Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X₂ channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel''s transition between a closed and an open state, we showed that the gating of P2X₂ is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X₂ even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X₂-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].     

Effectiveness of a WHO self‐help psychological intervention for preventing mental disorders among Syrian refugees in Turkey: a randomized controlled trial

Refugees are at high risk of developing mental disorders. There is no evidence from randomized controlled trials (RCTs) that psychological interventions can prevent the onset of mental disorders in this group. We assessed the effectiveness of a self‐help psychological intervention developed by the World Health Organization, called Self‐Help Plus, in preventing the development of mental disorders among Syrian refugees experiencing psychological distress in Turkey. A two‐arm, assessor‐masked RCT was conducted in two Turkish areas. Eligible participants were adult Syrian refugees experiencing psychological distress (General Health Questionnaire ≥3), but without a diagnosis of mental disorder. They were randomly assigned either to the Self‐Help Plus arm (consisting of Self‐Help Plus combined with Enhanced Care as Usual, ECAU) or to ECAU only in a 1:1 ratio. Self‐Help Plus was delivered in a group format by two facilitators over five sessions. The primary outcome measure was the presence of any mental disorder assessed by the Mini International Neuropsychiatric Interview at six‐month follow‐up. Secondary outcome measures were the presence of mental disorders at post‐intervention, and psychological distress, symptoms of post‐traumatic stress disorder and depression, personally identified psychological outcomes, functional impairment, subjective well‐being, and quality of life at post‐intervention and six‐month follow‐up. Between October 1, 2018 and November 30, 2019, 1,186 refugees were assessed for inclusion. Five hundred forty‐four people were ineligible, and 642 participants were enrolled and randomly assigned to either Self‐Help Plus (N=322) or ECAU (N=320). Self‐Help Plus participants were significantly less likely to have any mental disorders at six‐month follow‐up compared to the ECAU group (21.69% vs. 40.73%; Cramer''s V = 0.205, p<0.001, risk ratio: 0.533, 95% CI: 0.408‐0.696). Analysis of secondary outcomes suggested that Self‐Help Plus was not effective immediately post‐intervention, but was associated with beneficial effects at six‐month follow‐up in terms of symptoms of depression, personally identified psychological outcomes, and quality of life. This is the first prevention RCT ever conducted among refugees experiencing psychological distress but without a mental disorder. Self‐Help Plus was found to be an effective strategy for preventing the onset of mental disorders. Based on these findings, this low‐intensity self‐help psychological intervention could be scaled up as a public health strategy to prevent mental disorders in refugee populations exposed to ongoing adversities.     

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1^- and lpg5A^-/lpg5B^- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.     

Lidocaine alters the input resistance and evokes neural activity in crayfish sensory neurons

Lidocaine, a use-dependent Na(+) channel blocker, paradoxically evokes neural activation in the slowly adapting stretch receptor organ of crayfish at 5-10 mmol/l concentration. For elucidating the underlying mechanisms of this paradoxical effect, a series of conventional electrophysiological experiments were performed in the stretch receptor neurons of crayfish. In the presence of tetrodotoxin, lidocaine did not evoke impulse activity, however, a slowly developing and dose-dependent depolarization occurred in both the rapidly and slowly adapting stretch receptors. Similar effects were observed by perfusion of equivalent concentrations of benzocaine but not of procaine or prilocaine. Lidocaine did not evoke neural activity in the rapidly adapting neuron which fires action potential(s) in response to rapid changes in membrane potential. Slowly developing mode of the depolarization indicated the reason why only depolarization but not action potential responses were observed in the rapidly adapting neuron. The depolarizing effect of lidocaine was independent from any ionic channel or exchanger system. However, lidocaine and benzocaine but not procaine and prilocaine evoked a dose-dependent alteration in the input resistance of the neuron. It was proposed that the principal mechanism of the effect could stem from a change in the physical properties of the neuronal membrane.     

Targeting CD4 coreceptor expression to postselection thymocytes reveals that CD4/CD8 lineage choice is neither error-prone nor stochastic

The mechanism by which CD4/CD8 lineage choice is coordinated with TCR specificity during positive selection remains an unresolved problem in immunology. The stochastic/selection model proposes that CD4/CD8 lineage choice in TCR-signaled CD4(+)CD8(+) thymocytes occurs randomly and therefore is highly error-prone. This perspective is strongly supported by "coreceptor rescue" experiments in which transgenic CD4 coreceptors were ectopically expressed on thymocytes throughout their development and caused significant numbers of cells bearing MHC-II-specific TCR to differentiate into mature, CD8 lineage T cells. However, it is not known if forced coreceptor expression actually rescued positively selected thymocytes making an incorrect lineage choice or if it influenced developing thymocytes into making an incorrect lineage choice. We have now reassessed coreceptor rescue and the concept that lineage choice is highly error-prone with a novel CD4 transgene (referred to as E8(I)-CD4) that targets expression of transgenic CD4 coreceptors specifically to thymocytes that have already undergone positive selection and adopted a CD8 lineage fate. Unlike previous CD4 transgenes, the E8(I)-CD4 transgene has no effect on early thymocyte development and cannot itself influence CD4/CD8 lineage choice. We report that the E8(I)-CD4 transgene did in fact induce expression of functional CD4 coreceptor proteins on newly arising CD8 lineage thymocytes precisely at the point in thymic development that transgenic CD4 coreceptors would putatively rescue MHC-II-specific thymocytes that incorrectly adopted the CD8 lineage. However, the E8(I)-CD4 transgene did not reveal any MHC-II-selected thymocytes that adopted the CD8 lineage fate. These results demonstrate that CD4/CD8 lineage choice is neither error-prone nor stochastic.     

内蒙古黑里河自然保护区蝴蝶种类初报

2004~2006年初步查清内蒙古黑里河自然保护区的蝴蝶种类,隶属于7科,37种,其中凤蝶科Papilionidae 2种、粉蝶科Pieridae 7种、眼蝶科Satyridae 6种、蛱蝶科Nymphalidae 16种、灰蝶科Lycaenidae 2种、弄蝶科Hesperiidae 3种、绢蝶科Parnassiidae 1种。