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第二章：恒温動物（哺乳類）, 哺乳類には，白色と褐色の2種類の脂肪組織が存在する．白色脂肪組織はエネルギーを中性脂肪として貯蔵する役割を担うのに対し，褐色脂肪組織はミトコンドリアの脱共役タンパク質1（UCP1）により熱を産生する非震え熱産生の部位である．哺乳類は寒冷刺激を受けると交感神経を介して褐色脂肪組織の熱産生を活性化し，体温の低下を防ぐ．寒冷刺激が長期に渡ると褐色脂肪組織が増生するとともに，白色脂肪組織中にUCP1を発現するベージュ脂肪細胞が誘導され（白色脂肪の褐色化），個体レベルの熱産生能が増大して寒冷環境に適応する．本稿では，寒冷適応における脂肪組織の変化とその分子機序を概説するとともに，動物種による褐色脂肪組織の発達や機能の違いについて紹介する．, Brown adipose tissue is a site for non-shivering thermogenesis by mitochondrial uncoupling protein 1 (UCP1). Cold stimulation activates heat production in brown adipose tissue via the sympathetic nervous system, preventing a decrease in body temperature. Prolonged cold stimulation causes the hyperplasia of brown adipose tissue, and induces UCP1-expressing beige adipocytes in white adipose tissue. This remodeling of adipose tissues increases the thermogenic capacity at the individual level and improves adaptation to the cold environment. In this section, the molecular mechanisms of adipose tissue remodeling during cold adaptation and species-dependent differences in the development and the function of brown adipose tissue in mammals are discussed.

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to −25°C. P355′s stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (kcat) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

The Fuegians, ancient inhabitants of Tierra del Fuego, are an exemplary case of a cold-adapted population, since they were capable of living in extreme climatic conditions without any adequate clothing. However, the mechanisms of their extraordinary resistance to cold remain enigmatic. Brown adipose tissue (BAT) plays a crucial role in this kind of adaptation, besides having a protective role on the detrimental effect of low temperatures on bone structure. Skeletal remains of 12 adult Fuegians, collected in the second half of XIX century, were analyzed for bone mineral density and structure. We show that, despite the unfavorable climate, bone mineral density of Fuegians was close to that seen in modern humans living in temperate zones. Furthermore, we report significant differences between Fuegians and other cold-adapted populations in the frequency of the Homeobox protein Hox-C4 (HOXC4) rs190771160 variant, a gene involved in BAT differentiation, whose identified variant is predicted to upregulate HOXC4 expression. Greater BAT accumulation might therefore explain the Fuegians extreme cold-resistance and the protection against major cold-related damage. These results increase our understanding of how ecological challenges have been important drivers of human–environment interactions during Humankind history.

Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. Using molecular evolution techniques, this study aimed to identify which functional classes of genes changed during the cryonotothenioid radiation in a polar ocean. The influences of both positive and relaxed selective pressures were isolated following two major physiological transitions: the onset of freezing temperatures, and the loss of hemoproteins. Looking at the influence of cold temperatures, positive selective pressure was most prominently found to act on biosynthetic processes (the machinery of transcription and translation) as well as on protein polymerization, cell adhesion, and cell cycle progression, suggesting these are key challenges to life in freezing waters. Relaxation of selective pressure produced a more complex pattern of change, affecting several homeostatic processes, suggesting their attenuation in cold-stable and oxygen-rich waters, but also seemingly acting as a route to key genetic change behind the morphological and ecological diversification seen in the clade. Finally, while chronic cold-water temperatures appear to have instigated clear genetic change, the loss of hemoproteins led to little observable change relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection show that long-term exposure to cold has led to profound changes in cryonotothenioid genomes, which may make it challenging for them to respond to unpredictable heat waves and to adapt to a rapidly changing climate.

Global warming effects in temperate and polar regions include higher average temperatures and a decrease in snow cover, which together lead to an increase in the number of freeze–thaw cycles (FTC). These changes could affect the fitness of both terrestrial and aquatic species. In this study, we tested how tardigrades, ubiquitous microscopic invertebrates, face FTC. Tardigrades are amongst the most resistant animals to unfavorable conditions, including long and deep freezing periods, and are an emerging model group for invertebrate ecology and evolution. We used 12 populations of tardigrades, representing different families within order Parachela, inhabiting different ecosystems (glaciers, snow, terrestrial, aquatic), found in various substrates (mosses, sediments in lakes, cryoconite on glaciers, and snow), and originating from different latitudes and altitudes. We estimated the number of cycles required to kill 50% of individuals and tested for its association with ecological characteristics of the natural habitat (e.g., number of months with predicted FTC), while accounting for phylogeny. The most resistant tardigrades to FTC were the ones from mountain areas and glaciers. The estimated number of cycles required to kill 50% of individuals was the highest for mountainous species inhabiting rock pools and cryoconite holes on glaciers (30 and 14 FTC, respectively). Tardigrades from lowlands were the most sensitive to changes, with 50% of individuals dying after three FTC, while lacustrine and subtropical tardigrades required only one FTC to reach 50% mortality. Our study shows that the response to recurrent freezing stress is taxon dependent and related to the local environmental conditions. The predicted increase of FTC cycles will negatively impact tardigrade populations. Considering the abundance and various trophic roles of tardigrades, reduction in population sizes or the disappearance of some fragile species could affect the functioning of both aquatic and terrestrial ecosystems. Tardigrades are candidate indicators of how freeze–thaw cycles impact ubiquitous microscopic metazoans with similar physiological capabilities. peerReviewed

There are a large number of active cold-adapted microorganisms in the perennial cold environment. Due to their high-efficiency and energy-saving catalytic properties, cold-adapted microorganisms have become valuable natural resources with potential in various biological fields. In this study, a series of cold response strategies for microorganisms were summarized. This mainly involves the regulation of cell membrane fluidity, synthesis of cold adaptation proteins, regulators and metabolic changes, energy supply, and reactive oxygen species. Also, the potential of biocatalysts produced by cold-adapted microorganisms including cold-active enzymes, ice-binding proteins, polyhydroxyalkanoates, and surfactants was introduced, which provided a guidance for expanding its application values. Overall, new insights were obtained on response strategies of microorganisms to cold environments in this review. This will deepen the understanding of the cold tolerance mechanism of cold-adapted microorganisms, thus promoting the establishment and application of low-temperature biotechnology.

Cold stress is an important factor affecting cattle health, production performance, and reproductive efficiency. Understanding of the potential mechanism underlying genetic adaptation to local environments, particularly extreme cold environment, is limited. Here, by using FLK and hapFLK methods, we found that the Zinc finger CCCH-type containing 10 (ZC3H10) gene underwent positive selection in the Menggu, Fuzhou, Anxi, and Shigatse humped cattle breeds that are distributed in the cold areas of China. Furthermore, ZC3H10 expression significantly increased in bovine fetal fibroblast (BFF) cells at 28 °C for 4 h. ZC3H10 knockout BFFs were generated using CRISPR/Cas9. Wild and ZC3H10-deleted BFFs were treated at two temperatures and were divided into four groups (WT, wild and cultured at 38 °C; KO, ZC3H10−/− and 38 °C; WT_LT, wild, and 28 °C for 4 h; and KO_LT, ZC3H10−/− and 28 °C for 4 h. A total of 466, 598, 519, and 650 differently expressed genes (two-fold or more than two-fold changes) were identified by determining transcriptomic difference (KO_LT vs. KO, WT_LT vs. WT, KO vs. WT, and KO_LT vs. WT_LT, respectively). Loss of ZC3H10 dysregulated pathways involved in thermogenesis and immunity, and ZC3H10 participated in immunity-related pathways induced by cold stress and regulated genes involved in glucose and lipid metabolism and lipid transport (PLTP and APOA1), thereby facilitating adaptability to cold stress. Our findings provide a foundation for further studies on the function of ZC3H10 in cold stress and development of bovine breeding strategies for combatting the influences of cold climate.

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at low temperatures down to − 0.4 °C. Maintaining cytoplasmic membrane fluidity by changing the lipid membrane composition is important during growth at low temperatures. In Listeria monocytogenes, the dominant adaptation effect is the fluidization of the membrane by shortening of fatty acid chain length. In some strains, however, an additional response is the increase in menaquinone content during growth at low temperatures. The increase of this neutral lipid leads to fluidization of the membrane and thus represents a mechanism that is complementary to the fatty acid-mediated modification of membrane fluidity. This study demonstrated that the reduction of menaquinone content for Listeria monocytogenes strains resulted in significantly lower resistance to temperature stress and lower growth rates compared to unaffected control cultures after growth at 6 °C. Menaquinone content was reduced by supplementation with aromatic amino acids, which led to a feedback inhibition of the menaquinone synthesis. Menaquinone-reduced Listeria monocytogenes strains showed reduced bacterial cell fitness. This confirmed the adaptive function of menaquinones for growth at low temperatures of this pathogen.

Atriplex halimus grows in humid and saline warm regions of Mediterranean area showing high adaptability but less information is available on resistance to chilling temperatures. Potted plants of fi...

Arcobacter butzleri isolates showed strain‐dependent growth patterns at 8 °C. In addition, 13 genes homologous to cold shock‐related genes of Escherichia coli were identified in the genome of the A. butzleri reference strain, and similar expression patterns of these genes were observed by reverse transcription‐quantitative PCR in the early phase after cold shock., Arcobacter (A.) butzleri is an emerging zoonotic pathogen associated with gastrointestinal diseases, such as abdominal cramps and diarrhea, and is widely detected in animals, showing a high prevalence in poultry and seafood. The survival and adaptation of A. butzleri to cold temperatures remains poorly studied, although it might be of interest for food safety considerations. To address this, growth patterns of eight A. butzleri isolates were determined at 8 °C for 28 days. A. butzleri isolates showed strain‐dependent behavior: six isolates were unculturable after day 18, one exhibited declining but detectable cell counts until day 28 and one grew to the stationary phase level. Out of 13 A. butzleri cold shock‐related genes homologous to Escherichia coli, 10 were up‐regulated in response to a temperature downshift to 8 °C, as demonstrated by reverse transcription‐quantitative PCR. Additionally, we compared these data with the cold‐shock response in E. coli. Overall, we provide a deeper insight into the environmental adaptation capacities of A. butzleri, which we find shares similarities with the E. coli cold‐shock response.

In recent years, the prevalence of obesity has increased dramatically and has become a 21st century epidemic. Obesity is associated with the development of many diseases, and therefore treatments that can reduce body mass are actively sought. The aim of this study was to examine the effect of 20 cryostimulation sessions on body composition in obese/high body mass (HBM, n = 12) males and normal body mass (NBM, n = 9) controls. The HBM group had a mean age = 29.08 ± 4.19 years, body fat percentage = 32.08 ± 6.16%, body mass index = 36.23 ± 8.13 kg/m2) and NBM group had a mean age = 22.00 ± 2.45 years, body fat percentage = 12.14 ± 4.93%, body mass index = 23.58 ± 2.00 kg/m2. Kilocalorie intake was similar for both groups. All participants received 20 sessions of systemic cryostimulation at −120°C for 2–3 min in a cryochamber. Blood samples were collected before the first session, 1 h after the 10th session, and 1 h after the 20th cryostimulation session. C-reactive protein (CRP) plasma concentrations, and expression of the heat shock protein genes (HSPA1A, HSPB1) and CRP mRNA in leukocytes were evaluated after 10 and 20 cryostimulation sessions. In both groups, 20 sessions were associated with a significant decrease in body mass, fat mass and the percentage of body fat. CRP concentrations were significantly higher in obese people before the first session and after 10 treatments, but not at the end of study. Expression of HSPA1A and HSPB1 mRNA gradually decreased with the number of cryostimulation sessions. A significant difference in HSPA1A expression was found after 20 sessions (NBM > HBM) and for HSPB1 at baseline and after 20 sessions (HBM > NBM). Our results show that cryostimulation influences body composition and that cryostimulation-induced HSP genes expression depends on the number of cryosessions and baseline body mass, and is differentially altered in HBM individuals. Further research on the interaction between body mass and cold adaptation is warranted.

Introduction: Extreme-altitudes (5500 m/18045 ft and higher) pose environmental, psychophysiological, infrastructural, logistic, and ergonomic challenges that question explorer’s adaptability and mission-efficiency due to isolation, monotony, intimidating environment and terse health conditions. The assessment of an explorer’s comprehensive adaptability in extreme-altitudes is of paramount importance in ensuring mission-preparedness. Knowledge, attitude, and practices (KAP) of explorers staying on extreme-altitudes was assessed through personal interview technique. Methods: 125 healthy, acclimatized, mountain-trained explorers staying above 4570 m/15000 ft in winter-season for at least 30 days on extreme-altitudes were assessed by a single cross-sectional study through personal interviews on KAP related to extreme-altitude and cold-adaptation. Results: Mean duration of stay on extreme-altitude was 55.7 days. All explorers knew about difficulties and health-problems at extremealtitude. All explorers felt that mountain-training and acclimatization were beneficial. 92% felt that mission tenure of 90 days on the extreme-altitude was adequate. 92.8% felt they were adequately trained for the extreme-altitude; however, only 52% felt confident about health-training. 66.4% did preventive rewarming of extremities. 66.4% regularly smoked/chewed tobacco. 57.6% had sleep problems and 64% had altered appetite. 26.4% felt difficulties related to living conditions, infrastructure, and logistics. Conclusion: Explorers at extreme-altitude exhibited adequate knowledge and performed activities with progressive attitude and healthy practices. Explorers endured altered psychophysiology at extreme-altitude and regarded the utility of training and acclimatization programs. Knowledge-practice gap existed towards tobacco consumption. There was a felt need for the improvement of living conditions, infrastructure, logistics, and health-related training.

The aim of this study was isolation and characterization of heterotrophic bacteria capable of ammonium and nitrite removal at 15 °C (optimal temperature for growing rainbow trout Oncorhynchus mykiss). Environmental isolates were grown in liquid media containing ammonium or nitrite, and best strains in terms of growth and ammonium or nitrite removal were identified via 16S rRNA sequencing. Dyadobacter sp. (no. 68) and Janthinobacterium sp. (no. 100) were selected for optimal adaptation to growth at 15 °C and best ammonium and nitrite removal (P P P

[cat] Els fruits climatèrics es caracteritzen per un procés de maduració comercial estretament vinculat a la producció d’etilè, que comporta la maduració i sobre-maduració del fruit al llarg de la cadena de producció, tot portant a pèrdues que afecten a la qualitat i a la seva apreciació pels consumidors. Diverses estratègies com l’emmagatzematge en fred s’implementen actualment per tal de frenar la maduració comercial i allargar la vida útil del fruit i, així, millorar-ne la comercialització. Per aquesta raó, l’estudi de processos fisiològics desencadenats en resposta a l’exposició a temperatures fredes i l’impacte en la qualitat és essencial per comprendre els avantatges d’aquesta tècnica i les seves limitacions com, per exemple, els danys per fred que tant afecten a la vida post-collita. En aquest estudi, es van seleccionar fruits d’alvocat de la varietat Bacon (Persea americana Mill. cv. Bacon) amb l’objectiu d’aprofundir en el coneixement sobre el procés de maduració comercial, determinar l’impacte de l’emmagatzematge en fred sobre el contingut d’antioxidants en el fruit i estudiar tècniques innovadores per augmentar la qualitat del fruit en la post- collita. En base als resultats, la maduració comercial dels alvocats està regulada, a part de l’etilè, per altres fitohormones estretament relacionades entre si i l’emmagatzematge en fred altera la resposta hormonal tot retardant aquest procés. Ara bé, les baixes temperatures indueixen una resposta d’aclimatació regulada principalment per jasmonats, els quals tenen un rol crucial en la tolerància al fred i en evitar el desordre de danys per fred en el fruit. Per una banda, la resposta dels jasmonats contra els danys per fred pot potenciar-se mitjançant l’aplicació directa en el fruit de metil jasmonat (100µM) en esprai en la post-collita sense afectar les propietats antioxidants del fruit i, per altra banda, el contingut antioxidant, principalment en vitamines E i B6, es pot incrementar notablement durant un llarg termini de temps d’emmagatzematge en fred (30d) amb tractaments post-collita de piridoxal 5’-fosfat (9mM) per immersió, els quals tenen a més un impacte positiu en fruits exposats a temperatura ambient. En conclusió, cal destacar la varietat ‘Bacon’ pel seu elevat contingut en vitamina E. A més, s’ha establert que les baixes temperatures són eficients per retardar la resposta hormonal implicada en la senyalització de maduració de l’alvocat. No obstant, el fred pot arribar a ocasionar danys al fruit que afecten negativament en el mercat. Així mateix, aplicacions de metil jasmonat en esprai i tractaments de piridoxal 5’-fosfat per immersió poden afavorir l’emmagatzematge i qualitat del fruit al llarg de la cadena de producció., [eng] Climacteric fruit are characterized by a ripening process tightly related to ethylene production, thus inducing ripening and overripening along the supply chain causing quality losses that affect consumers acceptance. Some strategies are implemented currently aiming to delay or inhibit ripening to prolong shelf life and increase avocado marketability. For this reason, the knowledge in physiological events triggered by cold exposure and its impact on fruit quality is of paramount importance to understand the advantages and disadvantages of the technique, for instance, chilling injury disorder that highly affects postharvest quality in avocados. Here, ‘Bacon’ avocados (Persea americana Mill. cv. Bacon) were selected aiming to deepen into our understanding of hormonal response regulating ripening, to determine the impact of low temperature exposure on antioxidants and to study some innovative techniques to increase postharvest quality on the fruit. According to the results, avocado ripening is regulated, rather than by ethylene alone, by orchestrated phytohormonal interactions and, cold storage alters these hormonal modulations, therefore delaying ripening. Differently, cold storage induced an acclimation response modulated by jasmonates participating in cold tolerance events for preventing chilling injury. In this sense, jasmonates response against chilling injury can be enhanced by spraying harvested fruit with methyl jasmonate (100µM) solutions, which resulted in a reduced number of damaged fruits without negatively affecting antioxidant properties due to vitamin E. Moreover, antioxidant content related to vitamins E and B6 can be triggered in long-term cold-stored fruit (30d) by submersing avocados in pyridoxal 5’-phosphate (9mM) solutions which, at room temperature, slightly delayed early ripening of fruit. In conclusion, ‘Bacon’ variety has higher amounts of vitamin E than the widely commercialised ‘Hass’ or ‘Fuerte’ avocados. Moreover, cold temperatures prevent hormonal responses related to ripening and activates cold tolerance mechanisms but, long term exposure can induce chilling injury in ‘Bacon’ avocados, thus affecting its marketability. Noteworthy, methyl jasmonate and pyridoxal 5’-phosphate treatments can improve ‘Bacon’ avocado storage and quality along the supply chain.

Despite advances in research and therapy, cardiovascular diseases are still the leading cause of death worldwide. A closer understanding of the endogenous protective mechanism may improve pharmacological interventions for the treatment of heart diseases. Cold acclimation or hardening has strong potential for reducing cardiovascular risk and the literature shows that it stimulates the β-adrenergic and thyroid systems in tissues. At the same time, the adrenergic system in the heart is one of the main regulators of cardiac activity. However, these signaling pathways have surprisingly not been studied at the protein level in the heart yet, and no studies can be found on the subject matter in current literature. Our results show a reduced infarct size induced by ischemic injury in cold-acclimated rats (CA) at 8 ř C for 5 weeks and then returning to normothermic conditions for 2 weeks (CAR). The aim of this dissertation is to determine, the degree of involvement of the adrenergic system in the myocardium during acclimation after 3 days, 10 days, 5 weeks of CA and subsequent CAR at the level of all three β-adrenergic receptor isoforms (β-ARs) and their signaling pathways. The results show unchanged signaling of β1-AR-Gs-adenylyl cyclase-protein kinase A in the cardioprotective regimes CA and CAR, whose...

The eco-evolutionary history of penguins is characterised by shifting from temperate to cold environments. Breeding in Antarctica, the Emperor penguin appears as an extreme outcome of this process, with unique features related to insulation, heat production and energy management. However, whether this species actually diverged from a less cold-adapted ancestor, more ecologically similar to its sister species, the King penguin, is still an open question. As the Antarctic colonisation likely resulted in vast changes in selective pressure experienced by the Emperor penguin, the relative quantification of the genomic signatures of selection, unique to each sister species, could answer this question. Applying phylogeny-based selection tests on 7651 orthologous genes, we identified a more pervasive selection shift in the Emperor penguin than in the King penguin, supporting the hypothesis that its extreme cold adaptation is a derived state. Furthermore, among candidate genes under selection, four (TRPM8, LEPR, CRB1, and SFI1) were identified before in other cold-adapted homeotherms, like the woolly Mammoth, while other 161 genes can be assigned to biological functions relevant to cold adaptation identified in previous studies. Location and structural effects of TRPM8 substitutions in Emperor and King penguin lineages support their functional role with putative divergent effects on thermal adaptation. We conclude that extreme cold adaptation in the Emperor penguin largely involved unique genetic options which, however, affect metabolic and physiological traits common to other cold-adapted homeotherms.

Cold temperature is prevalent across the biosphere and slows the rates of chemical reactions. Increased catalysis has been predicted to be a dominant adaptive trait of enzymes to reduced temperature, and this expectation has informed physical models for enzyme catalysis and influenced bioprospecting strategies. To systematically test rate enhancement as an adaptive trait to cold, we paired kinetic constants of 2223 enzyme reactions with their organism’s optimal growth temperature (TGrowth) and analyzed trends of rate constants as a function of TGrowth. These data do not support a general increase in rate enhancement in cold adaptation. In the model enzyme ketosteroid isomerase (KSI), there is prior evidence for temperature adaptation from a change in an active site residue that results in a tradeoff between activity and stability. Nevertheless, we found that little of the rate constant variation for 20 KSI variants was accounted for by TGrowth. In contrast, and consistent with prior expectations, we observed a correlation between stability and TGrowth across 433 proteins. These results suggest that temperature exerts a weaker selection pressure on enzyme rate constants than stability and that evolutionary forces other than temperature are responsible for the majority of enzymatic rate constant variation.

The CCCryo Culture Collection of Cryophilic Algae is a diverse bioresource of mainly cold-adapted microalgae from polar environments. It currently comprises 518 strains of 178 species in 101 genera. Most strains were isolated from field samples collected during seven polar expeditions, mainly to Svalbard (Norway) and Antarctica. Almost 90% of the strains are cryophilic, with 33% of all strains being psychrophilic (obligate cryophilic) representing the “true” snow algae and 56% being psychrotrophic (non-obligate cryophilic), comprising other snow and permafrost algae from cold habitats. There are also some cultures of mosses, cyanobacteria, fungi and bacteria from cold or thermal environments. These strains are publicly available and serve as a rare bioresource for fundamental studies as well as for the development of commercial products. Basic studies address taxonomic and phylogenetic as well as physiological questions and genome and transcriptome research, whereas applied studies mostly address the capabilities of cryophilic algae to produce commercially interesting products for markets like food, feed and supplements, cosmetics, tools for molecular biology and diagnostics or food processing. Examples of strains studied for the production of valuable polyunsaturated omega-3 fatty acids like EPA, secondary carotenoids like astaxanthin, ice-structuring proteins, cold-active enzymes, UV-absorbing mycosporine-like amino acids and extracellular polymeric substances as form stabilizers are given and discussed in the context of customers’ acceptance, legal regulations for specific markets, technical feasibility of an industrial production process, potential profit and chances for the successful development of a commercial product.

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum., This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2016-78002-R) and the Ministry of Science and Innovation PID2019-108595RB-I00/AEI/10.13039/501100011033. We also acknowledge financial support from the “Severo Ochoa Program for Centers of Excellence in R&D” (SEV-2015-0533 and CEX2019-000902-S), and the CERCA Program from the Catalan Government (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PS held an INPHINIT Programme fellowship from Fundació “La Caixa” and AK was a recipient of a Netaji Subhas - Indian Council of Agricultural Research (ICAR) International Fellowship. RP-J received FI and FPU fellowships from Generalitat de Catalunya and the Ministry of Education and Professional Formation, respectively. BD have received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement No. 945043.

International audience; CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence CC A at tRNA 3'-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to À15°C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation-a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme.

Carotenoids have several crucial biological functions and are part of the cold adaptation mechanism of some bacteria. Some pink-pigmented Arthrobacter species produce the rare C50 carotenoid bacterioruberin, whose function in these bacteria is unclear and is found mainly in halophilic archaea. Strains Arthrobacter agilis DSM 20550T and Arthrobacter bussei DSM 109896T show an increased bacterioruberin content if growth temperature is reduced from 30 down to 10 °C. In vivo anisotropy measurements with trimethylammonium-diphenylhexatriene showed increased membrane fluidity and a broadening phase transition with increased bacterioruberin content in the membrane at low-temperature growth. Suppression of bacterioruberin synthesis at 10 °C using sodium chloride confirmed the function of bacterioruberin in modulating membrane fluidity. Increased bacterioruberin content also correlated with increased cell resistance to freeze–thaw stress. These findings confirmed the adaptive function of bacterioruberin for growth at low temperatures for pink-pigmented Arthrobacter species.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni–NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0–50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20–30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

(1) Background: Chitooligosaccharides (COS) have numerous applications due to their excellent properties. Chitosan hydrolysis using chitosanases has been proposed as an advisable method for COS preparation. Although many chitosanases from various sources have been identified, the cold-adapted ones with high stability are still rather rare but required. (2) Methods: A novel chitosanase named CsnY from marine bacterium Renibacterium sp. Y82 was expressed in Escherichia coli, following sequence analysis. Then, the characterizations of recombinant CsnY purified through Ni–NTA affinity chromatography were conducted, including effects of pH and temperature, effects of metal ions and chemicals, and final product analysis. (3) Results: The GH46 family chitosanase CsnY possessed promising thermostability at broad temperature range (0–50 °C), and with optimal activity at 40 °C and pH 6.0, especially showing relatively high activity (over 80% of its maximum activity) at low temperatures (20–30 °C), which demonstrated the cold-adapted property. Common metal ions or chemicals had no obvious effect on CsnY except Mn2+ and Co2+. Finally, CsnY was determined to be an endo-type chitosanase generating chitodisaccharides and -trisaccharides as main products, whose total concentration reached 56.74 mM within 2 h against 2% (w/v) initial chitosan substrate. (4) Conclusions: The results suggest the cold-adapted CsnY with favorable stability has desirable potential for the industrial production of COS.

Despite the economic importance of creating cold resilient cattle breeds, our knowledge of the genetic basis of adaptation to cold environments in cattle is still scarce compared to information on other economically important traits. Herein, using whole-genome resequencing of animals showing contrasting phenotypes on temperature maintenance under acute cold stress combined with the existing SNP (single nucleotide polymorphism) functional annotations, we report chromosomal regions and candidate SNPs controlling body temperature in the Siberian cattle populations. The SNP ranking procedure based on regional FST calculations, functional annotations, and the allele frequency difference between cold-tolerant and cold-sensitive groups of animals pointed to multiple candidate genes. Among these, GRIA4, COX17, MAATS1, UPK1B, IFNGR1, DDX23, PPT1, THBS1, CCL5, ATF1, PLA1A, PRKAG1, and NR1I2 were previously related to thermal adaptations in cattle. Other genes, for example KMT2D and SNRPA1, are known to be related to thermogenesis in mice and cold adaptation in common carp, respectively. This work could be useful for cattle breeding strategies in countries with harsh climates, including the Russian Federation.

In this study, we characterize the response of the free-living oligotrophic alphaproteobacterium Caulobacter crescentus to low temperatures by global transcriptomic analysis. Our results showed that 656 genes were upregulated and 619 were downregulated at least 2-fold after a temperature downshift. The identified differentially expressed genes (DEG) belong to several functional categories, notably inorganic ion transport and metabolism, and a subset of these genes had their expression confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Several genes belonging to the ferric uptake regulator (Fur) regulon were downregulated, indicating that iron homeostasis is relevant for adaptation to cold. Several upregulated genes encode proteins that interact with nucleic acids, particularly RNA: cspA, cspB, and the DEAD box RNA helicases rhlE, dbpA, and rhlB. Moreover, 31 small regulatory RNAs (sRNAs), including the cell cycle-regulated noncoding RNA (ncRNA) CcnA, were upregulated, indicating that posttranscriptional regulation is important for the cold stress response. Interestingly, several genes related to transport were upregulated under cold stress, including three AcrB-like cation/multidrug efflux pumps, the nitrate/nitrite transport system, and the potassium transport genes kdpFABC. Further characterization showed that kdpA is upregulated in a potassium-limited medium and at a low temperature in a SigT-independent way. kdpA mRNA is less stable in rho and rhlE mutant strains, but while the expression is positively regulated by RhlE, it is negatively regulated by Rho. A kdpA-deleted strain was generated, and its viability in response to osmotic, acidic, or cold stresses was determined. The implications of such variation in the gene expression for cold adaptation are discussed. IMPORTANCE Low-temperature stress is an important factor for nucleic acid stability and must be circumvented in order to maintain the basic cell processes, such as transcription and translation. The oligotrophic lifestyle presents further challenges to ensure the proper nutrient uptake and osmotic balance in an environment of slow nutrient flow. Here, we show that in Caulobacter crescentus, the expression of the genes involved in cation transport and homeostasis is altered in response to cold, which could lead to a decrease in iron uptake and an increase in nitrogen and high-affinity potassium transport by the Kdp system. This previously uncharacterized regulation of the Kdp transporter has revealed a new mechanism for adaptation to low temperatures that may be relevant for oligotrophic bacteria.

The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that Ubqln1, 2 and 4 are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. In conclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at −1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water, pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

It is known that adaptive evolution in permanently cold environments drives cold adaptation in enzymes. However, how the relatively high enzyme activities were achieved in cold environments prior to cold adaptation of enzymes is unclear. Here we report that an Antarctic strain of Chlorella vulgaris, called NJ-7, acquired the capability to grow at near 0 °C temperatures and greatly enhanced freezing tolerance after systematic increases in abundance of enzymes/proteins and positive selection of certain genes. Having diverged from the temperate strain UTEX259 of the same species 2.5 (1.1–4.1) to 2.6 (1.0–4.5) Ma, NJ-7 retained the basic mesophilic characteristics and genome structures. Nitrate reductases in the two strains are highly similar in amino acid sequence and optimal temperature, but the NJ-7 one showed significantly higher abundance and activity. Quantitative proteomic analyses indicated that several cryoprotective proteins (LEA), many enzymes involved in carbon metabolism and a large number of other enzymes/proteins, were more abundant in NJ-7 than in UTEX259. Like nitrate reductase, most of these enzymes were not upregulated in response to cold stress. Thus, compensation of low specific activities by increased enzyme abundance appears to be an important strategy for early stage cold adaptation to Antarctica, but such enzymes are mostly not involved in cold acclimation upon transfer from favorable temperatures to near 0 °C temperatures.

Purpose We summarized the evidence on the effects of heat and cold exposures on mortality in China. We included studies published on this topic in both Chinese and English, thereby filling a gap in knowledge using data from a country that consists of one-fifth of the world's population. Methods We conducted a systematic search of peer-reviewed studies on the association between daily mean temperature and mortality published from 2001 up to July 2018. We searched one Chinese database (China National Knowledge infrastructure, http://www.cnki.net ) and three English databases (PubMed, Scopus, Web of Science). We converted the effect estimates of heat/cold to rate ratios (RRs) associated with 1° increase/decrease beyond the heat/cold reference temperatures. For studies that provided lag-specific estimates, we used both the maximum and minimum of RR estimates. We calculated summary effect estimates for all-cause and cause-specific mortalities, as well as RRs stratified by sex, age, and socioeconomic status. We also investigated patterns of heat and cold adaptation at different latitudes, and at different reference temperatures. Results In total, 45 articles were included in this systematic review. For every 1° temperature increase/decrease beyond reference points, the rate of non-accidental mortality increased by 2% (RR, 1.02; 95% confidence interval (95% CI [1.01–1.02]) for heat and 4% (RR, 1.04; 95% CI [1.03–1.04]) for cold, respectively; the rate of cardiovascular mortality increased 3% (RR, 1.03; 95% CI [1.03–1.04]) for heat and 6% (RR, 1.06; 95% CI [1.04–1.07]) for cold; the rate of respiratory mortality increased 2% (RR, 1.02; 95% CI [1.01–1.03]) for heat and 2% (RR, 1.02; 95% CI [1.00–1.04]) for cold; the rate of cerebrovascular mortality increased 2% (RR, 1.02; 95% CI [1.02–1.03]) for heat and 3% (RR, 1.03; 95% CI [1.02–1.04]) for cold. We identified a variation in optimal temperature range related to latitude of the residential area, and differences in people's capability to adapt to heat versus cold. Conclusion We found consistent evidence of the association between temperature and mortality, as well as evidence of patterns in human adaptation, and we discussed the implications of our findings.

As global temperatures rise, the mechanistic links between temperature, physiology and behaviour will increasingly define predictions of ecological change. However, for many taxa, we currently lack consensus about how thermal performance traits vary within and across populations, and whether and how locally adaptive trait plasticity can buffer warming effects. The metabolic cold adaptation hypothesis posits that cold environments (e.g. high elevations and latitudes) select for high metabolic rates (MR), even after controlling for body size differences, and that this enables high activity levels when an organism is near its cold lower thermal limits. Steep MR reaction norms are further predicted at cold temperatures to enable rapid behavioural activation with rising temperatures needed to exploit brief thermal windows suitable for performing eco-evolutionary tasks. We tested these predictions by performing common garden experiments comparing thermal reaction norms of MR (from 15 to 32°C) and behaviour (from 10 to 40°C) across populations of the ant Aphaenogaster iberica sampled from a 2 km elevation gradient in the Sierra Nevada Mountains of southern Spain. As predicted, high-elevation ants had higher MR and steeper MR-temperature reaction norms. However, higher rates of energy use did not yield the predicted benefits of steeper activity-level reaction norms. The evidence for locally adaptive metabolic physiology only became apparent at intermediate temperatures, highlighting the importance of testing thermal performance hypotheses across thermal gradients, rather than focusing only on performance at thermal limits (i.e. critical thermal values). The partial support for the metabolic cold adaptation hypothesis highlights that while organisms likely show a wealth of unexplored metabolic temperature plasticity, the physiological mechanisms and eco-evolutionary trade-offs underlying such local adaptation remain obscure., J.Z.S. was supported by a postdoctoral fellowship from the Smithsonian Institution Competitive Grants Program, by a Marie Curie International Incoming Fellowship (327940 INSEAME) and by the Centre for Social Evolution at the University of Copenhagen. X.A. was supported by a Ramón y Cajal research contract by the Spanish Ministry of Economy and Competitiveness (RYC-2015-18448). X.C. and R.B. were funded by the Spanish Ministry of Economy and Competitiveness and FEDER (CGL2015-65807-P).

We present here a review of the literature analyzing the link between the physiological mechanisms of cold adaptation and cold-induced prooxidant and compensatory antioxidant processes. Cold adaptation has been shown to increase the number and activity of mitochondria to support increases in ATP consumption; this leads to increased generation of reactive oxygen species (ROS), as mitochondria are among the main sources of ROS in physiological conditions. Studies in recent decades have shown that protein PGC-1α – one of the main regulators of mitochondrial biogenesis – also affects the synthesis of antioxidant enzymes, thus controlling the mitochondrial generation of reactive oxygen species. At the whole-body level, systematic cold stimulates intrinsic protective resources by enhancing oxidative processes, which in turn initiates activation of the antioxidant systems and increases the overall resistance of the body to stress factors of different types.

Pedobacter cryoconitis BG5 is an obligate psychrophilic bacterium that was first isolated on King George Island, Antarctica. Over the last 50 years, the West Antarctic, including King George Island, has been one of the most rapidly warming places on Earth, hence making it an excellent area to measure the resilience of living species in warmed areas exposed to the constantly changing environment due to climate change. This bacterium encodes a genome of approximately 5694 protein-coding genes. However, 35% of the gene models for this species are found to be hypothetical proteins (HP). In this study, three conserved HP genes of P. cryoconitis, designated pcbg5hp1, pcbg5hp2 and pcbg5hp12, were cloned and the proteins were expressed, purified and their functions and structures were evaluated. Real-time quantitative PCR analysis revealed that these genes were expressed constitutively, suggesting a potentially important role where the expression of these genes under an almost constant demand might have some regulatory functions in thermal stress tolerance. Functional analysis showed that these proteins maintained their activities at low and moderate temperatures. Meanwhile, a low citrate synthase aggregation at 43 °C in the presence of PCBG5HP1 suggested the characteristics of chaperone activity. Furthermore, our comparative structural analysis demonstrated that the HPs exhibited cold-adapted traits, most notably increased flexibility in their 3D structures compared to their counterparts. Concurrently, the presence of a disulphide bridge and aromatic clusters was attributed to PCBG5HP1’s unusual protein stability and chaperone activity. Thus, this suggested that the HPs examined in this study acquired strategies to maintain a balance between molecular stability and structural flexibility. Conclusively, this study has established the structure–function relationships of the HPs produced by P. cryoconitis and provided crucial experimental evidence indicating their importance in thermal stress response.

The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at −1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

In order to investigate the effect of intermittent mild cold stimulation (IMCS) on immune function of spleens and adaptability to cold stress in broilers, 400 healthy 1-day-old Ross-308 chickens were divided into 5 groups: CC (control) reared in normal thermal environment from 1 to 49 d; CS3, CS4, CS5, and CS6 (treatments) raised at 3°C below the temperature of CC for 3, 4, 5, or 6 h at 1-d intervals from 15 to 35 d, respectively. Subsequently, CS3-6 was raised at 20°C from 36 to 49 d. At 50 d, all groups were exposed to acute cold stress (ACS) for 12 h. The spleen immunity index at 22, 29, 36, 43, and 49 d, expression levels of toll-like receptors (TLRs), cytokines and immunoglobulins at 22, 43, and 49 d and heat shock proteins (HSPs) before and after ACS at 50 d were examined. The spleen index of broilers aged 22 to 49 d did not differ between CS and CC (P > 0.05), and the spleen index of CS5 was higher than that of CS3 at 49 d (P < 0.05). The mRNA levels of TLR5, TLR15, TLR21, and IL-2 in CS3, TLR3, TLR4, TLR15, TLR21, IL-2, IL-6, and IFN-ϒ in CS4, TLR1, TLR3, TLR4, TLR21, IL-2, IFN-a, IFN-ϒ, IgA, and IgG in CS6, but all TLRs, immunoglobulins and cytokines except IFN-ϒ in CS5 differential expressed stably compared with CC at 43 and 49 d (P < 0.05). Compared with Pre-ACS, the mRNA levels of HSP60, HSP70, and HSP90 were upregulated in CS after ACS (P < 0.05). Except for HSP90 mRNA and HSP70 protein in CS6, and HSP90 protein in CS3, the levels of HSPs after ACS in all treatment groups were higher than those in CC (P < 0.05), and the highest HSPs levels after ACS were found in CS5. We concluded that IMCS could enhance immunity of spleens and adaptability to ACS in broilers, besides CS5 was the optimal program.

The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that Ubqln1, 2 and 4 are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. Inconclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6–7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Graphical abstract, Highlights • A high-resolution structure of a psychrophilic RNA polymerase contributes to our knowledge of cold adaptation. • While catalytic core motifs are conserved, at least one shows cold adaptation. • Loss of helix-capping increases structural flexibility in a catalytic core motif. • Overall reduction of alpha-helical elements appears as a strategy for cold adaptation., CCA-adding enzymes are highly specific RNA polymerases that add and maintain the sequence C-C-A at tRNA 3‘-ends. Recently, we could reveal that cold adaptation of such a polymerase is not only achieved at the expense of enzyme stability, but also at the cost of polymerization fidelity. Enzymes from psychrophilic organisms usually show an increased structural flexibility to enable catalysis at low temperatures. Here, polymerases face a dilemma, as there is a discrepancy between the need for a tightly controlled flexibility during polymerization and an increased flexibility as strategy for cold adaptation. Based on structural and biochemical analyses, we contribute to clarify the cold adaptation strategy of the psychrophilic CCA-adding enzyme from Planococcus halocryophilus, a gram-positive bacterium thriving in the arctic permafrost at low temperatures down to −15 °C. A comparison with the closely related enzyme from the thermophilic bacterium Geobacillus stearothermophilus reveals several features of cold adaptation - a significantly reduced amount of alpha-helical elements in the C-terminal tRNA-binding region and a structural adaptation in one of the highly conserved catalytic core motifs located in the N-terminal catalytic core of the enzyme.

Psychrophilic organisms possess several adaptive strategies which allow them to sustain life at low temperatures between −20 to 20 °C. Studies on Antarctic psychrophiles are interesting due to the multiple stressors that exist on the permanently cold continent. These organisms produce, among other peculiarities, cold-active enzymes which not only have tremendous biotechnological potential but are valuable models for fundamental research into protein structure and function. Recent innovations in omics technologies such as genomics, transcriptomics, proteomics and metabolomics have contributed a remarkable perspective of the molecular basis underpinning the mechanisms of cold adaptation. This review critically discusses similar and different strategies of cold adaptation in the obligate psychrophilic yeast, Glaciozyma antarctica PI12 at the molecular (genome structure, proteins and enzymes, gene expression) and physiological (antifreeze proteins, membrane fluidity, stress-related proteins) levels. Our extensive studies on G. antarctica have revealed significant insights towards the innate capacity of- and the adaptation strategies employed by this psychrophilic yeast for life in the persistent cold. Furthermore, several cold-active enzymes and proteins with biotechnological potential are also discussed.

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

International audience; Temperate species, contrary to their tropical counterparts, are exposed not only to thermally variable environments with low temperatures but also to long winters. Different selective pressures may have driven divergent physiological adaptations in closely related species with different biogeographic origins. To survive unfavourable winter conditions, Drosophila species in temperate areas generally undergo a period of reproductive dormancy, associated with a cold-induced cessation of oogenesis and metabolic reorganization. This work aims to compare cold tolerance and metabolic signatures of cold-exposed females exhibiting different reproductive maturity status (mature and immature females) of four Drosophila species from tropical vs. temperate origins. We expected that the capacity for delayed reproduction of immature females could result in the redirection of the energy-related metabolites to be utilized for surviving the cold season. To do so, we studied an array of 45 metabolites using quantitative target GC–MS profiling. Reproductively immature females of temperate species showed the lower CTmin and the faster chill coma recovery time (i.e. the most cold-tolerant group). Principal component analysis captured differences across species, but also between reproductive maturity states. Notably, temperate species exhibited significantly higher levels of glucose, alanine, and gluconolactone than tropical ones. As proline and glycerol showed higher abundances in immature females of temperate species compared to the levels exhibited by the rest of the groups, we reasoned that glucose and alanine could serve as intermediates in the synthesis of these compatible solutes. All in all, our findings suggest that cold-exposed females of temperate species accumulate energy-related and protective metabolites (e.g. glycerol and proline) while delaying reproduction, and that these metabolites are relevant to cold tolerance even at modest concentrations.