In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada.

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca2+ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

Factors Predicting the Quality of Life of University Students in Japan Amidst COVID-19: A Cross-Sectional Study.

Owing to the COVID-19 pandemic, classes and club activities in Japanese universities have been canceled; this may increase students' perceived stress and adversely affect their quality of life. This study investigated the factors that influence Japanese university students' quality of life during the pandemic. An online questionnaire collating data related to demographic characteristics, the perceived stress scale (PSS), sense of coherence (SOC), number of social supports, and quality of life (Short Form Health Survey; SF-8) was distributed to university students. Participants were divided into two groups: those who entered university before (seniors) and after (juniors) the onset of the pandemic. Their scores on the two sub-domains (physical and mental) of the SF-8 were evaluated. Multiple regression analysis was performed to identify factors associated with the composite mental summary of the SF-8. Regression analysis indicated that the predictor model of the composite mental summary differed between juniors and seniors. Among seniors, the composite mental summary was significantly indicated by the composite physical summary (ß = 0.549, p < 0.0001) and PSS (ß = 0.422, p < 0.0001). Among juniors, it was significantly indicated by the composite physical summary (ß = 0.531, p < 0.0001), PSS (ß = 0.390, p < 0.0001), and number of social supports (ß = -0.148, p = 0.006). The factors associated with quality of life differed between seniors and juniors. Universities must provide opportunities for students to find more friends, especially for juniors who have limited socialization opportunities owing to the pandemic.

Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades.

BACKGROUND: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. RESULTS: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. CONCLUSIONS: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Deciphering the Biological Enigma-Genomic Evolution Underlying Anhydrobiosis in the Phylum Tardigrada and the Chironomid Polypedilum vanderplanki.

Anhydrobiosis, an ametabolic dehydrated state triggered by water loss, is observed in several invertebrate lineages. Anhydrobiotes revive when rehydrated, and seem not to suffer the ultimately lethal cell damage that results from severe loss of water in other organisms. Here, we review the biochemical and genomic evidence that has revealed the protectant molecules, repair systems, and maintenance pathways associated with anhydrobiosis. We then introduce two lineages in which anhydrobiosis has evolved independently: Tardigrada, where anhydrobiosis characterizes many species within the phylum, and the genus Polypedilum, where anhydrobiosis occurs in only two species. Finally, we discuss the complexity of the evolution of anhydrobiosis within invertebrates based on current knowledge, and propose perspectives to enhance the understanding of anhydrobiosis.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade.

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada.

BACKGROUND: Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. RESULTS: In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. CONCLUSIONS: Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).

The association between subjective health perception and lifestyle factors in Shiga prefecture, Japan: a cross-sectional study.

BACKGROUND: The Ministry of Health has reported that in Japan, the Shiga prefecture has the highest life expectancy. Subjective health perception is a predictive indicator of mortality. For this study, we examined the association between subjective health perception and multiple lifestyle factors. METHODS: Data were obtained from the 2015 Health and Nutrition Survey in Shiga prefecture. The analytic sample comprised 6057 adults aged 20 or older. Information on subjective health perception and lifestyle behaviors was obtained from a self-administered questionnaire. As for subjective health perception, participants were divided into 2 groups: (1) Excellent or Good and (2) Average, Poor, or Very Poor. A 1-day dietary survey was also administered. The health behaviors score (HBS) was calculated based on 5 factors: consuming a healthy diet, never smoking, low-risk alcohol drinking, regular exercise, and moderate sleep duration. HBS scores ranged from 0 to 5. Multiple logistic regression was used to calculate the sex-, age- BMI- and energy intake-adjusted odds ratios (ORs) of poor subjective health across HBS, with 0 points as the reference. RESULTS: Among all participants, 2397 (39.6%) individuals were classified into the good subjective health group. Participants with an HBS of 3 (OR 0.59, 95% CI 0.37-0.96), 4 (OR 0.40, 95% CI 0.24-0.65) or 5 (OR 0.33, 95% CI 0.19-0.59) had a lower OR of rating themselves as being average/poor health compared with those having zero. The association with a higher HBS was remarkable (p for trend: < 0.001). Additional analyses revealed that the combinations including regular exercise were particularly associated with a lower risk of subjective average/poor health. CONCLUSIONS: This study showed that the higher the number of healthy lifestyle factors, the lower risk of subjective average/poor health. Combinations of healthy lifestyle factors, especially those involving exercise, suggest good subjective health for individuals living in the Shiga prefecture.

The Association between Combined Lifestyle Factors and All-Cause and Cause-Specific Mortality in Shiga Prefecture, Japan.

BACKGROUND: To decrease mortality, the benefit of combined healthy lifestyles has been suggested but is still unclear, especially for cause-specific mortality. We examined the relationship between combined lifestyle factors and all-cause and cause-specific mortality in Shiga prefecture, Japan. METHODS: This was an ecological study of 19 municipalities, using the data from the 2008-2012 standard mortality ratio (SMR) reported by the Ministry of Health and Welfare and the 2015 Health and Nutrition Survey in Shiga prefecture. The health behaviors score was calculated based on five factors (ranging from 0 to 5): diet quality (assessed adherence to dietary reference intake for Japanese), smoking, alcohol drinking, regular exercise, and sleep duration. In the multiple linear regression, the relationships between the health behaviors score and SMR of all-cause, cancer, heart diseases, and cerebrovascular diseases were estimated by sex. RESULTS: The health behaviors score was negatively associated with the cancer SMR in women (ß = -0.968, p = 0.011). For other causes, no significant association was found for either sex. A greater proportion of those who never smoked (ß = -0.780, p = 0.016) and those who had a higher quality diet (ß = -0.703, p = 0.048) were associated with lower cancer SMR in women. Women's intake of some micronutrients, particularly fruits, was higher than men. This study suggests that a combination of health behaviors, especially never smoking and high-quality diet intake are associated with lower cancer SMR in women and could be helpful in prolonging life expectancy.

Quantification of anomeric structural changes of glucose solutions using near-infrared spectra.

Glucose is the most abundant carbohydrate found in living organisms. It exists as two anomers: α-D-glucose and ß-D-glucose, which differ in how the hydroxyl group on the C1 carbon is directed. In solutions, the ratio between α- and ß-D-glucose is typically 4:6 but can vary depending on the surrounding ions or temperature. In this study, we obtained near-infrared (NIR) spectra of the glucose anomers based on concentration, and analyzed the spectral difference between each anomer by spectra subtraction and principal component analysis, respectively. Moreover, by simultaneously measuring the optical rotation and NIR spectra from dissolution to equilibration, we showed that NIR spectra quantitatively estimated the specific rotations of glucose solutions using partial least-squares regression in the 1100-1800 nm wavelength range. All the analytical results indicated that the absorption at 1742 nm possess the potential to distinguish each glucose anomer quantitatively. Therefore, we addressed the prediction of the specific rotation by the absorption at 1742 nm, and demonstrated that the absorption normalized by line subtraction showed the high correlation with measured specific rotation. The absorption at 1742 nm reflects structural changes of the glucose anomers in solution. Our spectroscopy study not only provides spectral information about glucose anomers, which are the most fundamental chemical compounds in organisms, but also shows the possibility to detect the anomer ratio in vivo for the fields of agriculture and medicine by taking advantage of NIR.

[Studyon Renal Cell Carcinoma Detected by Abdominal Ultrasonography during Routine Health Check-Up].

We performed a study on renal cell carcinoma (RCC) detected by abdominal ultrasonography during routine health check-up at Sapporo Teishin Hospital. The study included a total of 13,127 ultrasonographic examinations performed on 4,339 patients who visited our hospital for routine health check-ups during a 21- year period from April 1995 to March 2016. RCC was suspected from ultrasonographic findings and later histopathologically diagnosed in 11 patients in this group (detection rate 0.25%). Patients were male, aged 37 to 64 (median, 52.1 years). None of the patients had subjective symptoms, and all were negative for microscopic hematuria. Greatest dimensions of tumors were 18 to 40 mm (mean, 28 mm) ; anechoic rims, which are a characteristic ultrasonographic finding in RCC, were identified in 8 patients (72. 7%), intratumoral cysts were identified in 8 patients (72.7%), and protrusions from the surface of the kidney were identified in 9 patients (81. 8%). Regarding vascular distribution of renal tumors during Doppler ultrasonography, peripheral patterns or mixed penetrating and peripheral patterns were identified in 100% of cases (8/8). All 11 patients were diagnosed histopathologically with clear cell renal cell carcinoma, and the histological grade was G1 in 6 patients, G1＞G2, in 1 patient G2＞G1, in 1 patient and G2 in 3 patients. All 11 tumors were classified as pT1a pathological stage according to the TNM classification, and no regional lymph node metastases or distant metastases were identified. We consider that ultrasonography plays an integral role in the early detection of RCC during check-ups and examinations.

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

What Kinds of Skills Are Necessary for Physicians Involved in International Disaster Response?

UNLABELLED: Introduction Physicians are key disaster responders in foreign medical teams (FMTs) that provide medical relief to affected people. However, few studies have examined the skills required for physicians in real, international, disaster-response situations. Problem The objectives of this study were to survey the primary skills required for physicians from a Japanese FMT and to examine whether there were differences in the frequencies of performed skills according to demographic characteristics, previous experience, and dispatch situations to guide future training and certification programs. METHODS: This cross-sectional survey used a self-administered questionnaire given to 64 physicians with international disaster-response site experience. The questionnaire assessed demographic characteristics (sex, age, years of experience as a physician, affiliation, and specialty), previous experience (domestic disaster-relief experience, international disaster-relief experience, or disaster medicine training experience), and dispatch situation (length of dispatch, post-disaster phase, disaster type, and place of dispatch). In addition, the frequencies of 42 performed skills were assessed via a five-point Likert scale. Descriptive statistics were used to assess the participants' characteristics and total scores as the frequencies of performed skills. Mean scores for surgical skills, health care-related skills, public health skills, and management and coordination skills were compared according to the demographic characteristics, previous experience, and dispatch situations. RESULTS: Fifty-two valid questionnaires (81.3% response rate) were collected. There was a trend toward higher skill scores among those who had more previous international disaster-relief experience (P=.03). The more disaster medicine training experience the participants had, the higher their skill score was (P<.001). Physicians reported involvement in 23 disaster-relief response skills, nine of which were performed frequently. There was a trend toward higher scores for surgical skills, health care-related skills, and management and coordination skills related to more disaster medicine training experience. CONCLUSION: This study's findings can be used as evidence to boost the frequency of physicians' performed skills by promoting previous experience with international disaster relief and disaster medicine training. Additionally, these results may contribute to enhancing the quality of medical practice in the international disaster relief and disaster training curricula. Noguchi N , Inoue S , Shimanoe C , Shibayama K , Matsunaga H , Tanaka S , Ishibashi A , Shinchi K . What kinds of skills are necessary for physicians involved in international disaster response? Prehosp Disaster Med. 2016;31(4):397-406.

Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in ß-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.