Abstract: Detection and characterization of reactive ions, whether intermediates in chemical reactions or elusive species proposed under extreme conditions, is a challenge. The obstacles are low population and low stability of these reactive ions which often precludes the use of classical methods. We have developed an approach to transfer the reactive ions from solution to the gas phase at low temperatures or generate these ions in situ of mass spectrometer. Cryotrapping of the reactive ions in the gas phase provides sufficient time for their full spectroscopic characterization. I will show what fundamentally new information can be obtained by this approach on several examples.

Abstract: Our research uses chemistry principles to address questions of importance in life sciences and molecular medicine. This lecture will cover recent examples of emerging areas in our group in:

• building linkerless antibody-drug conjugates through direct and site-selective conjugation of anti-cancer drugs to antibodies [1];
• construction of artificial metalloproteins for controlled and tissue specific carbon monoxide (CO) delivery and their use for CO-immunotherapy in cancer [2];
• harnessing the power of natural product architectures in cancer chemical biology. By identifying on- and off-targets for anti-cancer entities and unveiling the underlying molecular mechanisms of target recognition, we explore the use of natural products as cancer modulators and ligands for the selective delivery of cytotoxic payloads [3].

References:
[1] Freedy AM, et al. Bernardes GJL: J. Am. Chem. Soc. 2017, 139, 18365.
[2] Ferreira MC et al., Bernardes GJL: Angew. Chem. Int. Ed. 2015, 54, 1172.
[3] Rodrigues T et al., Bernardes GJL: Angew. Chem. Int. Ed. 2016, 55, 11077.

Abstract: The brain controls adiposity via central and peripheral neural circuits. We used molecular genetic tools such as optogenetics to probe the connection between peripheral sympathetic neurons and adipocytes. Further, we found this neuro-adipose junction to drive lipolysis via norepinephrine (NE) signaling (1) and that the SNS is necessary and sufficient for fat mass reduction (1,2). As obesity is a chronic inflammatory state, we set to define neuroimmune mechanisms that link inflammation to SNS neurons (3). We report the discovery of Sympathetic neuron-Associated Macrophages (SAMs) that directly regulate the extracellular availability of norepinephrine (NE). We identified the molecular mechanism by which SAMs import and metabolize norepinephrine (NE). Abrogation of the mechanism for the uptake of NE by SAMs increases NE availability, which in turn promotes thermogenesis and browning, and long-term amelioration of obesity independently of food intake (3). The role of SAMs at steady state and obesity will be discussed.

References:

• [1] Zeng W, Pirzgalska RM, Pereira MMA, Kubasova N, Barateiro A, Seixas E, Lu YH, Kozlova A, Voss H, Martins GG, Friedman JM, Domingos AI: Sympathetic Neuro-Adipose Connections Mediate Leptin-Driven Lipolysis. Cell 163 (1): 84–94, 2015
• [2] Pereira MMA, Mahú I, Seixas E, Martinéz-Sánchez N, Kubasova N, Pirzgalska RM, Cohen P, Dietrich MO, López M, Bernardes GJL, Domingos, AI: A brain-sparing diphtheria toxin for chemical genetic ablation of peripheral cell lineages. Nature Communications 8: 15673, 2017
• [3] Pirzgalska RM, Seixas E, Seidman JS, Link VM, Sánchez NM , Mahú I, Mendes R, Gres V, Kubasova N, Morris I, Arús BA, Larabee CM, Vasques M, Tortosa F, Sousa AL , Anandan S, Tranfield E, Hahn MK, Iannacone M, Spann NJ, Glass CK, Domingos AI: Sympathetic neuron–associated macrophages contribute to obesity by importing and metabolizing norepinephrine. Nature Medicine 23 (11): 1309-1318, 2017.

Jonathan K. POKORSKI

Abstract: Biopharmaceuticals are the main growth area in pharmaceutical research and development and, most often, proteins are the active pharmaceutical ingredient. Recombinant protein production can be inexpensively scaled to multi-kilogram scales since the molecular biotechnology field is rapidly improving. Of particular interest are viral nanoparticles. The self-assembled, protein-derived nanostructures show exceptional promise in both vaccine development and tissue specific delivery of cargo. One technological hurdle, however, is the formulation of functional proteins either as injectables or into therapeutic reservoirs, also known as depots.

This talk will detail two central areas in developing viral nanoparticles for administration 1) chemical modification to lessen carrier-specific immune responses and 2) melt-processing of viruses into depot devices and the effect on macromolecular structure of the processed proteins. The first portion of the talk correlates a fundamental understanding of an immune-shielding polymer conformation, when attached to viruses, and their ability to evade carrier specific immune responses. Polynorbornenes have been developed as an alternative to linear PEG and were found to exhibit unique conformations, that better shielded the protein from antibody recognition. The second portion of the talk will describe melt processing of viral particles into slow release implants. Melt processing is exceptional scalable, with commercial extruders reaching throughputs of 1000 kg/h and 100% of the active protein is encapsulated. Melt processing is thought to be possible because of the reduced hydration state in the melt, thus eliminating the driving force to form amorphous protein aggregates. The primary focus of this portion of the talk will be a discussion of virus like nanoparticles (VLPs) derived from bacteriophage Qß. Qß is a combinatorial vaccine platform that has seen success in vaccine development for influenza, HIV, and hypertension. Melt processing conditions, physical models of processing, and biological data will be described in which Qß is processed into slow-release depot delivery formulations.

Abstract: Nucleic acids are perhaps the most significant biopolymers on this planet. They have played the decisive role in the spontaneous creation of life on the Earth four billion years ago. Since that, they continue in storing the genetic information in all forms of life and play key roles in the cellular regulatory processes. We will briefly overview three selected topics from our nucleic acids research.
How the first molecules capable of Darwinian evolution could have been created from “nothing” in a formamide-based scenario? Formamide pathway to the origin of life is a newly emerging "Universe-wide" chemical concept concurrent to the traditional “water-based” HCN scenario and can straightforwardly lead to biological molecules while avoiding the "water paradox”.
What is the role of dynamical recognition in protein-RNA complexes and how molecular dynamics simulations complement static data from X-ray crystallography and NMR?
How DNA guanine quadruplexes with only ~20-30 residues can achieve folding times of hour to months, why they comprise the most versatile nucleic acids class that nature has ever evolved and what consequences could this have for their biochemical roles?

The most recent reviews:

• Šponer J.E. et al.: Emergence of the First Catalytic Oligonucleotides in a Formamide-Based Origin Scenario. Chemistry-A European Journal 2016, 22, 3572-3586
• Šponer J.E. et al.: New Evolutionary Insights Into the Non-enzymatic Origin of RNA Oligomers. Wiley Interdisciplinary Reviews: RNA 2017, 8, e1400
• Šponer J. et al.: Folding of Guanine Quadruplex Molecules–funnel-like Mechanism or Kinetic Partitioning? An Overview from MD Simulation Studies. Biochimica et Biophysica Acta - General Subjects 2017, 1861, 1246–1263
• Šponer J. et al.: RNA Structural Dynamics as Captured by Molecular Simulations: A Comprehensive Overview: Chemical Reviews, 2018, DOI: 10.1021/acs.chemrev.7b00427

Abstract: Diplonemids are unicellular eukaryotes related to pathogenic trypanosomes. Unexpectedly, they were recently shown to be the 3rd most diverse and 6th most abundant marine eukaryotes. This reflects how little do we know about oceanic microorganisms, which produce half of all oxygen and are critical for the well-being of this planet. I will introduce this group of protozoans, until recently considered rare and obscure, in a way digestable for chemists.

References:

• Flegontova O., Flegontov P., Malviya S., Audic S., Wincker P., de Vargas C., Bowler C., Lukeš J. & Horák A. (2016) Unexpected diversity and abundance of planktonic diplonemids in the world ocean. Curr. Biol. 26, 3060-3065.
• Gawryluk R.M.R., del Campo J., Okamoto N., Strassert J.F.H., Lukeš J., Richards T.A., Worden A.Z., Santoro A.E. & Keeling P.J. (2016) Morphological identification and single-cell genomics of marine diplonemids. Curr. Biol. 26, 3053-3059.
• de Vargas C. et al. (2015) Eukaryotic plankton diversity in the sunlit global ocean. Science 348, 1261605. Lukeš J., Flegontova O. & Horák A. (2015) Diplonemids. Curr. Biol. 25, R702-R704.

Abstrakt: Detection and characterization of reactive ions, whether intermediates in chemical reactions or elusive species proposed under extreme conditions, is a challenge. The obstacles are low population and low stability of these reactive ions which often precludes the use of classical methods. We have developed an approach to transfer the reactive ions from solution to the gas phase at low temperatures or generate these ions in situ of mass spectrometer. Cryotrapping of the reactive ions in the gas phase provides sufficient time for their full spectroscopic characterization. I will show what fundamentally new information can be obtained by this approach on several examples.

Abstrakt: Our research uses chemistry principles to address questions of importance in life sciences and molecular medicine. This lecture will cover recent examples of emerging areas in our group in:

• building linkerless antibody-drug conjugates through direct and site-selective conjugation of anti-cancer drugs to antibodies [1];
• construction of artificial metalloproteins for controlled and tissue specific carbon monoxide (CO) delivery and their use for CO-immunotherapy in cancer [2];
• harnessing the power of natural product architectures in cancer chemical biology. By identifying on- and off-targets for anti-cancer entities and unveiling the underlying molecular mechanisms of target recognition, we explore the use of natural products as cancer modulators and ligands for the selective delivery of cytotoxic payloads [3].

References:
[1] Freedy AM, et al. Bernardes GJL: J. Am. Chem. Soc. 2017, 139, 18365.
[2] Ferreira MC et al., Bernardes GJL: Angew. Chem. Int. Ed. 2015, 54, 1172.
[3] Rodrigues T et al., Bernardes GJL: Angew. Chem. Int. Ed. 2016, 55, 11077.

Abstrakt: The brain controls adiposity via central and peripheral neural circuits. We used molecular genetic tools such as optogenetics to probe the connection between peripheral sympathetic neurons and adipocytes. Further, we found this neuro-adipose junction to drive lipolysis via norepinephrine (NE) signaling (1) and that the SNS is necessary and sufficient for fat mass reduction (1,2). As obesity is a chronic inflammatory state, we set to define neuroimmune mechanisms that link inflammation to SNS neurons (3). We report the discovery of Sympathetic neuron-Associated Macrophages (SAMs) that directly regulate the extracellular availability of norepinephrine (NE). We identified the molecular mechanism by which SAMs import and metabolize norepinephrine (NE). Abrogation of the mechanism for the uptake of NE by SAMs increases NE availability, which in turn promotes thermogenesis and browning, and long-term amelioration of obesity independently of food intake (3). The role of SAMs at steady state and obesity will be discussed.

Reference:

• [1] Zeng W, Pirzgalska RM, Pereira MMA, Kubasova N, Barateiro A, Seixas E, Lu YH, Kozlova A, Voss H, Martins GG, Friedman JM, Domingos AI: Sympathetic Neuro-Adipose Connections Mediate Leptin-Driven Lipolysis. Cell 163 (1): 84–94, 2015
• [2] Pereira MMA, Mahú I, Seixas E, Martinéz-Sánchez N, Kubasova N, Pirzgalska RM, Cohen P, Dietrich MO, López M, Bernardes GJL, Domingos, AI: A brain-sparing diphtheria toxin for chemical genetic ablation of peripheral cell lineages. Nature Communications 8: 15673, 2017
• [3] Pirzgalska RM, Seixas E, Seidman JS, Link VM, Sánchez NM , Mahú I, Mendes R, Gres V, Kubasova N, Morris I, Arús BA, Larabee CM, Vasques M, Tortosa F, Sousa AL , Anandan S, Tranfield E, Hahn MK, Iannacone M, Spann NJ, Glass CK, Domingos AI: Sympathetic neuron–associated macrophages contribute to obesity by importing and metabolizing norepinephrine. Nature Medicine 23 (11): 1309-1318, 2017.

Jonathan K. POKORSKI

Abstrakt: Biopharmaceuticals are the main growth area in pharmaceutical research and development and, most often, proteins are the active pharmaceutical ingredient. Recombinant protein production can be inexpensively scaled to multi-kilogram scales since the molecular biotechnology field is rapidly improving. Of particular interest are viral nanoparticles. The self-assembled, protein-derived nanostructures show exceptional promise in both vaccine development and tissue specific delivery of cargo. One technological hurdle, however, is the formulation of functional proteins either as injectables or into therapeutic reservoirs, also known as depots.

This talk will detail two central areas in developing viral nanoparticles for administration 1) chemical modification to lessen carrier-specific immune responses and 2) melt-processing of viruses into depot devices and the effect on macromolecular structure of the processed proteins. The first portion of the talk correlates a fundamental understanding of an immune-shielding polymer conformation, when attached to viruses, and their ability to evade carrier specific immune responses. Polynorbornenes have been developed as an alternative to linear PEG and were found to exhibit unique conformations, that better shielded the protein from antibody recognition. The second portion of the talk will describe melt processing of viral particles into slow release implants. Melt processing is exceptional scalable, with commercial extruders reaching throughputs of 1000 kg/h and 100% of the active protein is encapsulated. Melt processing is thought to be possible because of the reduced hydration state in the melt, thus eliminating the driving force to form amorphous protein aggregates. The primary focus of this portion of the talk will be a discussion of virus like nanoparticles (VLPs) derived from bacteriophage Qß. Qß is a combinatorial vaccine platform that has seen success in vaccine development for influenza, HIV, and hypertension. Melt processing conditions, physical models of processing, and biological data will be described in which Qß is processed into slow-release depot delivery formulations.

Abstract: Nucleic acids are perhaps the most significant biopolymers on this planet. They have played the decisive role in the spontaneous creation of life on the Earth four billion years ago. Since that, they continue in storing the genetic information in all forms of life and play key roles in the cellular regulatory processes. We will briefly overview three selected topics from our nucleic acids research.
How the first molecules capable of Darwinian evolution could have been created from “nothing” in a formamide-based scenario? Formamide pathway to the origin of life is a newly emerging "Universe-wide" chemical concept concurrent to the traditional “water-based” HCN scenario and can straightforwardly lead to biological molecules while avoiding the "water paradox”.
What is the role of dynamical recognition in protein-RNA complexes and how molecular dynamics simulations complement static data from X-ray crystallography and NMR?
How DNA guanine quadruplexes with only ~20-30 residues can achieve folding times of hour to months, why they comprise the most versatile nucleic acids class that nature has ever evolved and what consequences could this have for their biochemical roles?

Recentní reviews:

• Šponer J.E. et al.: Emergence of the First Catalytic Oligonucleotides in a Formamide-Based Origin Scenario. Chemistry-A European Journal 2016, 22, 3572-3586
• Šponer J.E. et al.: New Evolutionary Insights Into the Non-enzymatic Origin of RNA Oligomers. Wiley Interdisciplinary Reviews: RNA 2017, 8, e1400
• Šponer J. et al.: Folding of Guanine Quadruplex Molecules–funnel-like Mechanism or Kinetic Partitioning? An Overview from MD Simulation Studies. Biochimica et Biophysica Acta - General Subjects 2017, 1861, 1246–1263
• Šponer J. et al.: RNA Structural Dynamics as Captured by Molecular Simulations: A Comprehensive Overview: Chemical Reviews, 2018, DOI: 10.1021/acs.chemrev.7b00427

Abstrakt: Diplonemids are unicellular eukaryotes related to pathogenic trypanosomes. Unexpectedly, they were recently shown to be the 3rd most diverse and 6th most abundant marine eukaryotes. This reflects how little do we know about oceanic microorganisms, which produce half of all oxygen and are critical for the well-being of this planet. I will introduce this group of protozoans, until recently considered rare and obscure, in a way digestable for chemists.

Literatura:

• Flegontova O., Flegontov P., Malviya S., Audic S., Wincker P., de Vargas C., Bowler C., Lukeš J. & Horák A. (2016) Unexpected diversity and abundance of planktonic diplonemids in the world ocean. Curr. Biol. 26, 3060-3065.
• Gawryluk R.M.R., del Campo J., Okamoto N., Strassert J.F.H., Lukeš J., Richards T.A., Worden A.Z., Santoro A.E. & Keeling P.J. (2016) Morphological identification and single-cell genomics of marine diplonemids. Curr. Biol. 26, 3053-3059.
• de Vargas C. et al. (2015) Eukaryotic plankton diversity in the sunlit global ocean. Science 348, 1261605.
• Lukeš J., Flegontova O. & Horák A. (2015) Diplonemids. Curr. Biol. 25, R702-R704.