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Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural “flexibility.” However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on K M of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis—termed mobile regions 1 and 2 (MR1 and MR2), respectively—showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Remote ischemic preconditioning (IPreC) is an effective strategy to defend against cerebral ischemia/reperfusion (IR) injury; however, its mechanisms remain to be elucidated. The aim of the present study was to investigate the effect of IPreC on brain tissue following cerebral ischemia, as well as the underlying mechanisms. Adult male Sprague-Dawley rats were treated with IPreC for 72 h prior to the induction of transient cerebral ischemia and reperfusion. The results demonstrated that IPreC reduced the area of cerebral infarction in the IR rats by 2,3,5-triphenyl-tetrazolium chloride staining. In addition, cell apoptosis was markedly suppressed by IPreC with an increased expression of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associatd X protein using Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay and western blot analysis. IR induced a decrease in the level of superoxide dismutase, and IPreC significantly suppressed increased levels of malondialdehyde, lactate dehydrogenase and nitric oxide. The expression of CD11b and CD18 was markedly inhibited by IpreC unsing flow cytometry. Furthermore, IPreC markedly decreased the release of pro-inflammatory factors interleukin (IL)-6 and IL-1β, and enhanced the level of anti-inflammatory factors (IL-10 and IL-1 receptor antagonist) by ELISA assay. Finally, IPreC reduced the levels of transforming growth factor-β-activated kinase 1, phosphorylated-P65/P65, and tumor necrosis factor-α, indicating that the nuclear factor-κB pathway was involved in IPreC-mediated protection against cerebral ischemia. Taken together, the results suggested that IPreC decreased ischemic brain injury through alleviating free radical injury and the inflammatory response in cerebral IR rats.

Rationale Cervical spondylotic radiculopathy (CSR) is a common sensory, motor, and reflex disorder. Numbness, a common subjective symptom of CSR, lacks objective quantitative indicators and recognized effective treatments, but is also difficult to recover from. We present a case report describing a traditional acupuncture treatment for CSR, utilizing a special acupuncture method and point, namely the Yunmen point. Patient concerns A 40-year-old woman presented with unilateral arm numbness caused by CSR. Diagnoses A diagnosis of CSR was made in the orthopedic department of a local hospital. Interventions We attempted acupuncture at the Yunmen (LU 2) acupoint combined with neck-seven-acupoint under computed tomographic guidance. Outcomes After 10 times treatment sessions, the patient no longer experienced weakness, coldness, or numbness in the affected upper limb. In addition, the stiffness in the neck and shoulders was reduced. On physical examination, the patient's left brachial plexus traction test was negative; reassessment of the CSR-20-point score scale showed a perfect score, and the visual analog scale score was 0. Lessons Our report indicates that acupuncture at the LU 2 acupoint combined with neck-seven-acupoint is effective in treating numbness and coldness of the arm, and other neurological symptoms caused by cervical spondylosis. Moreover, with the appropriate acupuncture technique, the risk of acupuncture at the LU 2 acupoint can be minimized.

Rationale According to the literature reports and clinical studies on alopecia areata (AA) from 2008 to 2018, most clinical treatments have been oral drugs and external ointments. At present, systemic immunosuppressive therapy has been widely used in AA, but there are various side effects such as elevated liver enzymes, gastrointestinal discomfort, poor drug compliance, and repeated illness. We present a case report describing a traditional medicine treatment for AA that uses an ethnic therapy of Zhuang medicine, a kind of Traditional Chinese Medicine, namely, medicated thread moxibustion. Patient concerns A 36-year-old man endured AA after going through a family misfortune. Half a year ago, his father passed away suddenly. Since then, he suffered continuous anguish, alcoholism and hair loss, especially in the past 2 months. A coin-shaped area of hair loss began to appear at the top of his head and gradually expanded to the surrounding region. Diagnoses A diagnosis of AA was made in the dermatology department of a local hospital. Interventions The patient was treated with the medicated thread moxibustion method of Traditional Zhuang Medicine at the Kuihua (special points of Zhuang medicine), Zusanli (ST 36), Xuehai (SP 10), Baihui (DU 20), and Taichong (LR 3) points every other day for 4 weeks. Outcomes The area of hair loss showed slight improvement after 1 week of treatment. Only just a sprinkling of wooly hairs, whose color and thickness were similar to those of fine facial hairs, began to emerge sporadically from the follicles; they could be seen only in a bright light. When the patient saw the obvious curative effect, we continued the treatment for 2 weeks with the patient's consent. Three weeks later, the patchy AA area was covered with small cotton-like hairs of different lengths and uneven colors. Lessons The medicated thread moxibustion method of Zhuang medicine can be an effective alternative treatment in patients with AA.

We studied thermal tolerance limits, heat-hardening, and Hsp70 to elucidate the difference in thermotolerance between two color variants of the sea cucumber Apostichopus japonicus. Green and Red variants occupy different habitats and have different aestivation responses to high temperature in summer. In the absence of heat-hardening, the variants showed no difference in the temperature at which 50% of the individuals died: Green, 31.49 degrees C; Red, 31.39 degrees C. However, Green specimens acquired higher thermotolerance than Red after a prior sublethal heat exposure. After 72 h of recovery from a heat-hardening treatment (30 degrees C for 2 h), the survival of Green variants was more than 50% and that of Red was less than 5% when they were treated at 33 degrees C for 2 h. Levels of mRNA and protein for Hsp70 were significantly higher in Green than Red after the heat shock of 30 degrees C, and the stability of hsp70 mRNA of Green was significantly higher than that of Red. Our findings suggest that within the same species, different variants that have similar thermal limits in the absence of heat-hardening can acquire different thermotolerances after a prior sublethal heat shock. The difference in induced thermotolerance between Green and Red is closely related to the expression pattern of Hsp70, which was partly governed by the stability of hsp70 mRNA.

Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme function, to temperature change. A series of studies using two structurally and catalytically related enzymes, A4-lactate dehydrogenase (A4-LDH) and cytosolic malate dehydrogenase (cMDH) have been especially valuable in determining the functional attributes of enzymes most sensitive to temperature, and identifying amino acid substitutions that lead to changes in those attributes. The results of these efforts indicate that ligand binding affinity and catalytic rate are key targets during temperature adaptation: ligand affinity decreases during cold adaptation to allow more rapid catalysis. Structural changes causing these functional shifts often comprise only a single amino acid substitution in an enzyme subunit containing approximately 330 residues; they occur on the surface of the protein in or near regions of the enzyme that move during catalysis, but not in the active site; and they decrease stability in cold-adapted orthologs by altering intra-molecular hydrogen bonding patterns or interactions with the solvent. Despite these structure–function insights, we currently are unable to predict a priori how a particular substitution alters enzyme function in relation to temperature. A predictive ability of this nature might allow a proteome-wide survey of adaptation to temperature and reveal what fraction of the proteome may need to adapt to temperature changes of the order predicted by global warming models. Approaches employing algorithms that calculate changes in protein stability in response to a mutation have the potential to help predict temperature adaptation in enzymes; however, using examples of temperature-adaptive mutations in A4-LDH and cMDH, we find that the algorithms we tested currently lack the sensitivity to detect the small changes in flexibility that are central to enzyme adaptation to temperature.