A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.     

Modeling Harvest Intensity of Sooty Shearwater Chicks by Rakiura Māori in New Zealand

Abstract: Cultural evidence suggests that sooty shearwater (Puffinus griseus) chicks have been harvested by Rakiura Māori on islands in southern New Zealand since prehistoric times. Concerns exist that modern harvests may be impacting sooty shearwater abundance. We modeled human-related and ecological determinants of harvest (total no. of individuals harvested) of sooty shearwater chicks on 11 islands and examined the relationship between shearwater abundance and harvesting rates (chicks/hr) and harvester behavior throughout the harvesting season. Models best explaining variation in harvest between harvesting areas (manu), for both the early and late parts of the harvesting season, included harvester-days (included in all models with change in deviance information criteria [ΔDIC], ΔDIC < 8.36 and ΔDIC < 11.5, for the early and late periods, respectively). Other harvest determinants included shearwater density, size of the manu, and number of people helping harvesters (all included in the top 5 models within ΔDIC = 2.25 for the late period). Areas harvested by several families under a common-property harvesting system had higher harvest intensity for their size (24% points higher, 95% credible interval 11–36%) than those managed as an exclusive resource for one family. The slowest harvesters spent more time harvesting but on average only harvested 36% (95% credible interval 15–65%) and 34% (95% credible interval 12–63%) of the harvest taken by the fastest harvesters during the early and late periods, respectively. Our results highlight the possibility of elevated harvest intensity as the population of harvesters increases. However, our models suggested that a corresponding reduction in harvesting rate at low prey densities during the most productive period could potentially regulate harvest intensity. Future research will integrate these results into prospective shearwater demographic models to assess the utility of a range of harvesting strategies in ensuring harvest sustainability.     

On multistability of delayed genetic regulatory networks with multivariable regulation functions

Many genetic regulatory networks (GRNs) have the capacity to reach different stable states. This capacity is defined as multistability which is an important regulation mechanism. Multiple time delays and multivariable regulation functions are usually inevitable in such GRNs. In this paper, multistability of GRNs is analyzed by applying the control theory and mathematical tools. This study is to provide a theoretical tool to facilitate the design of synthetic gene circuit with multistability in the perspective of control theory. By transforming such GRNs into a new and uniform mathematical formulation, we put forward a general sector-like regulation function that is capable of quantifying the regulation effects in a more precise way. By resorting to up-to-date techniques, a novel Lyapunov-Krasovskii functional (LKF) is introduced for achieving delay dependence to ensure less conservatism. New conditions are then proposed to ensure the multistability of a GRN in the form of linear matrix inequalities (LMIs) that are dependent on the delays. Our multistability conditions are applicable to several frequently used regulation functions especially the multivariable ones. Two examples are employed to illustrate the applicability and usefulness of the developed theoretical results.     

Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: Role of DNA methylation and histone acetylation

Thyroid hormones have long been known to play important roles in the development and functions of the central nervous system, however, the precise molecular mechanisms that regulate thyroid hormone-responsive gene expression are not well understood. The present study investigated the role of DNA methylaion and histone acetylation in the effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor (BDNF) gene expression in rat hippocampus. The findings indicated that the activities of DNA methyltransferase (DNMT), methylated reelin and BDNF genes were up-regulated, whereas, the activities of histone acetylases (HAT), the levels of global acetylated histone 3 (H3) and global acetylated histone 4 (H4), and acetylated H3, acetylated H4 at reelin promoter and at BDNF gene promoter for exon II were down-regulated in the hippocampus at the developmental stage of the hypothyroid animals. These results suggest that epigenetic modification of chromatin might underlie the mechanisms of hypothyroidism-induced down-regulation of reelin and BDNF gene expression in developmental rat hippocampus.     

关于我国医疗质量监管体系的探讨

通过文献研究和典型现场调查,对国内外医疗质量监管体系进行比较研究,进而提出我国医疗质量监管体系的框架：以政府为主导,政府授权的专业监管机构、医疗保险机构和社会监督力量共同参与的多元化的监管体系。     

醛固酮对大鼠主动脉bax基因表达的影响

目的研究醛固酮对大鼠主动脉bax基因表达的影响。方法32只SD大鼠随机分为空白对照组、腺瘤组、腺瘤＋依普利酮组和腺瘤＋肼苯哒嗪组。在每只大鼠皮下埋植的微量渗透泵内注入空白溶剂或醛固酮。8周后通过免疫组化、RT—PCR和Western印迹检测主动脉bax基因的表达。结果与对照组相比,腺瘤组大鼠主动脉bax mRNA和蛋白表达都显著上调（P〈0．05）;依普利酮能够抑制醛固酮对bax基因的诱导作用（P〈0．05）;而肼苯哒嗪虽然可以使大鼠收缩压下降,但不能阻止醛固酮对bax基因的作用。结论醛固酮通过诱导bax基因表达,调节血管平滑肌细胞凋亡和干预细胞周期进程,可能是其导致血管重构的机制之一。     

Opioid regulation of CNS dopaminergic pathways: A review of methodology,receptor types,regional variations and species differences

Biochemical measurements of DOPAC, HVA, DA and 3-MT allow the assessment of both DA metabolism in nerve endings and DA release into the synaptic cleft. Using these biochemical indices of DA neuronal activity, we have examined the actions of mu, delta and kappa opiate receptor agonists on the nigrostriatal pathway. These studies indicate that delta and mu₂ isoreceptors regulate activity in the nigrostriatal pathway of the rat, mouse, gerbil and hamster. In general, increased DA metabolism is observed; however, increased DA release is only evident in pathways devoid of a presynaptic clamping action. As indicated with enkephalinase inhibitors, these enkephalinergic inputs to dopaminergic neurons possess a phasic ongoing activity.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.