These results suggest that upon further study, EGCG and theaflavin can be potentially useful to treat COVID-19.

Lopinavir-ritonavir is not an effective inhibitor of the main protease activity of SARS-CoV-2 in vitro

COVID-19 has caused over 900,000 deaths worldwide as of September 2020, and effective medicines are urgently needed. Lopinavir was identified as an inhibitor of the HIV protease, and a lopinavir-ritonavir combination therapy was reported to be beneficial for the treatment of SARS and MERS. However, recent clinical tests could not prove that lopinavir-ritonavir therapy was an effective treatment for COVID-19. In this report, we examined the effect of lopinavir and ritonavir to the activity of the purified main protease (Mpro) protein of SARS- CoV-2, the causative virus of COVID-19. Unexpectedly, lopinavir and ritonavir did not inhibit Mpro activity. These results will aid the drug candidate selection for ongoing and future COVID-19 clinical trials.

Co-administration of everolimus and N-acetylcysteine attenuates hepatic stellate cell activation and hepatic fibrosis.

The accelerated course of hepatic fibrosis that occurs in some patients after liver transplantation is an important clinical problem. Activation of hepatic stellate cell (HSCs) is the dominant event in hepatic fibrosis. Previous studies have shown that treatment with mammalian target of rapamycin (mTOR) inhibitors was more effective in reducing the progression of fibrosis than treatment with calcineurin inhibitors, suggesting that mTOR could be a crucial target for inhibition of fibrosis. In addition, N-acetylcysteine (NAC) has been shown to effectively suppress HSC activation-dependent expression of alpha-smooth muscle actin in HSCs, suggesting that NAC could be a candidate for the clinical treatment of hepatic fibrosis. Here, we have evaluated the effects of immunosuppressive drugs and NAC in a mice model of hepatic fibrosis and on HSC activation in vitro. We demonstrated that an mTOR inhibitor significantly inhibited fibrogenic genes in cultured HSCs until day 14. In addition, co-administration of NAC with everolimus further reduced the expression of fibrogenic genes and improved the characteristic of HSCs via blockage of HSC activation and up-regulation of fibrolytic gene. Moreover, in vivo studies showed that everolimus inhibited collagen deposition and inflammation in a mouse model of fibrogenesis, as determined by histological analysis, and everolimus treatment, in combination with NAC, significantly decreased extracellular matrix deposition and improved liver histology. These findings indicated that everolimus, combined with NAC, synergistically inhibited hepatic fibrosis and thus may become a valuable option in immunosuppressant therapy.

Explorative study of serum biomarkers of liver failure after liver resection.

Conventional biochemical markers have limited usefulness in the prediction of early liver dysfunction. We, therefore, tried to find more useful liver failure biomarkers after liver resection that are highly sensitive to internal and external challenges in the biological system with a focus on liver metabolites. Twenty pigs were divided into the following 3 groups: sham operation group (n = 6), 70% hepatectomy group (n = 7) as a safety margin of resection model, and 90% hepatectomy group (n = 7) as a liver failure model. Blood sampling was performed preoperatively and at 1, 6, 14, 30, 38, and 48 hours after surgery, and 129 primary metabolites were profiled. Orthogonal projection to latent structures-discriminant analysis revealed that, unlike in the 70% hepatectomy and sham operation groups, central carbon metabolism was the most significant factor in the 90% hepatectomy group. Binary logistic regression analysis was used to develop a predictive model for mortality risk following hepatectomy. The recommended variables were malic acid, methionine, tryptophan, glucose, and γ-aminobutyric acid. Area under the curve of the linear combination of five metabolites was 0.993 (95% confidence interval: 0.927-1.000, sensitivity: 100.0, specificity: 94.87). We proposed robust biomarker panels that can accurately predict mortality risk associated with hepatectomy.