Biophysical Reviews’ “Meet the Councilor Series”—a profile of Peter Pohl

It is my pleasure to write a few words to introduce myself to the readers of Biophysical Reviews as part of the “Meet the Councilor Series.” Currently, I am serving the second period as IUPAB councilor after having been elected first in 2017. Initially, I studied Biophysics in Moscow (Russia) and later Medicine in Halle (Germany). My scientific carrier took me from the Medical School of the Martin Luther University of Halle-Wittenberg, via the Leibniz Institute for Molecular Pharmacology (Berlin) and the Institute for Biology at the Humboldt University (Berlin) to the Physics Department of the Johannes Kepler University in Linz (Austria). My key research interests lie in the molecular mechanisms of transport phenomena occurring at the lipid membrane, including (i) spontaneous and facilitated transport of water and other small molecules across membranes in reconstituted systems, (ii) proton migration along the membrane surface, (iii) protein translocation, and (iv) bilayer mechanics. Training of undergraduate, graduate, and postdoctoral researchers from diverse academic disciplines has been—and shall remain—a consistent part of my work.

     

Academic motherhood – what happens when you can't make it happen?

We need more openness about age‐related infertility as it is a particular risk for many female scientists in academia who feel that they have to delay having children. Subject Categories: S&S: Careers & Training, Genetics, Gene Therapy & Genetic Disease

Balancing motherhood and a career in academic research is a formidable challenge, and there is substantial literature available on the many difficulties that scientists and mothers face (Kamerlin, 2016). Unsurprisingly, these challenges are very off‐putting for many female scientists, causing us to keep delaying motherhood while pursuing our hypercompetitive academic careers with arguments “I’ll wait until I have a faculty position”, “I’ll wait until I have tenure”, and “I’ll wait until I’m a full professor”. The problem is that we frequently end up postponing getting children based on this logic until the choice is no longer ours: Fertility unfortunately does decline rapidly over the age of 35, notwithstanding other potential causes of infertility.This column is therefore not about the challenges of motherhood itself, but rather another situation frequently faced by women in academia, and one that is still not discussed openly: What if you want to have children and cannot, either because biology is not on your side, or because you waited too long, or both? My inspiration for writing this article is a combination of my own experiences battling infertility in my path to motherhood, and an excellent piece by Dr. Arghavan Salles for Time Magazine, outlining the difficulties she faced having spent her most fertile years training to be a surgeon, just to find out that it might be too late for motherhood when she came out the other side of her training (Salles, 2019). Unfortunately, as academic work models remain unsupportive of parenthood, despite significant improvements, this is not a problem faced only by physicians, but also one faced by both myself and many other women I have spoken to.I want to start by sharing my own story, because it is a bit more unusual. I have a very rare (~ 1 in 125,000 in women (Laitinen et al, 2011)) congenital endocrine disorder, Kallmann syndrome (KS) (Boehm et al, 2015); as a result, my body is unable to produce its own sex hormones and I don’t have a natural cycle. It doesn’t take much background in science to realize that this has a major negative impact on my fertility—individuals with KS can typically only conceive with the help of fertility treatment. It took me a long time to get a correct diagnosis, but even before that, in my twenties, I was being told that it is extremely unlikely I will ever have biological children. I didn’t realize back then that KS in women is a very treatable form of infertility, and that fertility treatments are progressing forward in leaps and bounds. As I was also adamant that I didn’t even want to be a mother but rather focus on my career, this was not something that caused me too much consternation at the time.In parallel, like Dr. Salles, I spent my most fertile years chasing the academic career path and kept finding—in my mind—good reasons to postpone even trying for a child. There is really never a good time to have a baby in academia (I tell any of my junior colleagues who ask to not plan their families around “if only X…” because there will always be a new X). Like many, I naïvely believed that in vitro fertilization (IVF) would be the magic bullet that can solve all my fertility problems. I accordingly thought it safe to pursue first a faculty position, then tenure, then a full professorship, as I will have to have fertility treatment anyhow. In my late twenties, my doctors suggested that I consider fertility preservation, for example, through egg freezing. At the time, however, the technology was both extravagantly expensive and unreliable and I brushed it off as unnecessary: when the time comes, I would just do IVF. In reality, the IVF success rates for women in their mid‐to‐late 30s are typically only ~ 40% per egg retrieval, and this only gets worse with age, something many women are not aware of when planning parenthood and careers. It is also an extremely strenuous process both physically and emotionally, as one is exposed to massive doses of hormones, multiple daily injections, tremendous financial cost, and general worries about whether it will work or not.Then reality hit. What I believed would be an easy journey turned out to be extremely challenging, and took almost three years, seven rounds of treatment, and two late pregnancy losses. While the driving factor for my infertility remained my endocrine disorder, my age played an increasing role in problems responding to treatment, and it was very nearly too late for me, despite being younger than 40. Despite these challenges, we are among the lucky ones and there are many others who are not.I am generally a very open person, and as I started the IVF process, I talked freely about this with female colleagues. Because I was open about my own predicament, colleagues from across the world, who had never mentioned it to me before, opened up and told me their own children were conceived through IVF. However, many colleagues also shared stories of trying, and how they are for various—not infrequently age‐related—reasons unable to have children, even after fertility treatment. These experiences are so common in academia, much more than you could ever imagine, but because of the societal taboos that still surround infertility and pregnancy and infant loss, they are not discussed openly. This means that many academic women are unprepared for the challenges surrounding infertility, particularly with advanced age. In addition, the silence surrounding this issue means that women lose out on what would have otherwise been a natural support network when facing a challenging situation, which can make you feel tremendously alone.There is no right or wrong in family planning decisions, and having children young, delaying having children or deciding to not have children at all are all equally valid choices. However, we do need more openness about the challenges of infertility, and we need to bring this discussion out of the shadows. My goal with this essay is to contribute to breaking the silence, so that academics of both genders can make informed choices, whether about the timing of when to build a family or about exploring fertility preservation—which in itself is not a guaranteed insurance policy—as relevant to their personal choices. Ultimately, we need an academic system that is supportive of all forms of family choices, and one that creates an environment compatible with parenthood so that so many academics do not feel pressured to delay parenthood until it might be too late.     

Syncytia formation by SARS‐CoV‐2‐infected cells

In the supporting information of the article, the authors noticed that there was an error in Movie EV1. The right panel (SARS‐CoV‐2 + IFITM1) showed the same PI channel data (red) as the middle panel (SARS‐CoV‐2). This mistake occurred during the assembly of the merged movie file and does not change the interpretation of the data. A corrected version of the movie is herewith updated.     

After 2 years of a pandemic,students are struggling to find strong reference letters

Writing and receiving reference letters in the time of COVID. Subject Categories: Careers

“People influence people. Nothing influences people more than a recommendation from a trusted friend. A trusted referral influences people more than the best broadcast message.” —Mark Zuckerberg.

I regularly teach undergraduate courses in genetics and genomics. Sure enough, at the end of each semester, after the final marks have been submitted, my inbox is bombarded with reference letter requests. “Dear Dr. Smith, I was a student in your Advanced Genetics course this past term and would be forever grateful if you would write me a reference for medical school…” I understand how hard it can be to find references, but I have a general rule that I will only write letters of support for individuals that I have interacted with face‐to‐face on at least a few occasions. This could include, for example, research volunteers in my laboratory, honors thesis students that I have supervised, and students who have gone out of their way to attend office hours and/or been regularly engaged in class discussions. I am selective about who I will write references for, not because I am unkind or lazy, but because I know from experience that a strong letter should include concrete examples of my professional interactions with the individual and should speak to their character and their academic abilities. In today''s highly competitive educational system, a letter that merely states that a student did well on the midterm and final exams will not suffice to get into medical or graduate school.However, over the past 2 years many, if not most, students have been attending university remotely with little opportunity to foster meaningful relationships with their instructors, peers, and mentors, especially for those in programs with large enrollments. Indeed, during the peak of Covid‐19, I stopped taking on undergraduate volunteers and greatly reduced the number of honors students in my laboratory. Similarly, my undergraduate lectures have been predominantly delivered online via Zoom, meaning I did not see or speak with most of the students in my courses. It did not help that nearly all of them kept their cameras and microphones turned off and rarely attended online office hours. Consequently, students are desperately struggling to identify individuals who can write them strong letters of reference. In fact, this past spring, I have had more requests for reference letters than ever before, and the same is true for many of my colleagues. Some of the emails I have received have been heartfelt and underscore how taxing the pandemic has been on young adults. With permission, I have included an excerpt from a message I received in early May:Hi Dr. Smith. You may not remember me, but I was in Genome Evolution this year. I enjoyed the class despite being absent for most of your live Zoom lectures because of the poor internet connection where I live. Believe it or not, my mark from your course was the highest of all my classes this term! Last summer, I moved back home to rural Northern Ontario to be closer to my family. My mom is a frontline worker and so I''ve been helping care for my elderly grandmother who has dementia as well as working part‐time as a tutor at the local high school to help pay tuition. All of this means that I''ve not paid as much attention to my studies as I should have. I''m hoping to go to graduate school this coming fall, but I have yet to find a professor who will write a reference for me. Would you please, please consider writing me a letter?I am sympathetic to the challenges students faced and continue to face during Covid‐19 and, therefore, I have gone out of my way to provide as many as I can with letters of support. But, it is no easy feat writing a good reference for someone you only know via an empty Zoom box and a few online assignments. My strategy has been to focus on their scholarly achievements in my courses, providing clear, tangible examples from examinations and essays, and to highlight the notable aspects of their CVs. I also make a point to stress how hard online learning can be for students (and instructors), reiterating some of the themes touched upon above. This may sound unethical to some readers but, in certain circumstances, I have allowed students to draft their own reference letters, which I can then vet, edit, and rewrite as I see fit.But it is not just undergraduates. After months and months of lockdowns and social distancing, many graduate students, postdocs, and professors are also struggling to find suitable references. In April, I submitted my application for promotion to Full Professor, which included the names of 20 potential reviewers. Normally, I would have selected at least some of these names from individuals I met at recent conferences and invited to university seminars, except I have not been to a conference in over 30 months. Moreover, all my recent invited talks have been on Zoom and did not include any one‐on‐one meetings with faculty or students. Thus, I had to include the names of scientists that I met over 3 years ago, hoping that my research made a lasting impression on them. I have heard similar anecdotes from many of my peers both at home and at other universities. Given all of this, I would encourage academics to be more forthcoming than they may have traditionally been when students or colleagues approach them for letters of support. Moreover, I think we could all be a little more forgiving and understanding when assessing our students and peers, be it for admissions into graduate school, promotion, or grant evaluations.Although it seems like life on university campuses is returning to a certain degree of normality, many scholars are still learning and working remotely, and who knows what the future may hold with regard to lockdowns. With this uncertainty, we need to do all we can to engage with and have constructive and enduring relationships with our university communities. For undergraduate and graduate students, this could mean regularly attending online office hours, even if it is only to introduce yourself, as well as actively participating in class discussions, whether they are in‐person, over Zoom, or on digital message boards. Also, do not disregard the potential and possibilities of remote volunteer research positions, especially those related to bioinformatics. Nearly, every laboratory in my department has some aspect of their research that can be carried out from a laptop computer with an Internet connection. Although not necessarily as enticing as working at the bench or in the field, computer‐based projects can be rewarding and an excellent path to a reference letter.If you are actively soliciting references, try and make it as easy as possible on your potential letter writers. Clearly and succinctly outline why you want this person to be a reference, what the letter writing/application process entails, and the deadline. Think months ahead, giving your references ample time to complete the letter, and do not be shy about sending gentle reminders. It is great to attach a CV, but also briefly highlight your most significant achievements in bullet points in your email (e.g., Dean''s Honours List 2021–22). This will save time for your references as they will not have to sift through many pages of a CV. No matter the eventual result of the application or award, be sure to follow up with your letter writers. There is nothing worse than spending time crafting a quality support letter and never learning the ultimate outcome of that effort. And, do not be embarrassed if you are unsuccessful and need to reach out again for another round of references—as Winston Churchill said, “Success is stumbling from failure to failure with no loss of enthusiasm.”     

Biophysical Reviews ‘Meet the Editors Series’ — a profile of Sabrina Leslie

It is my pleasure to introduce myself to the readers of Biophysical Reviews as part of the ‘Meet the Editors Series’.     

Meet the IUPAB Councilor—Hans-Joachim Galla

As one of the twelve Councilors, it is my pleasure to provide a short biographical sketch for the readers of Biophys. Rev. and for the members of the Biophysical Societies. I have been a member of the council in the former election period. Moreover, I served since decades in the German Biophysical Society (DGfB) as board member, secretary, vice president, and president. I hold a diploma degree in chemistry as well as PhD from the University of Göttingen. The experimental work for both qualifications has been performed at the Max Planck Institute for Biophysical Chemistry in Göttingen under the guidance of Erich Sackmann and the late Herman Träuble. When E. Sackmann moved to the University of Ulm, I joined his group as a research assistant performing my independent research on structure and dynamics of biological and artificial membranes and qualified for the “habilitation” thesis in Biophysical Chemistry. I have spent a research year at Stanford University supported by the Deutsche Forschungsgemeinschaft (DFG) and after coming back to Germany, I was appointed as a Heisenberg Fellow by the DFG and became Professor in Biophysical Chemistry in the Chemistry Department of the University of Darmstadt. Since 1990, I spent my career at the Institute for Biochemistry of the University of Muenster as full Professor and Director of the institute. I have trained numerous undergraduate, 150 graduate, and postdoctoral students from chemistry, physics, and also pharmacy as well as biology resulting in more than 350 published papers including reviews and book articles in excellent collaboration with colleagues from different academic disciplines in our university and also internationally, e.g., as a guest professor at the Chemistry Department of the Chinese Academy of Science in Beijing.

     

The evolution of a cell biologist

I am honored and humbled to receive the E. B. Wilson Medal and happy to share some reflections on my journey as a cell biologist. It took me a while to realize that my interest in biology would center on how cells are spatially and dynamically organized. From an initial fascination with cellular structures I came to appreciate that cells exhibit dynamism across all scales—from their molecules, to molecular complexes, to organelles. Uncovering the principles of this dynamism, including new ways to observe and quantify it, has been the guiding star of my work.

Jennifer Lippincott-Schwartz     

Biophysical Reviews’ “Meet the Councilor”—a profile of Anastasia A. Anashkina

As one of the twelve Councilors of the International Union of Pure and Applied Biophysics elected in summer 2021, I have been asked to provide this short biographical sketch for the journal readers. I am a new member of the IUPAB Council. I hold a specialist degree in Applied Physics and Mathematics from the Moscow Institute of Physics and Technology and PhD in Biophysics from Moscow State University. I have spent my entire professional career at Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences in Moscow, where I am currently a senior researcher. I am Associate Professor at the Digital Health Institute of the I.M. Sechenov First Moscow State Medical University since 2018, and have trained undergraduate students in structural biology, biophysics, and bioinformatics. In addition, I serve as the Guest Editor of special journal issues of International Journal of Molecular Sciences and Frontiers in Genetics BMC genomics. Now I joined Biophysical Reviews Editorial Board as IUPAB Councilor. I am a Secretary of National Committee of Russian Biophysicists, and have helped to organize scientific conferences and workshops, such as the VI Congress of Russian Biophysicists.

     

Exercise‐induced α‐ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1‐dependent adrenal activation

The authors approached the journal to correct a mistake in the data presented in Appendix␣Fig S3D. The authors state that the mouse images in Appendix␣Fig S3D mistakenly displayed images from Fig 2F and Appendix␣Fig S1F. The images in Appendix␣Fig S3D are herewith corrected. The authors state that this change does not affect the conclusions or the statistics. The source data for these panels have been added to the original publication.The authors note that the following sentence needs to be corrected from: Appendix Figure S3D. Original. Appendix Figure S3D. Corrected. “Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by endurance exercise (Figs 1D and EV1A–D)”.to“Interestingly, several well‐established accumulation signatures of succinate, malate, hypoxanthine, and xanthine induced by endurance exercise (Lewis et␣al, 2010) were found to be decreased by resistance exercise (Figs 1D and EV1A–D)”.Further, the authors requested to amend the legend of Appendix␣Fig S3R to indicate that the same sample for the iWAT group, “WT+2%AKG” treatment, is shown in Fig 3P. The corrected legend reads: “(R‐S). Representative images (R) and quantification (S) of p‐HSL DAB staining from male OXGR1OE^AG mice treated with AKG for 12 weeks (n = 6 per group). The same sample is shown as in Fig 3P ”.The authors regret these errors and any confusion they may have caused. All authors approve of this correction.     

First person – Chady Hakim

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Chady Hakim is first author on ‘ Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs’, published in DMM. Chady is a Research Assistant Professor in the lab of Dongsheng Duan at the University of Missouri, Colombia, MO, USA, investigating the preclinical development of gene therapy for Duchenne muscular dystrophy (DMD), with a particular interest in using the canine DMD model.

Chady Hakim How would you explain the main findings of your paper to non-scientific family and friends? DMD is a severe muscle disease caused by dystrophin deficiency. Loss of dystrophin leads to muscle degeneration and remodeling, and eventually to muscle death and replacement by fatty and fibrotic tissues. The canine DMD model shares clinical and pathophysiological similarities to that of human patients. Therefore, studies performed with the canine model provide critical insight into understanding muscle disease in DMD. In this study, we were first interested in developing a force assay platform to evaluate the contractile force and characterize the kinetic properties of a single muscle in the canine DMD model. We focused on the extensor carpi ulnaris (ECU) muscle from the forelimb muscle group. As expected, we saw a loss of muscle force in affected dogs. Surprisingly, we observed an unexpected contractile kinetic profile. It has been well established that the dystrophic muscle undergoes a fast-to-slow fiber-type switch. This led us to predict that the affected muscle would exhibit slow contraction and relaxation. Surprisingly, we saw just the opposite. There was a decrease in the time taken to reach peak tension and relax the affected ECU muscle, indicating a faster contraction and relaxation. Additional characterization of myofiber-type composition in the normal and affected ECU muscle confirmed the kinetic assay results.

“[…] studies performed with the canine model provide critical insight into understanding muscle disease in DMD.”

What are the potential implications of these results for your field of research? The unexpected slow-to-fast myofiber-type switch highlights the complexity of muscle remodeling in dystrophic large mammals and paves the way for better utilizing dystrophic canines as a preclinical model in the study of DMD pathogenesis. Additionally, the fiber-type switch phenomenon offers a unique entry point for (1) investigating the molecular mechanism(s) that lead to this phenomenon and how it directly correlates to the loss of dystrophin, and (2) evaluating the pathophysiological implications for muscle strength and in determining whether this is unique to canine muscle. Most importantly, these results have significant implications for therapeutic approaches to DMD, such as gene replacement and editing, and evaluating their efficacy in correcting the fiber-type switch. What are the main advantages and drawbacks of the model system you have used as it relates to the disease you are investigating? When initiating this study, our goal was to evaluate muscle strength in the affected dogs. To achieve this goal, we developed an all-in-one automated in situ force assay platform. This novel platform has several advantages. First, we designed all the components to be adjustable to meet the need for studying muscles at different anatomic locations or with different sizes. Our design was also made with the consideration to adopt the platform to accommodate other large animal models besides the canine. Second, we developed a detailed protocol to optimize the stimulation parameters, allowing the muscle to reach its optimal force during contraction. This allowed the comprehensive evaluation of the contractile and kinetic properties of a single muscle. Together, this novel platform offers a unique ability to correlate the physiological findings with the molecular, cellular, biochemical and histological changes in a single muscle. This ability is critical for evaluating preclinical intervention studies. Unfortunately, this is a terminal assay, limiting the investigators to follow disease progression and therapeutic response in the same animal over time. What has surprised you the most while conducting your research? It is well established that the dystrophic muscle undergoes a fast-to-slow, rather than a slow-to-fast, transition in fiber type. In this study, we observed the opposite in affected canine muscles as they were mainly composed of the fast fiber type. The underlying mechanism of this fiber-type switch needs to be further investigated so it can be determined whether it is unique to canine muscle. Furthermore, muscles that are mainly composed of the fast fiber type are characterized by a higher force, higher contraction and relaxation rate, and less time needed to achieve full contraction and relaxation. In affected dogs, we noticed a reduction in the time taken for contraction and relaxation. Surprisingly, the force, contraction rate and relaxation rate were significantly reduced in the affected muscle compared to the normal muscle. Open in a separate windowRepresentative myosin heavy chain isoform immunostaining photomicrographs of a normal (left) and an affected (right) ECU muscle. Blue, type I myofiber; red, type IIa myofiber; magenta, type I/IIa hybrid myofiber; green, laminin immunostaining Describe what you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years? Unlike other genetic diseases, DMD is very challenging to treat. First, it is caused by mutations in the second largest gene in the body. The large size of the dystrophin gene makes it impossible to replace it through the gene replacement approach unless a truncated gene with a similar function to the full-length gene is used. Second, DMD affects every muscle type in the body, making a whole-body treatment necessary to achieve a complete cure. Gene therapy using adeno-associated virus (AAV)-mediated CRISPR/Cas9 gene editing shows much promise for treating DMD. It allows for the restoration of a near full-length dystrophin protein without the need for using a highly truncated microgene that can only result in limited function rescue (Hakim et al. 2018). We recently showed that AAV CRISPR therapy resulted in efficient dystrophin restoration in affected dogs but resulted in a Cas9-specific immune response that eliminated the edited cell. This unfortunate response is a critical barrier to advancing CRISPR therapy into clinics. With further advances in gene therapy, combined with advances in the understanding of CRISPR genome editing and how to evade the Cas9-specific T-cell response, AAV CRISPR therapy will be suitable for treating DMD. What changes do you think could improve the professional lives of early-career scientists? I believe it is critical for early-career scientists to collaborate and interact with other related research fields. This empowers and extends their knowledge, and also has a positive influence on their research focus. Before I started my PhD, I had gained some knowledge about muscle physiology. I wanted to extend this knowledge in my PhD studies by building a bridge between muscle physiology and molecular biology, and the perfect application was the field of DMD gene therapy. Through my previous experience, I was able to develop tools, such as the platform presented in this study, to answer critical molecular questions in the field of gene therapy. As a matter of fact, the outcome observation of the fiber-type switch was a result of analyzing the kinetic properties of the affected muscle force. This observation will now become an important biomarker in the evaluation of the efficacy of novel therapy.

“I believe it is critical for early-career scientists to collaborate and interact with other related research fields.”

What''s next for you? With the novelty of the data presented in this study, I''m excited about finding out whether gene therapy approaches would correct the fiber-type switch and reverse the remodeling observed in the affected canine muscle. I''m currently working with my mentor Dr Dongsheng Duan to evaluate fiber-type composition-affected canine muscles treated with gene therapy.     

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T_m value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t_1/2 values of>24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t_1/2=1631min) compared with FRNA (t_1/2=53.2min) and MeRNA (t_1/2=187min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.     

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.Keywords: PBP2, Neisseria gonorrhoeae, beta-lactam, conformational dynamics, antibiotic resistance

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3, 4, 5). However, treatment failures have been reported for both agents, and in 2018, a strain with high-level resistance to both ceftriaxone and azithromycin was identified (6, 7). Concern about azithromycin resistance led the Centers for Disease Control and Prevention recently to drop the recommendation of dual therapy in favor of an increased dose (500 mg) of ceftriaxone alone (8). Both penicillin and ceftriaxone inhibit cell wall biosynthesis in N. gonorrhoeae by targeting penicillin-binding protein 2 (PBP2).PBP2 is an essential peptidoglycan transpeptidase (TPase) that crosslinks the peptide chains from adjacent peptidoglycan strands during cell-wall synthesis (9). β-lactam antibiotics, including the extended-spectrum cephalosporin (ESC) ceftriaxone, are analogs of the d-Ala-d-Ala C terminus of the peptidoglycan substrate and as such target PBP2 by binding to and reacting with the active-site serine nucleophile (Ser310 in N. gonorrhoeae PBP2) to form a covalently acylated complex (10, 11). The acylation reaction (Equation 1) proceeds first through formation of a noncovalent complex with the β-lactam (defined by the equilibrium constant, Ks), which is then attacked by the serine nucleophile to form a covalent acyl-enzyme complex (k₂). For PBPs, hydrolysis of the acyl-enzyme (k₃) is very slow compared with its formation, and the enzyme is essentially irreversibly inactivated. The acylation of PBPs by β-lactam antibiotics is therefore defined by a second-order rate constant, k₂/Ks (M⁻¹ s⁻¹), which reflects both the noncovalent binding affinity (Ks) and the first-order acylation rate (k₂):E+S↔KsE⋅S→k2E−S→k3E+P(1)The emergence of resistance to penicillin and ceftriaxone in N. gonorrhoeae occurs primarily via the acquisition of mutant alleles of the penA gene encoding PBP2 (12). These alleles are referred to as mosaic because they arise through multiple homologous recombination events with DNA released by commensal Neisseria species. PBP2 from the high-level ceftriaxone-resistant strain, H041, contains 61 mutations compared with PBP2 from the antibiotic-susceptible strain, FA19 (13, 14). Determining how these mutations lower the k₂/Ks of ceftriaxone for PBP2 by over 10,000-fold while still preserving essential TPase activity is fundamental for understanding the evolution of antibiotic resistance.Toward this goal, we have identified a subset of these mutations that, when incorporated into the penA gene from FA19, confer ∼80% of the increase in minimum inhibitory concentration for ceftriaxone relative to that of the penA gene from H041 (penA41) (15, 16). We recently reported the structures of apo and ceftriaxone-acylated PBP2 at high resolution and have detailed conformational changes in β3 and the β3–β4 loop involved in antibiotic binding and acylation (17). Intriguingly, although present in the active site region, most of the mutations conferring resistance are not in direct contact with ceftriaxone in the crystal structure of acylated PBP2 (17, 18). We have proposed that these mutations alter the binding and acylation kinetics of PBP2 with ceftriaxone by restricting protein dynamics (18).To understand further the structural and biochemical mechanisms by which these mutations lower the acylation rates of β-lactam antibiotics, we utilized a combination of solution ¹⁹F NMR, X-ray crystallography, and biochemical approaches to investigate PBP2. We report that the β3–β4 loop in the TPase domain of WT PBP2, which is known to adopt markedly different conformations in the apo versus acylated crystal structures (17), samples two major conformational states in solution. Substitutions of WT PBP2 residues with mutations in H041 that confer ceftriaxone resistance alter the conformational landscape of PBP2 by destabilizing the high-affinity state containing the inward conformation of the β3–β4 loop and stabilizing a low-affinity conformation containing an extended β3–β4 loop conformation, thereby restricting access to the inward conformation required for high-affinity drug binding. Our combined solution NMR and crystallographic analyses of PBP2 and its preacylation drug complexes further support the notion that mutations in PBP2 from ceftriaxone-resistant strains of N. gonorrhoeae confer antibiotic resistance by hindering conformational changes required to form a productive drug-binding state (18).     

Dan Salah Tawfik (1955‐2021)—A giant of protein evolution

It was with great sorrow that we have learned of the untimely death of our friend, mentor, collaborator, and hero, Dan Tawfik. Danny was a true legend in the field of protein function and evolution. He had an incredibly creative mind and a breadth of knowledge—his interests spanned chemistry and engineering to genetics and evolution—that allowed him to see connections that the rest of us could not. More importantly, he made solving biochemical mysteries fun: He was passionate about his work, and his face lit up with joy whenever he talked about scientific topics that excited him (of which there were a lot). Conversations with Danny made us all smarter by osmosis.Danny’s own evolution in science began with physical organic chemistry and biochemistry. His PhD at the Weizmann Institute of Science, awarded in 1995, was on catalytic antibodies under the supervision of Zelig Eshhar and Michael Sela. It was followed by a highly productive period at the University of Cambridge’s Centre for Protein Engineering, first as a postdoctoral fellow with Alan Fersht and Tony Kirby, and then as a senior researcher. Among his many achievements during his time in Cambridge was the demonstration that off‐the‐shelf proteins—the serum albumins—could rival the best catalytic antibodies in accelerating the Kemp elimination reaction due to non‐specific medium effects. This work was an early example of unexpected catalytic promiscuity, and it sowed the seed for Danny’s later fascination with “esoteric, niche enzymology” that went far beyond convenient model systems.It was also in Cambridge where Danny first realized the power of the then new field of directed evolution, both for biotechnology and for elucidating evolutionary processes. He and Andrew Griffiths pioneered emulsion‐based in vitro compartmentalization. The idea of controlling biochemical reactions in separate aqueous droplets inspired emulsion PCR and next‐generation sequencing technologies, whereas Danny used it to solve a long‐standing problem in directed evolution; in vitro selection techniques had always been good at identifying ligand‐binding proteins, but compartmentalization finally enabled the directed evolution of ultra‐fast catalysts.Danny returned to Israel in 2001 to join the faculty of the Weizmann Institute of Science where his scientific trajectory further evolved, diverged, and even “drifted”. He developed new methods for enzyme engineering and applied his evolutionary insights into de novo protein design efforts. In this context, Danny’s interest was always focused on how proteins evolve, particularly the connection between promiscuity, conformational diversity, and evolvability. His depth of understanding underpinned both applied research, such as engineering enzymes to detoxify nerve agents, and fundamental research, such as the evolution of enzymes from non‐catalytic scaffolds.Through it all, Danny retained his sense of joy and wonder at the “beautiful aspects of Nature’s chemistry”. This includes his discovery of an exquisite molecular specificity mechanism mediated by a single, short H‐bond that enables microbes to scavenge phosphate in arsenate‐rich environments. In recent years, he deciphered the biosynthetic mechanism of dimethyl sulfide, “the smell of the sea”, and homed in on the interplay between the evolution of an enzyme, its host organism, and environmental complexity. His insights into how the first proteins emerged caused tremendous excitement in the field. He established the roots of two common enzyme lineages, the Rossmann and P‐loop NTPases, as simple polypeptides, and suggested ornithine as the first cationic amino acid. Prior to his death, he published the results of another tour de force: evidence that the first organisms to utilize oxygen may have appeared much earlier than thought.His work impacted many research fields, and he won many significant awards. Most recently, Danny was awarded the EMET Prize for Art, Science and Culture (2020), informally dubbed “Israel’s Nobel Prize”. He was an active and valued member of the EMBO community, having been elected in 2009, and, until his passing, served on the Editorial Advisory Board of EMBO Reports.Danny was also a superb science communicator. Both his research articles and reviews are a joy to read. What stood out just as much as his brilliance was his personality, as he embodied the Yiddish concept of being a true “mensch”. Danny was humble, was down‐to‐earth, and treated all his colleagues—including the most junior members of our research teams—as equals. He championed the careers of others, both those who worked directly for him and those who were lucky enough to be “just” his friends and collaborators. He believed in us even when we did not believe in ourselves, and he was always there to answer questions both scientific and professional. While he loved to share his own ideas, he would be just as excited about ours. Despite his own busy schedule, he always found the time to help others. He was also excellent company, with a great, very dry, sense of humor, and endless interesting stories, including from his own colorful life. In the days after his untimely death, an often‐repeated phrase was “he was my best friend”. Danny’s former group members have gone on to be highly successful in both industry and academia, including more than 15 former doctoral and postdoctoral researchers who are now faculty. The network of researchers Danny has trained, mentored, or influenced is broad, and this legacy is testament to his qualities as both a scientist and a person.Danny was born in Jerusalem to an Iraqi Jewish family, and his Arabic Jewish identity was important to him. He believed strongly in coexistence and peace, and very much valued the Arabic part of his heritage. In his own words: “I am an Israeli, a Jew, an Arab, but first and foremost a human being”. He would often speak of the achievements of his children with immense pride. Danny also had a passion for being outdoors, especially climbing and hiking—when the best discussions were often to be had (Fig ​(Fig1).1). One of the easiest ways to persuade him to come for a seminar, a collaborative visit, or a conference was to have access to high‐quality climbing in the area. He passed away in a tragic rock‐climbing accident, doing what he loved most outside of science. Our thoughts are with his partner Ita and his children, and we join the much broader community of friends, collaborators, and colleagues whose hearts are broken by his sudden loss.Open in a separate windowFigure 1Dan Salah Tawfik (1955–2021)Photo courtesy of Prof. Joel Mackay, The University of Sydney.     

Editorial for ‘Issue focus on 2nd Costa Rica biophysics symposium — March 11th–12th, 2021’

This Editorial describes both the motivation for, and the five articles appearing in, the Issue Focus dedicated to the 2nd Costa Rica Biophysics Symposium which was held in March 2021. Some recent history about both the symposium and developments in science occurring within Costa Rica is described. 

The Costa Rica Biophysics Symposium was conceived as a forum for faculty, scholars and students interested on cutting-edge topics in biophysics and related fields. Following the success of the first event organized in 2019 (Solís et al (2020), the second edition of the symposium took place on March 2021 with the support of the Academia Nacional de Ciencias de Costa Rica (ANC, National Academy of Sciences of Costa Rica), the International Union of Pure and Applied Biophysics (IUPAB), the German Society of Biophysics (DGfB), and the Universidad Nacional of Costa Rica (UNA). The symposium aimed to reinforce and enhance the novel network of investigators established in the 2019 event. Participation of Costa Rican presenters, either located in the country or abroad, and foreign scientists from the USA, Germany, France, and Switzerland (Solís et al. (2021a) translated into an expansion and internationalization of the previous network. Moreover, the symposium attracted a broad international audience, which increases the opportunities of further international collaboration.The meeting was organized into 14 presentations and one keynote lecture. It was attended by researchers of the three main universities of Costa Rica: Universidad Nacional (UNA), Universidad de Costa Rica (UCR) and Tecnológico de Costa Rica (TEC). Presenters from international universities were also present, including UT Southwestern Medical Center, USA; Klinikum Nürnberg Medical School, Germany; École Polytechnique Fédérale de Lausanne, Switzerland; Institut de Neurosciences de Montpellier, France; University of California Berkeley, USA; and The University of Chicago, USA. The topics presented in the symposium were diverse and covered cutting-edge biophysical research areas. The presentations ranged from channel electrophysiology, machine learning focused on cellular microscopy, prediction of protein–protein interactions, channelopathies and novel biophysical techniques, among others (Solís et al., 2021a). Furthermore, each lecture was followed by questions from the audience, allowing discussion, engagement and interaction between researchers in spite of the limitations of a virtual symposium. The closing event for the symposium was a lecture by the world-renowned biophysicist Francisco Bezanilla from the University of Chicago, who engaged the audience into a master presentation of his vast research on protein voltage-sensor domains (VSD) with a focus on his recent work on the non-canonical mechanisms for VSD-mediated regulation of pore domains in voltage-gated potassium channels (Carvalho-de-Souza and Bezanilla 2019). After the consequent discussion, the symposium finished with a networking activity, where audience and presenters were able to socialize and share experiences.