INAUGURAL ARTICLE by a Recently Elected Academy Member:Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.Discovery and engineering of the microbial opsin genes not only has stimulated basic science investigation into the structure–function relationships of proteins involved in light-triggered ion flow but also has opened up opportunities for biological investigation (reviewed in ref. 1) via the technique of optogenetics, which involves targeting these genes and corresponding optical stimuli to control activity within specified types of cells within intact and functioning biological systems. For example, optogenetics has been used to identify causally the brain cells and projections involved in behaviors relevant to memory formation, affective states, and motor function, among many other discoveries (2–4). For the channelrhodopsins, an important member of this protein family widely used in optogenetics (5, 6), the light-activated cation-conducting channel pore has been the subject of structural investigation, both because of curiosity regarding the physical properties of its ion conduction and because the creation of inhibitory channels had been sought for optogenetic applications. Converging lines of work recently achieved the latter goal; resolving the high-resolution structure of channelrhodopsin (7) allowed a principled structure-guided approach to engineering for chloride selectivity by testing an electrostatic model for pore function (8, 9). Subsequently, by screening the genome of the Guillardia theta microbe, two naturally occurring light-gated chloride-conducting channelrhodopsins (10) were identified.Because optogenetic control of behavior has not yet been demonstrated with chloride channelrhodopsins, and to test further integrative ideas regarding pore function from structural considerations as shown here, we sought to design and test the next generation of enhanced chloride channels (iC++ and SwiChR++). Along the way, we provide the initial test of the hypothesis that light-activated channels will be more efficient tools than pumps for optogenetic neuronal inhibition at the cellular level, demonstrate the initial utility of light-gated chloride channels in controlling behavior in freely moving animals, and reveal key principles regarding the functional selectivity of light-gated ion channel pores.     

Benzene exposure among auto-repair workers from workplace ambience: A pioneer study from Pakistan

Objectives

In Pakistan, the reports on benzene exposure among workers in chemical industries are almost non-existing due to limited research work in the field of exposure science. This study aimed to investigate such exposure in a widely adopted occupation in Rawalpindi city.

Material and Methods

In this cross-sectional study, 60 blood samples (N = 20/group) of mechanics (MCs), spray painters (PNs) and control participants (CN) were analyzed. The socio-economic and demographic information of workers and that of workplaces was documented using a short questionnaire.

Results

We identified that the workers in spray-painting occupation are highly at risk of benzene exposure. The results showed that PNs were more at risk of exposure to benzene than MCs, and this exposure was significantly correlated with long working hours (r = 0.68, p < 0.001). Moreover, there are several limitations in workplace setups, which need to be addressed in order to mitigate workers health risk in this occupation. In addition to the reckless use of chemicals, other identified predictors of exposure included active and passive smoking, poor workplace hygiene and substandard ventilation.

Conclusions

To mitigate workplace exposure, it is necessary to reduce working hours and encourage regular use of self-protective equipments and adoption of proper hygiene in chemical workplaces.     

Overview of guidance for endoscopy during the coronavirus disease 2019 pandemic

From its beginning in December 2019, the coronavirus disease 2019 outbreak has spread globally from Wuhan and is now declared a pandemic by the World Health Organization. The sheer scale and severity of this pandemic is unprecedented in the modern era. Although primarily a respiratory tract infection transmitted by direct contact and droplets, during aerosol-generating procedures, there is a possibility of airborne transmission. In addition, emerging evidence suggests possible fecal–oral spread of the virus. Clinical departments that perform endoscopy are faced with daunting challenges during this pandemic. To date, multiple position statements and guidelines have been issued by various professional organizations to recommend practices in endoscopic procedures. This article aims to summarize and discuss available evidence for these practices, to provide guidance for endoscopy to enhance patient safety, avoid nosocomial outbreaks, protect healthcare personnel, and ensure rational use of personal protective equipment. Responses adapted to national recommendations and local infection control guidelines and tailored to the availability of medical resources are imminently needed to fight the coronavirus disease 2019 pandemic.     

Expression analysis of microRNA-155 in type 2 diabetes in Kashmiri population

International Journal of Diabetes in Developing Countries - The present study aimed to evaluate the expression of microRNA-155 (miR-155) in type 2 diabetes mellitus (T2DM) and assess its...     

Hysterectomy in a male? A rare case report

INTRODUCTION

Persistent Mullerian duct syndrome is a rare form of male pseudo-hermaphroditism characterized by the presence of Mullerian duct structures in an otherwise phenotypically, as well as genotypically, normal man; only a few cases have been reported in the worldwide literature. A great variety of organs have been found in indirect inguinal hernial sacs.

PRESENTATION OF CASE

We report a case of 70 year old man, father of 4 children with unilateral cryptorchidism on the right side and left-sided obstructed inguinal hernia containing uterus and fallopian tube (that is, hernia uteri inguinalis; type I male form of persistent Mullerian duct syndrome) coincidentally detected during an operation for an obstructed left inguinal hernia.

DISCUSSION

PMDS is usually coincidently detected during surgical operation, as was in our case. However pre-operative ultrasonography, computerized tomography and MRI allow possible pre-operative diagnosis.³

CONCLUSION

In cases of unilateral or bilateral cryptorchidism associated with hernia, as in our patient''s case, the possibility of PMDS should be kept in mind.     

Estimation of Cardiac Depressant Activity of Ten Medicinal Plant Extracts from Pakistan

Ten medicinal plants extracts were pharmacologically screened for their cardiac activity on isolated rabbit heart, and showed significant negative inotropic activity with negative chronotropic effects. In all cases alcohol dried extracts were used and reconstituted in either water or ethyl acetate for these tests. The plant extracts which produced cardiac depression were Cactus grandiflora , Carissa carandas Linn., Duranta repens Linn., Eclipta prostrata Roxb., Heliotropium ophioglossum , Prosopis glandulosa , Scheweinfurthia sphaerocarpa A. Br., Solanum surattense Burm. f., Tephrosia uniflora Pers., Nardostachys jatamansi Dc. (Syn. Valeriana jatamansi ).