Were medieval warm-season temperatures in Jämtland,central Scandinavian Mountains,lower than previously estimated?

Today few high-quality tree-ring based temperature reconstructions extending over the past millennium exist, and those have, in general, low replication in their early parts. Here we present a new and updated maximum latewood density (MXD) chronology extending over the last 1200 years, built from local Scots pine wood sources (living trees, drywood preserved the ground, and subfossil wood extracted from lakes) all collected within 20 km in the Scandinavian Mountains in Jämtland. The MXD data was used to reconstruct April-September mean temperatures, where 60% of the variance in observed temperatures could be explained. The reconstruction exhibited distinct multidecadal variability, with the coldest periods centred on ca. 900, 1450, 1600 and 1900 CE, and the warmest periods on ca. 1160, 1250, 1500, 1660 CE. The last part of the 20th – early part of 21st century was the warmest period throughout the whole record, and the reconstruction suggests that, on average, the Medieval Climate Anomaly (MCA, 950–1250 CE) was only slightly warmer than the Little Ice Age (LIA, 1450–1900 CE). In fact, compared to earlier reconstructions from the region, the new reconstruction suggested lower MCA warm-season temperatures. However, despite sufficient replication during that period, high inhomogeneity among the MXD series makes this period slightly uncertain. The unique drywood on which the chronology was built, displayed a distinct regeneration pattern, where changes in Scots pine establishment was interpreted as responses to changes in forest fire activity and climate throughout the past millennium.     

A 2700-year high resolution pollen record of climate change from varved Sugan Lake in the Qaidam Basin, northeastern Tibetan Plateau

A 2700-year high resolution pollen record from annually-varved Sugan Lake in the Qaidam Basin at 2793 m a.s.l was obtained to examine vegetation and climatic change on the NE Tibetan Plateau. Pollen data shows that Sugan Basin was constantly covered by open desert-steppe vegetation dominated by Chenopodiaceae, Artemisia, Poaceae and Ephedra. However, large variations in Artemisia/Chenopodiaceae (A/C) ratios suggest regional moisture fluctuations over the last 2700 years, including a dry and relatively stable climate prior to 300 AD, relatively wet climate from 300 to 1200 AD with variability during 1100-1200 AD, and unstable climate since 1200 AD with relatively moister climate during 1250-1400 AD and 1700-1800 AD. However, other proxies (varve thickness, Chironomid taxa, isotopes of oxygen in precipitated carbonate) show fresher water when regional moisture was lower inferred from A/C ratio. This inconsistency suggests the possible difference of in-lake lithology/environment and regional moisture change. Fresh water into the lake from ice melting on the surrounding mountains might have contributed to the in-lake lithology and environment variation. The effective moisture changes in the Basin are in opposite phases to snow accumulation records from Dunde ice core (5325 m a.s.l) and to the monsoon intensity inferred from Dongge Cave, suggesting that the regional topography might have played an important role in mediating moisture changes at regional scale. Pollen data from Sugan Lake shows the shift of moisture at 1200 AD, from stable to variable conditions. This event is well correlated with other paleoclimate proxies in China and other parts of the world; however, the mechanisms behind these patterns require further investigation.     

Simple, inexpensive attainment and measurement of very high cooling and warming rates

We have developed a simple, inexpensive system (<$300 US) for measuring cooling and warming rates of small (∼ 0.1 μl) aqueous samples at rates as high as 10⁵ °C/min. The measurement system itself, can track rates approaching one million °C/min. For temperature sensing, a Type T thermocouple with 50 μm wire was used. The thermocouple output voltage was read with an inexpensive USB based digital oscilloscope interfaced to a laptop computer, and the raw data were processed with MS Excel.     

Carbon/silica composite fabricated from rice husk by means of binderless hot-pressing

A carbon/silica composite designed for use under compressive loads was fabricated from rice husk (RH), an agricultural waste material. RH was pulverized by using a planetary ball mill, then carbonized and molded into the precursor by means of hot-pressing without using any binders. A compression of 100 MPa was intermittently applied to the RH powder heated from room temperature to 150 °C, and then to 280 °C. The precursor, the bulk density of which was 1.37 g/cm³, was sintered for further densification at up to 1400 °C without compression, in nitrogen gas. The smaller particle size of the pulverized RH was beneficial for densifying the carbon/silica composite and increasing its compressive strength. Sintering at 800 °C for 1 h in nitrogen gas provided the maximum bulk density of 1.52 g/cm³ and the maximum Vickers hardness at the surface of 343 HV. The maximum compressive strength was measured to be 55.7 MPa using a sintering temperature of 1200 °C.     

Paleoclimatic significance of lacustrine microbialites: A stable isotope case study of two lakes at Torres del Paine, southern Chile

Two Patagonian lakes studied here, Lago Sarmiento and Laguna Amarga, are located within the orographic rain shadow formed to the east of the Southern Patagonian Ice Field in the Andes Range. Major thrombolite colonies are present in Lago Sarmiento, whereas widespread stromatolites occur in Laguna Amarga. Based on the characterization of the hydrologic system of these two lakes, together with an estimation of the isotopic balance and an analysis of the equilibrium conditions between the water and biologically induced carbonates, it is concluded that the microbialites of Lago Sarmiento are better suited as paleotemperature indicators than those of Laguna Amarga. Lago Sarmiento thrombolites contain unique carbonate mineral species in which carbonate precipitation occurs close to isotopic equilibrium and where the variation in water temperature controls fractionation of the stable oxygen isotope.The results indicate that at 1215 cal yr Bp the level of the lake was at 85 m a.s.l with a temperature close to 9.3 °C, was at 82 m a.s.l. at 600 cal yr Bp with a temperature close to 8.5 °C. This coincides with the timing of the Northern Hemisphere Medieval Warming Period. At 183 cal yr Bp the level of the lake was at 80 m a.s.l with a cooler temperature close to 7.7 °C, representing a colder period coinciding with the timing of the Little Ice Age (LIA). An interesting outcome of this study is that it reinforces the idea that the δ¹³C signal in carbonate deposits can be an effective tool in distinguishing between inorganic and biologically induced precipitation.     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

Age-related differences in cutaneous warm sensation thresholds of human males in thermoneutral and cool environments

The purpose of this study was to investigate age-related differences in cutaneous temperature thresholds for warm thermal sensitivity in a thermoneutral (28 °C) and in a cool environment (22 °C). Peripheral warm thresholds were measured on nine body regions (cheek, chest, abdomen, upper arm, forearm, hand, thigh, shin, and foot) using a thermal stimulator in 12 young (22±1 years) and 13 elderly male subjects (67±3 years). The results showed that: (1) mean skin temperature did not differ by age in both environments; (2) the cutaneous warm thresholds for the hand, shin, and foot were significantly higher for the elderly than for the young in both environments (p<0.01), whereas the remaining body parts showed no age difference; (3) the most insensitive region for elderly males was the shin for both environments (p<0.01), while for young there was no statistical significant difference with T_a 28 °C; (4) the shin of the elderly was seven and nine times less sensitive to warmth when compared to those of the cheek at T_a 28 and 22 °C, respectively; and (5) warm thresholds were 3-4 °C greater at T_a 22 °C than at 28 °C, only for the elderly males' shin and foot (p<0.05), while for young the difference between T_a 22 and 28 °C was not statistically significant. The results indicate that age-related differences in cutaneous warm perception appear to be non-uniform over the body and significant on extremities; there is a greater bluntness of warm sensitivity in the cool environment for elderly males.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Interacting effect of thermoperiod and photoperiod on the eclosion rhythm in the onion fly, Delia antiqua supports the two-oscillator model

Daily light and temperature cycles entrain adult eclosion rhythms in many insect species, but little is known about their interaction. We studied this problem in the onion fly, Delia antiqua. Pupae were subjected to various combinations of a photoperiod of 12L:12D and thermoperiods. The thermoperiods consisted of 12 h warm phase (W) and 12 h cool phase (C), giving a mean temperature of 25 °C with different temperature steps of 8, 4 and 1 °C. As the phase relation of the two Zeitgebers was varied, the phase of eclosion rhythm was shifted, depending on the phase angle with the light cycle and the amplitude of the temperature cycle. When the temperature step in the thermoperiod was 8 °C (WC 29:21 °C), the eclosion rhythm was entrained mainly to thermoperiod rather than photoperiod. In the regime with a 4 °C temperature step (WC 27:23 °C), both thermoperiod and photoperiod affected eclosion rhythm, and a phase jump of the eclosion rhythm occurred when the warm phase of thermoperiod was delayed 15-18 h from light-on. In regimes with a 1 °C temperature step (WC 25.5:24.5 °C), the eclosion rhythm was completely entrained to photoperiod. The observed interacting effect of light and temperature cycle on the eclosion rhythm in D. antiqua can be explained by the two-oscillator model proposed by Pittendrigh and Bruce (1959).     

Vertical and horizontal movements of sailfish (Istiophorus platypterus) near Taiwan determined using pop-up satellite tags

Sailfish (Istiophorus platypterus) are of substantial economic importance to Taiwan because of their seasonal abundance (April to October, with a peak from May to July) off the island's eastern coast. They are harvested by drift gill nets, set nets, harpoons, and as incidental bycatch by inshore longline fisheries. Although the biology of sailfish has been investigated in eastern Taiwan, there is a paucity of data on movement patterns. Pop-up satellite archival tags (PSATs) were used to study the vertical and horizontal movements of three sailfish in 2008. The fish were tagged near the southeastern coast of Taiwan and linear displacements ranged from 1050 to 1400 km (~ 40 km day^− 1) from deployment to pop-up locations and all movements were confined to the East China Sea. Sailfish spent 88% of their time in the upper uniform mixed layer above 50 m, but made more extensive vertical movements during the nighttime ( = 60.61 m ± 48.24 SD) than daytime ( = 35.21 m ± 35.37 SD). Depths and ambient water temperatures visited ranged from 0 to 214 m and 30.0° to 17.8 °C, respectively. However in all cases, the depth distribution appeared primarily limited by an 8 °C change in water temperature. Diel diving patterns also suggested basking behavior. Sailfish behavior makes them particularly vulnerable to surface fishing gears.     

The paleoclimate of the Eastern Mediterranean during the transition from early to mid Pleistocene (900 to 700 ka) based on marine and non-marine records: An integrated overview

Climate change is frequently considered an important driver of hominin evolution and dispersal patterns. The role of climate change in the last phase (900-700 ka) of the Middle Pleistocene Transition (MPT) in the Levant and northeast Africa was examined, using marine and non-marine records. During the MPT the global climate system shifted from a linear 41 k.yr. into a highly non-linear 100 k.yr. system, considerably changing its global modulation. Northeast Africa aridity further intensified around 950 ka, as indicated by a sharp increase in dust flux, and a jump to overall higher levels thereafter, coinciding with a lack of sapropels in the deep eastern Mediterranean (930-690 ka). The increased dust flux centering at ∼800 ka corresponds to the minima in 400 k.yr. eccentricity, a minima in 65 °N solar forcing and in the weakest African monsoon precession periodicity. This resulted in expansion of hyper-arid conditions across North Africa, the lowest lake levels in eastern Africa and the lowest rainfall in the Nile River headwaters. In the eastern Mediterranean an increasing continental signature is seen in glacial stages 22 (∼880 ka) and 20 (∼800 ka). Lower arboreal pollen values also indicate arid conditions during these glacial stages. The southern and eastern parts of the Negev Desert, unlike its northern part, were hyper-arid during the MPT, making them highly unsustainable. The fluctuations in the stands of Lake Amora follow global climate variability but were more moderate than those of its last glacial Lake Lisan successor. In the northern Jordan-Valley Hula Lake, frequent fluctuations in lake level coincide with both global climate changes and minor changes in water salinity varying from fresh to oligohaline. It appears therefore that the most pronounced and widespread deterioration in climate occurred in northeast Africa from 900 to 700 ka, whereas in the Levant the corresponding climatic changes were more moderate.     

Relatively low upper threshold temperature in lizards from cool habitats

We used the slender forest skink (Scincella modesta) as a model animal to test for the hypothesis that the upper threshold of incubation temperature is relatively low in lizards using shaded (and thus, cool) habitats. Eight gravid females were collected in early May 2005 from a population in Hangzhou, Zhejiang (eastern China). All females laid a single clutch of 7–13 eggs between mid-May and early June. Eggs were incubated at 24, 28 and 30 (±0.2) °C. None of eggs incubated at 30 °C hatched. Eggs incubated at 24 and 28 °C differed in incubation length but not in hatching success. The incubation length at 24 and 28 °C averaged 22.3 and 20.3 days, respectively. Hatchlings from eggs incubated at 24 and 28 °C did not differ in all examined morphological traits, but hatchlings from eggs incubated at 28 °C performed apparently worse in the racetrack than did their counterparts from eggs incubated at 24 °C. The temperature of 28 °C is close to the upper thermal threshold for successful embryonic development in S. modesta. Compared to other oviparous lizards using open (and thus, warm) habitats, the upper thermal threshold and the range of optimal temperatures for embryonic development are both lower in S. modesta. Our study supports the previous conclusion that species living in thermally different habitats may differ in the upper thermal threshold and the range of optimal temperatures for embryonic development.     

The Extratropical Northern Hemisphere Temperature Reconstruction during the Last Millennium Based on a Novel Method

Large-scale climate history of the past millennium reconstructed solely from tree-ring data is prone to underestimate the amplitude of low-frequency variability. In this paper, we aimed at solving this problem by utilizing a novel method termed “MDVM”, which was a combination of the ensemble empirical mode decomposition (EEMD) and variance matching techniques. We compiled a set of 211 tree-ring records from the extratropical Northern Hemisphere (30–90°N) in an effort to develop a new reconstruction of the annual mean temperature by the MDVM method. Among these dataset, a number of 126 records were screened out to reconstruct temperature variability longer than decadal scale for the period 850–2000 AD. The MDVM reconstruction depicted significant low-frequency variability in the past millennium with evident Medieval Warm Period (MWP) over the interval 950–1150 AD and pronounced Little Ice Age (LIA) cumulating in 1450–1850 AD. In the context of 1150-year reconstruction, the accelerating warming in 20^th century was likely unprecedented, and the coldest decades appeared in the 1640s, 1600s and 1580s, whereas the warmest decades occurred in the 1990s, 1940s and 1930s. Additionally, the MDVM reconstruction covaried broadly with changes in natural radiative forcing, and especially showed distinct footprints of multiple volcanic eruptions in the last millennium. Comparisons of our results with previous reconstructions and model simulations showed the efficiency of the MDVM method on capturing low-frequency variability, particularly much colder signals of the LIA relative to the reference period. Our results demonstrated that the MDVM method has advantages in studying large-scale and low-frequency climate signals using pure tree-ring data.     

Hypothermia induction and recovery in free-ranging rats

1.

To avoid anesthesia confounders, free-ranging rats were exposed (4 h) to cool water (CW; 10 °C; 5 cm), warm water (WW; 35 °C; 5 cm) or temperate air (TA; 25 °C) to induce hypothermia, or control for water or novel environment stress, respectively.     

Pyrolytic characteristics of biomass acid hydrolysis residue rich in lignin

Pyrolytic characteristics of acid hydrolysis residue (AHR) of corncob and pinewood (CAHR, WAHR) were investigated using a thermo-gravimetric analyzer (TGA) and a self-designed pyrolysis apparatus. Gasification reactivity of CAHR char was then examined using TGA and X-ray diffractometer. Result of TGA showed that thermal degradation curves of AHR descended smoothly along with temperature increasing from 150 °C to 850 °C, while a “sharp mass loss stage” for original biomass feedstock (OBF) was observed. Char yield from AHR (42.64-30.35 wt.%) was found to be much greater than that from OBF (26.4-19.15 wt.%). In addition, gasification reactivity of CAHR char was lower than that of corncob char, and there was big difference in micro-crystallite structure. It was also found that CAHR char reactivity decreased with pyrolysis temperature, but increased with pyrolysis heating rate and gasification temperature at 850-950 °C. Furthermore, CAHR char reactivity performed better under steam atmosphere than under CO₂ atmosphere.     

Spike bursts generated by the thermosensitive (cold) neuron from the antennal campaniform sensilla of the ground beetle Platynus assimilis

Responses of the antennal thermosensitive neuron of the ground beetle Platynus assimilis to warming from 20 to 50 °C were measured and analysed. During warming, neurons switched from regular spiking to bursting. ISI analysis showed that the number of spikes in the burst and spike frequency within the burst were temperature dependent and may precisely encode unfavourably or dangerously high temperatures in a graded manner. In contrast, regular spikes of the neuron encode moderate temperatures at 20-30 °C. The threshold temperature of spike bursting varied in different neurons from 25 to 47 °C. As a result, the number of bursting neurons increased with temperature increase. Therefore, in addition to the burst characteristics, the total number of bursting neurons may also contain useful information on external temperature. A relationship between the spike bursts and locomotor activity of the beetles was found which may have importance in behavioural thermoregulation of the species. At 44.4 ± 0.6 °C, first indications of partial paralysis (of the hind legs) were observed. We emphasize, that in contrast to various sensory systems studied, the thermoreceptor neuron of P. assimilis has a stable and continuous burst train, no temporal information is encoded in the timing of the bursts.     

Effect of temperature on sporulation of Neozygites floridana isolates from different climates and their virulence against the tomato red spider mite, Tetranychus evansi

The fungal pathogen Neozygites floridana Weiser and Muma has been evaluated as a classical biological candidate for introduction into Africa against the invasive tomato red spider mite Tetranychus evansi Baker and Pritchard. In this study, the effect of temperature on sporulation, germination and virulence of three isolates of N. floridana collected from T. evansi in three climatically distinct regions of Brazil and Argentina was determined. Six constant temperatures of 13 °C, 17 °C, 21 °C, 25 °C, 29 °C and 33 °C were tested for their effect on the ability of the three fungal isolates to sporulate, germinate and kill the mites. Six alternating-temperature regimes of 17-13 °C, 21-13 °C, 29-13 °C, 33-13 °C, 33-23 °C, 33-28 °C under a 12 h photophase were also tested to estimate virulence of the three isolates against T. evansi. The Vipos isolate discharged more conidia than isolates from Recife or Piracicaba at all temperatures and sporulation was strongly temperature dependent. Optimal sporulation rates were observed at 25 °C while optimal germination rates were observed at 25 °C and 29 °C. At 29 °C, the shortest mean survival time of T. evansi (3.16 days, 95% CI of 3.05-3.27) was observed for the isolate from Vipos, while the longest LT₅₀ (3.47 days, 95% CI 3.34-3.59) was observed for the isolate from Piracicaba. Mortality of mites increased as the differences between alternating day and night temperatures increased from 8 °C (21-13 °C), to 10 °C (33-23 °C), to 16 °C (29-13 °C), with smallest and highest temperature differences of 4 °C (17-13 °C) and 20 °C (33-13 °C), both producing low mortalities. The overall results suggest that the Vipos isolate is better adapted to a wider range of temperatures than the other isolates tested.     

Heat coma and its relationship to ocean dynamics in the oceanic sea skaters of Halobates (Heteroptera: Gerridae) inhabiting Indian and Pacific Oceans

This study was performed to clarify how weather and current dynamics affect the resistance to temperature change in the oceanic sea skaters, Halobates. Heat coma temperature (HCT) was measured for the adults and 5th instar larvae of four Halobates species collected from a fixed sampling location (12°00′N, 135°00′E ) in western tropical Pacific Ocean and from 13 locations in the eastern area of the India Ocean ranging from 08°00′N-06°00′S and 86°00-76°00′E. Both the gap temperature for heat coma (GTHC, mean±SD: 7.83±1.86 °C, n=32) and the heat coma temperature (HCP, 35.03±1.80 °C, n=32) of individuals collected from the Pacific Ocean, during the first half (10 days) of the sampling period at the fixed sampling point, were significantly higher than those during the second half (GTHC: 5.10±2.05 °C, n=63; HCP: 34.03±2.02 °C, n=63). The reduction in heat tolerance shown in the second half of the 20 day period may have been caused by a decrease in air temperature due to rainfall that occurred around the sampling point accompanied with the arrival of Typhoon No. 6.In the study of individuals collected from the Indian Ocean, significantly higher average GTHCs of >8 °C were recorded for the adult H. micans collected at 02°00′S and 06°00′S (89°00′E) than those at 0°00-8°00′N in the eastern Indian Ocean. Dynamic mixture of water from northern and southern currents occurs at 02°00-6°00′S of the Indian Ocean and might relate to such high heat tolerance.Temperature dynamics in the ocean habitat might directly affect the temperature resistance of the oceanic sea skaters.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: II—Mathematical prediction and experimental validation of optimal cryopreservation protocols

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1 M propylene glycol, a cooling rate of 1 °C/minute, and plunging into liquid nitrogen at −41 °C, combined with subsequent warming in a 22 °C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5 °C/minute in the presence of 1.0 M dimethyl sulfoxide or 1.0 M propylene glycol, combined with plunge temperature of −80 °C was optimal.