Adaptation of sea turtles to climate warming: Will phenological responses be sufficient to counteract changes in reproductive output?

Sea turtles are vulnerable to climate change since their reproductive output is influenced by incubating temperatures, with warmer temperatures causing lower hatching success and increased feminization of embryos. Their ability to cope with projected increases in ambient temperatures will depend on their capacity to adapt to shifts in climatic regimes. Here, we assessed the extent to which phenological shifts could mitigate impacts from increases in ambient temperatures (from 1.5 to 3°C in air temperatures and from 1.4 to 2.3°C in sea surface temperatures by 2100 at our sites) on four species of sea turtles, under a "middle of the road" scenario (SSP2-4.5). Sand temperatures at sea turtle nesting sites are projected to increase from 0.58 to 4.17°C by 2100 and expected shifts in nesting of 26-43 days earlier will not be sufficient to maintain current incubation temperatures at 7 (29%) of our sites, hatching success rates at 10 (42%) of our sites, with current trends in hatchling sex ratio being able to be maintained at half of the sites. We also calculated the phenological shifts that would be required (both backward for an earlier shift in nesting and forward for a later shift) to keep up with present-day incubation temperatures, hatching success rates, and sex ratios. The required shifts backward in nesting for incubation temperatures ranged from -20 to -191 days, whereas the required shifts forward ranged from +54 to +180 days. However, for half of the sites, no matter the shift the median incubation temperature will always be warmer than the 75th percentile of current ranges. Given that phenological shifts will not be able to ameliorate predicted changes in temperature, hatching success and sex ratio at most sites, turtles may need to use other adaptive responses and/or there is the need to enhance sea turtle resilience to climate warming.

Chronic ethanol alters adrenergic receptor gene expression and produces cognitive deficits in male mice.

Hyperkateifia and stress-induced alcohol cravings drive relapse in individuals with alcohol use disorder (AUD). The brain stress signal norepinephrine (also known as noradrenaline) tightly controls cognitive and affective behavior and was thought to be broadly dysregulated with AUD. The locus coeruleus (LC) is a major source of forebrain norepinephrine, and it was recently discovered that the LC sends distinct projections to addiction-associated regions suggesting that alcohol-induced noradrenergic changes may be more brain region-specific than originally thought. Here we investigated whether ethanol dependence alters adrenergic receptor gene expression in the medial prefrontal cortex (mPFC) and central amgydala (CeA), as these regions mediate the cognitive impairment and negative affective state of ethanol withdrawal. We exposed male C57BL/6J mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and assessed reference memory, anxiety-like behavior and adrenergic receptor transcript levels during 3-6 days of withdrawal. Dependence bidirectionally altered mouse brain α1 and ß receptor mRNA levels, potentially leading to reduced mPFC adrenergic signaling and enhanced noradrenergic influence over the CeA. These brain region-specific gene expression changes were accompanied by long-term retention deficits and a shift in search strategy in a modified Barnes maze task, as well as greater spontaneous digging behavior and hyponeophagia. Current clinical studies are evaluating adrenergic compounds as a treatment for AUD-associated hyperkatefia, and our findings can contribute to the refinement of these therapies by increasing understanding of the specific neural systems and symptoms that may be targeted.

Anaesthesia of hatchling green sea turtles (Chelonia mydas) with intramuscular ketamine-medetomidine-tramadol.

OBJECTIVE: To characterise intramuscular ketamine-medetomidine-tramadol anaesthesia in hatchling green sea turtles (Chelonia mydas). STUDY DESIGN: Prospective clinical trial. ANIMALS: Ten hatchling green sea turtles. MATERIALS AND METHODS: Prior to anaesthesia, cardiopulmonary parameters, cloacal temperature, and venous blood gas and biochemistry were obtained from hatchling green sea turtles while they were being gently restrained. Animals were then anaesthetised with ketamine (5 mg kg-1 ), medetomidine (0.05 mg kg-1 ) and tramadol (5 mg kg-1 ) via intramuscular injection. Turtles were checked for the depth of anaesthesia at five-min intervals by recording reflexes (righting, palpebral, pinch, cloacal) and measuring heart rate, respiratory rate and cloacal temperature. After 20 min, a second venous blood sample was obtained for further blood gas and biochemical analysis and the medetomidine was antagonised using atipamezole (5:1 medetomidine, 0.25 mg kg-1 ). RESULTS: All turtles were successfully anaesthetised with a mean time to induction of 3.4 min (±1). In all animals, a loss of reflexes (except for palpebral reflex) and voluntary movement was observed for the entire 20 min. Anaesthesia resulted in marked apnoea for the duration of the procedure. Venous blood gas and biochemistry analysis indicated that a 20 min period of apnoea had no measurable effects on venous blood gas results. All turtles recovered uneventfully after atipamazole antagonisation, with a mean time to first breath 4.5 min (±3.7), and mean recovery time 15.5 min (±15.4). CONCLUSIONS AND CLINICAL RELEVANCE: Intramuscular ketamine-medetomidine-tramadol, antagonised with atipamazole appears to be an effective anaesthetic protocol in hatchling green sea turtles for short procedures with no deleterious effects on venous blood gases or biochemistry.

Design, synthesis, molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against Trypanosoma cruzi.

In this study, we designed and synthesized a series of thiophen-2-iminothiazolidine derivatives from thiophen-2-thioureic with good anti-Trypanosoma cruzi activity. Several of the final compounds displayed remarkable trypanocidal activity. The ability of the new compounds to inhibit the activity of the enzyme cruzain, the major cysteine protease of T. cruzi, was also explored. The compounds 3b, 4b, 8b and 8c were the most active derivatives against amastigote form, with significant IC50 values between 9.7 and 6.03µM. The 8c derivative showed the highest potency against cruzain (IC50=2.4µM). Molecular docking study showed that this compound can interact with subsites S1 and S2 simultaneously, and the negative values for the theoretical energy binding (Eb=-7.39kcal·mol(-1)) indicates interaction (via dipole-dipole) between the hybridized sulfur sp(3) atom at the thiazolidine ring and Gly66. Finally, the results suggest that the thiophen-2-iminothiazolidines synthesized are important lead compounds for the continuing battle against Chagas disease.

Resistance training suppresses intra-abdominal fatty acid synthesis in ovariectomized rats.

Ovarian hormone loss is associated with a shift in fat distribution to intra-abdomin al adipose tissue (intra-AAT) depots and with lipid metabolism disorders, which predisposes individuals to developing insulin resistance. Resistance training (RT) prevents increases in intra-AAT after ovarian hormone loss. However, the molecular mechanisms underlying these changes remain unclear. We investigated the effects of ovariectomy and RT on gene expression related to lipogenesis and fat oxidation in the intra-AAT of ovariectomized rats. Sprague-Dawley rats (n=6/group) were divided into the groups: sham-sedentary, ovariectomized-sedentary, sham-RT and ovariectomized-RT. RT groups performed a 10-week climbing program on a ladder with progressive overload. Intra-AAT was subjected to morphometric and mRNA analysis. Ovariectomized-sedentary group had larger adipocytes and higher expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element-binding protein-1c (SREBP-1c), stearoyl-CoA desaturase-1 (SCD-1), acetyl-CoA carboxylase (ACC), hormone-sensitive lipase (HSL) and lower expression of the oxidative carnitinepalmitoyltransferase-I (CPT-1). RT counteracted OVX-induced increases in PPAR-γ and SCD-1 and decreased SREBP-1c. ACC and HSL were downregulated in ovariectomized-RT compared with the ovariectomized-sedentary group. Ovariectomized-RT group had the highest CPT-1 gene expression. Adipocyte size decreased in ovariectomized-RT group. Results suggest that RT reduces intra-AAT adipocyte size in ovariectomized rats by suppressing intra-AAT fatty acid synthesis and enhancing fatty acid ß-oxidation.

Aspen shaving versus chip bedding: effects on breeding and behavior.

The choice of laboratory cage bedding material is often based on both practical and husbandry issues, whereas behavioral outcomes rarely appear to be considered. It has been noted that a breeding success difference appears to be associated with the differential use of aspen chip and aspen shaving bedding in our facility; therefore, we sought to analyze breeding records maintained over a 20-month period. In fact, in all four mouse strains analyzed, shaving bedding was associated with a significant increase in average weanlings per litter relative to chip bedding. To determine whether these bedding types also resulted in differences in behaviors associated with wellbeing, we examined nest building, anxiety-like, depressive-like (or helpless-like), and social behavior in mice housed on chip versus shaving bedding. We found differences in the nests built, but no overall effect of bedding type on the other behaviors examined. Therefore, we argue that breeding success, perhaps especially in more challenging strains, is improved on shaving bedding and this is likely due to improved nest-building potential. For standard laboratory practices, however, these bedding types appear equivalent.

(2E,4E)-1-(2-Hy-droxy-phen-yl)-5-phenyl-penta-2,4-dien-1-one.

In the structure of the title chalcone, C(17)H(14)O(2), derived from cinnamaldehyde, the olefine group has a trans configuration. The mol-ecular conformation is stabilized by an intra-molecular O-H⋯O hydrogen-bond inter-action with graph-set motif S(6).

Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits.

The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na(+)-K(+)-ATPase activity. We expressed sheep kidney Na(+)-K(+)-ATPase alpha- and beta-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na(+)-K(+)-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8-2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na(+) pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [(3)H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na(+)-K(+)-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive (86)Rb(+) uptake in whole cells as a means to specifically evaluate Na(+)-K(+)-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the alpha-subunit or beta-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the alpha-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the beta-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the alpha-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the alpha-beta heterodimer within the endoplasmic reticulum apparently does not require a Na(+) pump-specific chaperone.

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases.

Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.

Conformational coupling: the moving parts of an ion pump.

The Na,K-ATPase carries out the coupled functions of ATP hydrolysis and cation transport. These functions are performed by two distinct regions of the protein. ATP binding and hydrolysis is mediated by the large central cytoplasmic loop of about 430 amino-acids. Transmembrane cation transport is accomplished via coordination of the Na and K ions by side-chains of the amino-acids of several of the transmembrane segments. The way in which these two protein domains interact lies at the heart of the molecular mechanism of active transport, or ion pumping. We summarize evidence obtained from protein chemistry studies of the purified renal Na,K-ATPase and from bacterially expressed polypeptides which characterize these separate functions and point to various movements which may occur as the protein transits through its reaction cycle. We then describe recent work using heterologous expression of renal Na,K-ATPase in baculovirus-infected insect cells which provides a suitable system to characterize such protein motions and which can be employed to test specific models arising from recently acquired high resolution structural information on related ion pumps.

Real-time in vivo loading in the lumbar spine: part 1. Interbody implant: load cell design and preliminary results.

STUDY DESIGN: Instrumented interbody implants were placed into the disc space of a motion segment in two baboons. During the animal's activities, implants directly measured in vivo loads in the lumbar spine by telemetry transmitter. OBJECTIVES: Develop and test an interbody implant-load cell and use the implant to measure directly loads imposed on the lumbar spine of the baboon, a semiupright animal. SUMMARY OF BACKGROUND DATA: In vivo forces in the lumbar spine have been estimated using body weight calculations, moment arm models, dynamic chain models, electromyogram measurements, and intervertebral disc pressure measurements. METHODS: An analytical model was used to determine the force-strain relation in a customized interbody implant. After validation by finite element modeling, strain gauges were mounted onto the implant and connected to a telemetry transmitter. Implants were placed surgically into the L4-L5 disc space of skeletally mature baboons and the transmitter in the flank. After surgery, load data were collected from the animals during activities. Radiographs were taken monthly to assess fusion. RESULTS: The implant-load cell is sufficiently sensitive to monitor dynamic changes in strain and load. During extreme activity, highest measurable strain values were indicative of loads in excess of 2.8 times body weight. CONCLUSIONS: The study technique and technology are efficacious for measuring real-time in vivo loads in the spine. Measuring load on an intradiscal implant over the course of healing provides key information about the mechanics of this process. Loads may be used to indicate performance demands on the intervertebral disc and interbody implants for subsequent implant design.

In vivo pedicle screw placement: image-guided virtual vision.

Near-real-time frameless stereotaxy registering intraoperative anatomy to a preoperative three-dimensional computer model has been developed for use with in vivo pedicle screw placement. Eight patients underwent thoracolumbar and lumbar spine stabilization surgery using this new technology, and 32 pedicle screws were placed. Three additional patients had 12 pedicle screws removed during revision surgery, and they allowed the authors to estimate the accuracy of this navigational system. Accuracy was determined by comparing pedicle screw position on postoperative computed tomographs for the first eight patients and on preoperative computed tomographs for the latter three patients, with the intraoperative computer trajectory data gathered during operation. In the group of eight patients, all screws were intrapedicular. In evaluating all 11 patients, the overall accuracy was +/- 2 mm, but the greatest error of 5.4 mm was noted in the sagittal plane measurement. During the development phase of this technology, time constraints prolong surgery, but this may be addressed once the tool's accuracy has been confirmed and intraoperative radiographic confirmation becomes unnecessary. In vivo real-time frameless stereotaxy for pedicle screw placement offers promise for the future. Refinements are needed to improve accuracy and address time constraints.

BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity.

Matrix metalloproteinases (MMPs) have been implicated in tumor cell invasion, metastasis, and angiogenesis. BAY 12-9566, a novel, non-peptidic biphenyl MMP inhibitor, has shown preclinical activity on a broad range of tumor models and is currently in clinical development. The purpose of this study was to investigate the antiangiogenic activity of BAY 12-9566. In vitro, BAY 12-9566 prevented matrix invasion by endothelial cells in a concentration-dependent manner (IC50 = 8.4x10(-7) M), without affecting cell proliferation. In vivo, oral daily administration of BAY 12-9566 (50-200 mg/kg) inhibited angiogenesis induced by basic fibroblast growth factor in the Matrigel plug assay, reducing the hemoglobin content of the pellets. Histological analysis showed a reduction in the amount of functional vessels within the Matrigel. We conclude that the MMP inhibitor BAY 12-9566 inhibits angiogenesis, a property that further supports its clinical development as an antimetastatic agent.

Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.

Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Mesothelial cells induce the motility of human ovarian carcinoma cells.

The dissemination of ovarian carcinoma cells within the abdominal cavity involves interaction of tumor cells with the peritoneal mesothelium. The aim of our study was to investigate whether mesothelial cells might directly affect the spreading of this tumor by inducing motility and invasiveness of human ovarian carcinoma cells. Serum-free supernatants of cultured human mesothelial cells [conditioned medium (CM)] induced chemotaxis and invasiveness of the human ovarian carcinoma cell lines SK-OV-3, OVCAR-5 and A2780 in a Boyden chamber. Checkerboard analysis indicated that the stimulated motility was prevalently directional. Most of the chemotactic activity was retained by a heparin affinity column, indicating that the motility factor(s) is a heparin-binding protein. Using different monoclonal antibodies (MAbs) directed against chemotactic factors that are secreted by mesothelial cells, we found that chemotaxis was partially prevented (64.8% inhibition) by antibodies against fibronectin (FN). CM also induced haptotactic migration of ovarian carcinoma cells, and anti-FN antibodies significantly inhibited haptotaxis. The presence of FN in the CM was confirmed by Western blot analysis. Our findings suggest that mesothelium plays an active role in inducing the intraperitoneal spread of ovarian carcinoma cells, and point to FN as being one of the main mediators of mesothelium-induced ovarian carcinoma cell motility.

Effects of marijuana on equilibrium, psychomotor performance, and simulated driving.

Delta-9-tetrahydrocannabinol (THC) is frequently found in the blood of drivers involved in automobile accidents, and marijuana use has been associated with impaired field sobriety test performance. The present study used a within-subject design to compare the effects of marijuana (0, 1.77, or 3.95% THC) on equilibrium and simulated driving. Ten marijuana users (seven men, three women) smoked one marijuana cigarette at the beginning of each session. Then 2 min later, they began a 60-min test battery that included subjective effects scales, a computerized test of body sway, a rapid judgment task and brake latency measurement in a driving simulator, critical flicker fusion (CFF), and a choice reaction time task (CRT). Self-report ratings of 'high' and 'drug potency' increased comparably following both active doses. The high, but not the low, dose significantly increased body sway. The high dose also marginally increased brake latency by a mean of 55 ms (P < 0.10), which is comparable to an increase in stopping distance of nearly 5 feet at 60 mph Judgment, CFF, and CRT scores did not differ across dose conditions. The equilibrium and brake latency data with 3.95% THC are similar to prior results in our laboratory in participants with breath alcohol concentrations near 0.05%.

Structural changes associated with the coupling of ATP hydrolysis and cation transport by the Na pump.

Most of the residues associated with cation coordination seem to reside within transmembrane segments of the alpha-subunit of the Na,K-ATPase, whereas amino acids which appear to be involved in the coordination of ATP are found in the major cytoplasmic loop between transmembrane segments M4 and M5 (see Lingrel & Kuntzweiler, 1994; Lutsenko & Kaplan, 1995). The coupling of the two functions of cation transport and ATP hydrolysis involved in the active transport of Na and K ions must involve interactions between these two structural units. This paper summarizes recent experimental results and conclusions of studies on the renal Na,K-ATPase which have employed controlled proteolysis in the presence of physiological ligands, chemical modification with a range of reagents and a variety of functional assays. The data provide evidence for movements between specific transmembrane segments associated with cation-binding conformations and coupled changes which take place in the ATP binding domain. The binding of different cations in the cation-binding domain is sensed in the ATP binding domain and manifested as a change in reactivity. This occurs at amino acid residues which are widely spaced in primary structure. It is apparent that structural changes are transmitted through much of the ATP-binding domain as a consequence of the occupancy of the cation-binding domain. We also provide evidence that both the number and identity of cations bound are also sensed in the ATP-binding domain.