PubMed

Recent Publications

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1

Cell Chem Biol. 2021 Sep 11:S2451-9456(21)00357-3. doi: 10.1016/j.chembiol.2021.07.017. Online ahead of print.

ABSTRACT

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.

PMID:34520745 | DOI:10.1016/j.chembiol.2021.07.017



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Systematic engineering of optimized autonomous heavy-chain variable domains

J Mol Biol. 2021 Sep 8:167241. doi: 10.1016/j.jmb.2021.167241. Online ahead of print.

ABSTRACT

Autonomous heavy-chain variable (VH) domains are the smallest functional antibody fragments, and they possess unique features, including small size and convex paratopes, which provide enhanced targeting of concave epitopes that are difficult to access with larger conventional antibodies. However, human VH domains have evolved to fold and function with a light chain partner, and alone, they typically suffer from low stability and high aggregation propensity. Development of autonomous human VH domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human VH domain as a scaffold to construct phage-displayed synthetic libraries in which aspartate was systematically incorporated at different paratope positions. In selections, the library yielded many anti-EphA1 receptor VH domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 VH domain and an improved variant provided insights into the effects of aspartate and other substitutions on preventing aggregation while retaining function. Our naïve libraries and in vitro selection procedures offer a systematic approach to generating highly functional autonomous human VH domains that resist aggregation and could be used for basic research and biomedical applications.

PMID:34508727 | DOI:10.1016/j.jmb.2021.167241



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline

Nat Protoc. 2021 Sep 10. doi: 10.1038/s41596-021-00596-0. Online ahead of print.

ABSTRACT

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.

PMID:34508259 | DOI:10.1038/s41596-021-00596-0



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Reconfigurable multi-component micromachines driven by optoelectronic tweezers

Nat Commun. 2021 Sep 9;12(1):5349. doi: 10.1038/s41467-021-25582-8.

ABSTRACT

There is great interest in the development of micromotors which can convert energy to motion in sub-millimeter dimensions. Micromachines take the micromotor concept a step further, comprising complex systems in which multiple components work in concert to effectively realize complex mechanical tasks. Here we introduce light-driven micromotors and micromachines that rely on optoelectronic tweezers (OET). Using a circular micro-gear as a unit component, we demonstrate a range of new functionalities, including a touchless micro-feed-roller that allows the programming of precise three-dimensional particle trajectories, multi-component micro-gear trains that serve as torque- or velocity-amplifiers, and micro-rack-and-pinion systems that serve as microfluidic valves. These sophisticated systems suggest great potential for complex micromachines in the future, for application in microrobotics, micromanipulation, microfluidics, and beyond.

PMID:34504081 | DOI:10.1038/s41467-021-25582-8



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

ASO Visual Abstract: A Population-Based Analysis of Long-Term Outcomes Among Older Adults Requiring Unexpected Intensive Care Unit Admission After Cancer Surgery

Ann Surg Oncol. 2021 Sep 8. doi: 10.1245/s10434-021-10731-6. Online ahead of print.

NO ABSTRACT

PMID:34495447 | DOI:10.1245/s10434-021-10731-6



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Substrate-Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells

Bioelectricity. 2020 Sep 1;2(3):229-237. doi: 10.1089/bioe.2019.0037. Epub 2020 Sep 16.

ABSTRACT

Background: Neural precursor cells (NPCs) hold great promise for neural repair. Endogenous NPCs, found in the subventricular zone of the adult brain, proliferate and migrate toward lesion sites; however, it is not sufficient for neural repair. NPCs are electrosensitive cells that undergo directed migration in an electric field (EF). Here, we examined the EF-induced migration of a clinically relevant human NPC population. Materials & Methods: We examined the effects of different substrates and microenvironments on human NPC galvanotaxis. Results: Human NPCs increased their migration speed in the presence of an EF, and the direction of migration (anodal vs. cathodal) varied between substrates. The secretome and extracellular pH were not significant factors in EF-induced migration; however, our results are consistent with substrate stiffness playing a role in the direction of cell migration. Conclusion: These findings provide insight into the importance of the microenvironment on modulating human NPC migration and highlight substrate-dependent considerations for neurorepair.

PMID:34476355 | PMC:PMC8370329 | DOI:10.1089/bioe.2019.0037



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Novel Electrode Designs for Neurostimulation in Regenerative Medicine: Activation of Stem Cells

Bioelectricity. 2020 Dec 1;2(4):348-361. doi: 10.1089/bioe.2020.0034. Epub 2020 Dec 16.

ABSTRACT

Neural stem and progenitor cells (i.e., neural precursors) are found within specific regions in the central nervous system and have great regenerative capacity. These cells are electrosensitive and their behavior can be regulated by the presence of electric fields (EFs). Electrical stimulation is currently used to treat neurological disorders in a clinical setting. Herein we propose that electrical stimulation can be used to enhance neural repair by regulating neural precursor cell (NPC) kinetics and promoting their migration to sites of injury or disease. We discuss how intrinsic and extrinsic factors can affect NPC migration in the presence of an EF and how this impacts electrode design with the goal of enhancing tissue regeneration. We conclude with an outlook on future clinical applications of electrical stimulation and highlight technological advances that would greatly support these applications.

PMID:34471854 | PMC:PMC8370381 | DOI:10.1089/bioe.2020.0034



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene <em>STAG2</em>

Life Sci Alliance. 2021 Aug 30;4(11):e202101083. doi: 10.26508/lsa.202101083. Print 2021 Nov.

ABSTRACT

STAG2, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how STAG2 loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for STAG2-dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, STAG1, the paralog of STAG2, was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between STAG2 and the iron regulatory gene IREB2 Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.

PMID:34462321 | DOI:10.26508/lsa.202101083



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Peptide Platform as a Powerful Tool in the Fight against COVID-19

Viruses. 2021 Aug 23;13(8):1667. doi: 10.3390/v13081667.

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing over 195 million infections and more than 4 million fatalities as of July 2021.To date, it has been demonstrated that a number of mutations in the spike glycoprotein (S protein) of SARS-CoV-2 variants of concern abrogate or reduce the neutralization potency of several therapeutic antibodies and vaccine-elicited antibodies. Therefore, the development of additional vaccine platforms with improved supply and logistic profile remains a pressing need. In this work, we have validated the applicability of a peptide-based strategy focused on a preventive as well as a therapeutic purpose. On the basis of the involvement of the dipeptidyl peptidase 4 (DPP4), in addition to the angiotensin converting enzyme 2 (ACE2) receptor in the mechanism of virus entry, we analyzed peptides bearing DPP4 sequences by protein-protein docking and assessed their ability to block pseudovirus infection in vitro. In parallel, we have selected and synthetized peptide sequences located within the highly conserved receptor-binding domain (RBD) of the S protein, and we found that RBD-based vaccines could better promote elicitation of high titers of neutralizing antibodies specific against the regions of interest, as confirmed by immunoinformatic methodologies and in vivo studies. These findings unveil a key antigenic site targeted by broadly neutralizing antibodies and pave the way to the design of pan-coronavirus vaccines.

PMID:34452531 | PMC:PMC8402770 | DOI:10.3390/v13081667



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

A clinically applicable integrative molecular classification of meningiomas

Nature. 2021 Sep;597(7874):119-125. doi: 10.1038/s41586-021-03850-3. Epub 2021 Aug 25.

ABSTRACT

Meningiomas are the most common primary intracranial tumour in adults1. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas2. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.

PMID:34433969 | DOI:10.1038/s41586-021-03850-3



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄