Ben Padman writes:

Hi there, I heard your questions about the CLEM (Correlative Light Electron Microscopy) experiment from the transcytosis paper and wanted to clear up a few things:

1) I acquired the optical and electron images a week apart using 2 different microscopes: a Leica SP8 laser scanning confocal and a Hitachi H7500 Transmission Electron Microscope.

2) I used a modified version of my previously published protocol (DOI: 10.1371/journal.pone.0095967)

3) Kathy had it right: The sub-cellular locations were relocated after ultrathin sectioning of the resin embedded sample.

4) When I started the experiment, I was really surprised by how many phages there were inside the cells… yet none of them seemed to correlate directly with the optical data. That’s when we realised that SYBR was pH sensitive. This is a problem, because endosomes are slightly acidic (~pH6 for early endosomes; ~pH5 for late endosomes). The phages were leaking SYBR almost immediately after entering the cells. Hindsight is a cruel mistress; should have used a pH resistant dye.

Aaron writes:

Dear TWiV,

First of all, thank you for such an enlightening podcast—I only recently came across TWiV but have greatly enjoyed the last few episodes. As a bioengineer whose work occasionally intersects with immunity, being able to hear about virology from a community I wouldn’t otherwise be involved with has been enormously educational.

I felt compelled to write in about your response to a recent letter from Brandon in Fresno, CA, as I found your description of LGBT life experiences in academia (at Rockefeller) was problematic. There is nothing that needs be “done about” a colleague who is gay. Additionally, mentioning that an LGBT individual is “not a predator” only serves to promote a dangerous and inaccurate notion that gay people are more likely to be so.

To answer Brandon’s question, I can only describe my own experience, but as a gay man who has been out during my undergraduate studies through becoming a professor, I’ve only found the scientific communities I’ve been a part of to be entirely welcoming and inviting.  Whichever program you end up joining, I would encourage you to seek out mentors who are able to provide guidance about supportive labs, graduate, and scientific communities. Many campuses maintain an LGBT campus resource center, and organizations such as oSTEM have a wealth of resources and contacts.

Best of luck with your applications.

And thanks again TWiV for a wonderful podcast!

Arthur writes:

Hi y’all (did we ever decide on a gender neutral pronoun?!)

I’m a DPhil student working on oncolytic adenoviruses in Oxford UK.

Just a very quick note to say that I can thoroughly agree with the comments on being openly gay in science.

Universities tend to swing more towards liberal because of the demographic, and most universities have an incredibly strong and active LGBTQI+ society that you can easily search for online.

I have worked in a few labs and have been lucky to receive no discrimination, with my PIs often meeting my husband at work parties etc.

This isn’t to say that living in university cities that are liberal doesn’t include experiencing the homophobia, micro-aggressions and violence that are, unfortunately still a part of being an openly queer person.

I have collaborated with colleagues who are gay from more conservative (aka religious) countries such as Malaysia and RAE who live openly when they are here in the UK but, due to laws, cannot live openly in their home countries at work or at home so we still have some way to go.

In more liberal countries, however, I have a great many colleagues who are openly queer without fearing it will affect their careers so please don’t let that put anyone off!

Thanks so much for all you do as a team. There’s a tonne of work that goes into these podcasts and it truly pays off.



Oxford University

Ps I have a very embarrassing story about meeting a TWIVer at a conference. I was at the international adenovirus conference and started talking to a very friendly person who sat next to me and, as usual, I started my sales pitch on how fantastic TWIV is, only to have the person sitting next to me say “I actually am a contributor to that podcast, I’m Kathy Spindler” and I promptly turned the colour of borscht!

Andrew writes:

Dear TWIV Team,

I am a big fan of your podcast. You last heard from me on TWIV 185 when I was working on my Ph.D. and M.D. at Northwestern. The episode cover photo is actually me sitting in a fort built of plaque assays (HSV, not Polio). After finishing my MD and PhD at Northwestern I completed internal medicine residency at Johns Hopkins in Baltimore and am now a first year infectious disease (ID) fellow at Hopkins.

I am giving ID journal club next week on this paper (sorry it is likely behind a paywall) that I came across while listening to Mark Crislip’s Puscast. In it, the authors describe antibody dependent enhancement (ADE) of secondary dengue infections in humans. My understanding is that previously, this had only been worked out in animal models. I thought this was very relevant to the discussion in last week’s TWIV episode and helps to explain why the Sanofi vaccine data are problematic. Thank you for pointing me in the direction of the Halstead article which will certainly add to the discussion at the journal club. We are lucky to have Anna Durbin (mentioned in last weeks’ podcast in reference to TWIV 384) on faculty at Hopkins so I am excited to hear what she has to say about these papers.

It is currently 27F (-3C) in Maryland. Keep up the great work.

All the best,


Andrew H. Karaba M.D. Ph.D

Infectious Diseases Fellow

The Johns Hopkins Hospital

Andrew writes:

Dear Vincent and all the TWiV team,

I was just on a flight back from the nanopore sequencing conference in NYC, listening to the latest episode (TWiV 469). There were a number of issues that your listeners and TWiV stars have brought up during the last few episodes, surrounding the challenges of landing new PI positions and the pressure to work excessive hours or land ‘high-impact’ papers. I think I have a fairly unique perspective in that regard and felt compelled to write in.

Currently, I am an assistant professor in the Biochemistry and Molecular Biology department in University of Texas Medical Branch (UTMB) in Galveston and have just begun my third year. It has been a highly rewarding and challenging experience. I was previously a post-doc in Jack Johnson’s laboratory at The Scripps Research Institute in La Jolla. While Jack is renowned as a structural biologist solving the three-dimensional structure of many viruses and phages using electron microscopy and crystallography, I had carved out a small niche in his lab using next-generation sequencing to study how viruses package their genetic cargo. I always knew that I wanted to run my own laboratory, and so I had been eyeing-up opportunities to run my own lab studying virus evolution and structure.

However, during the time (early Sept ’14) that I was putting together my application materials to apply for new faculty positions, one day I was forced to go to the ER with abdominal pain, which was quickly found to involve septic colitis. Within a day or so, I crashed, going into septic shock, DIC and organ failure. I spent over two weeks intubated in the ICU (though, often conscious and able to interact with my care-team and friends). It was only a few days after an emergency colectomy it emerged that I had stage II colon cancer that likely allowed the unknown infection to take grip. It took around ten surgeries to successfully ‘restore domain’ of my abdomen and install an end-ileostomy. It is difficult to exaggerate the grimness and the trauma of this scenario that my wife (Brittany) and I now macabrely refer to as my ‘spa treatment’.

During this time, I received extraordinary care and concern, not just from my family and close-friends, but from all my colleagues and lab-mates at Scripps. My then PIs (Jack Johnson and Bruce Torbett) were often at my bedside, comforting me, making sure that I was receiving the best possible care. It was patently clear that I did not simply have a group of colleagues and co-workers, but a close-knit support network akin to a family. Due to the disorientation that is common among ICU patients, my friends and my (now) wife put together a collage of several drawings and pictures to adorn the walls of my hospital room. These images are encouraged by the nurses to conjure comfort and a sense of familiarity during distressed and confused wakeful periods. One of these images was a picture of an icosahedral particle of Flock House virus. This provoked much amusement and interest among my doctors and nursing staff. I remember trying to communicate through scribblings and an iPad that I was a virologist and did next-generation sequencing and that I would love to sequence myself to see what I had. It wasn’t till I was extubated that I was able to explain that Flock House virus is a simple yet fascinating insect virus and that we use it as a model system to study the lifecycle and molecular biology of other human pathogens.

Despite all expectations, and thanks to wonderful and compassionate care from many nurses and doctors at the UCSD Thornton hospital and support from my friends, colleagues and family, I pulled through. Within a month I was back at home, writing new code, checking out new datasets and binge-watching Downton Abbey. However, the challenges did not stop there. Stage II colorectal cancer carries around a 75-80% overall five-year survival rate. Despite this, it seemed natural to continue with the applications.

There were many details of my application and background that might not have screamed PI. Specifically, while I believe my CV was strong (highlights were 2 PNAS, 1 JMB and 1 NAR), I certainly had no ‘high impact’ CNS papers. Nor was I working on hot-button topics that might woo a headline-seeking search committee: I was not chasing Ebola, designing HIV vaccines, or determining the three dimensional structure of CRISPR-Cas. Rather, I was proposing to study Flock House virus as a model system in Drosophila cells to understand the mechanism of the evolution of defective-interfering viral genomes.

Nonetheless, I fielded a small number of applications. And was delighted when I was asked to interview at UTMB in February (only four months after leaving hospital). I contacted the chair of the department (Mariano Garcia-Blanco, ‘bumped’ in TWiV 458) and I told him about my health and that during my interview I would be in the middle of adjuvant chemotherapy (albeit mild and with few visual manifestations). Faculty interviews often occur over multiple days, so I requested to have extra time after the interview to recover and rest before heading home. I still had an ostomy device at the time and so I would also need extra time before long meetings or seminars and travelling might not be easy. Initially, I was nervous about bringing this up. I asked him keep this information private as I didn’t want to make needlessly dramatic first impressions during meetings. Mentioning the ‘cancer’ word can elicit a broad range of unpredictable responses. However, Mariano’s response was highly compassionate, reassuring and professional, which was strongly predictive of the supportive and kind chair that he has now become.

So off I went to Galveston for the first time. The experience turned out to be excellent. Given my interests in molecular virology and using next-generation sequencing strategies to study virus evolution, it was clear that UTMB would be highly fertile place in which to work and collaborate. Although I was studying model systems and had no CNS papers, I think my proposed research fit well within the desired path of the department. I instantly felt that this was a place I could work and thrive and a departmental community to which I would like to contribute. Admittedly, I didn’t know the institute well beforehand. And being a native Brit, I never imagined myself becoming a resident Texan. Nevertheless, I am delighted that I made the effort to seek out a new home in Galveston.

In the last two years since starting my position, I have thought very carefully about how to manage my work-life balance. I very rarely work the obscene hours I think has become the stereotype of super-star PIs and requirement for post-docs seeking that transition. Throughout my career, and now, I aim to put in 40-50 hours a week. I often tell my students and post-docs that I do not expect them to work greater than 40 hours weeks, but that there will certainly be times when you will need the capacity to do so.

I have had to be particularly sensitive to my own limitations, physically and mentally, to prevent burnout and to mediate diminishing returns in what is already a stressful career path. Due to the severity of my prolonged stay in ICU and as is very common among cancer-survivors receiving repeated CT scans, MRIs, echoes, blood tests etc, I found that I had to manage head-on the additional stress and anxiety of survivorship and the threat of recurrence. It is increasingly being recognized that prolonged ICU stays and cancer-treatment can induce post-traumatic stress issues in survivors and I do not feel I am any exception to this. I am now three-years past my original diagnosis, but if I do not recognize when I am experiencing especially irrational anxiety (i.e. beyond that which might be expected of a looming grant deadline for example), and take the time to address this, the mental issues will never heal. Sometimes, this simply means leaving work early, heading out for a jog, or persuading (well…corrupting) a colleague to join me for a mid-afternoon beer. I am fortunate in that I perform a lot of computational/bioinformatics work that I can do practically anywhere. But I have always felt that one of the great advantages to our work as laboratory scientists is the possibility to work flexible hours, taking time off when needed, and working hard at other times. I feel these ‘mental-health holidays’ are essential to me and I feel grateful that my line of work is tolerant to them. I would encourage my colleagues and employees to do the same.

I’m not really sure there is any specific moral to my story. I find it difficult to describe or write about these experiences and generally I don’t discuss them (indeed the majority of my colleagues have no knowledge of my medical history). I don’t think I have any specific advice for those seeking faculty positions based on my experiences. But when there is so much negativity and despondency surrounding the subject of becoming a PI, I am deeply concerned that there are those who are presented with apparently insurmountable barriers and will be discouraged to apply. Given my own PI-prospects and the effort involved in putting together applications, if I had not had the support and encouragement of my friends, colleagues and mentors, I may quite easily not have tried. I think it is incumbent upon us as faculty members to reduce barriers and to encourage up-coming young scientists seeking a long-term home to find fitting opportunities. I am not sure of the best way to do this, but I hope that by sharing my experience, I may encourage someone else to make that plunge. I was fortunate in that my proposed ideas fell on fertile ground. But you miss 100% of the shots you do not take. I would be delighted if my experiences and positive outcomes could influence some TWiV listeners and encourage them to seek out their dream jobs.

The temperature is a mild 70 degrees F/ 21 degrees C, with 94% humidity. It is overcast, yet bright and still. Just like a summer’s day back in the UK. Many thanks for all that you do. I have been a listener on and off for years, and greatly appreciate the discussions. I find it very helpful in keeping up with the latest zeitgeists, excitements and controversies in our field.

Apologies for the long letter – I tried to keep it concise while covering all the critical points. Please feel free to contact me if you have any other questions, or share my experiences if they can help.

With many kind regards,

Andrew Routh


Andrew L. Routh PhD

Assistant Professor


University of Texas Medical Branch

Hongwei writes:

Hello TWIV professors:

I was introduced to TWIV last December by my son who is working on his Ph.D in Duke University. I love your podcast and have not missed a single episode since then. I thoroughly enjoy every episode, especially love the way you discuss scientific papers! You have the talent to make the complicated paper so easy to understand! I am a self-taught virologist and start using plaque assays in my experiments. When I was having problems with my plaque assay back in March, TWIV came to the rescue! In the episode 435, you talked about what could inhibit plaques formation which helped me figuring out what was my problem very quickly!

I am a big fan and a happy patreon of TWIV!

I am looking forward for every new episode of TWIV and TWIV family of other podcast!

A main reason for me to write is I want to put my name in the hat to win the Viruses book!

The weather in Sacramento California is sunny 16 degree!

All the best,


Madalina writes:

Hi TWiV team,

I am writing to you all the way from ‘hyggeligt’ Denmark, having just got home from the lab after a chilly bike ride in 0°C​. I always listen to TWIV on my daily bike commute and I just realised that on Danish time, I have exactly 2 hours left until the deadline for the virus book competition, so here goes nothing!

I also wanted to mention how exciting it was, as a second year PhD student studying abroad in Denmark (I am originally from England, but born in Romania) to have my first paper that I have collaborated on mentioned on TWiV a few months ago (TWiV 456 – be careful of canons). As a TWiV listener and PhD student, this was extremely exciting!​ My PhD project is centred around studying patients with extreme varicella zoster virus infection, and trying to understand the potential genetic component behind this. You continually inspire me throughout the up’s and down’s of PhD life, and as a fairly ‘naive’ student at the beginning of my PhD, I particularly enjoy listening to your debates on highly relevant topics such as open access and research funding. Keep up the good work and as they say in Denmark, ‘tusind tak’ (literally translated as ‘a thousand thanks’) 🙂



Madalina Carter-Timofte

PhD Student

Department of Infectious Diseases Q

Research Basement

Aarhus University Hospital


Justin writes:

Hello Twivers,

Put me in the raffle for the book, I would love a copy.

You mentioned in episode 468 that you were looking for something “cool” to do in Austin. I don’t know how you could integrate this into a TWIV episode, but I have recently discovered a very active caving community in Texas. There are hundreds of caves of various sizes in and around the city of Austin. It might be neat to record a TWIV in a deep cavern under Austin if all the hosts are feeling somewhat adventurous. At any rate, it is certainly worth checking out if you are into that sort of thing.

The weather in College Station is at a sunny 23C (74F).

Have fun in Texas,


Scott writes:

Dear TWIV ninjas – It is with a terrible heaviness of heart that I have come to the end of binge listening just today to episodes 1-469 over the last 4 months at 1.5 speed, and now must adjust myself to only a weekly fix. I’ve started on TWIP, so that will be some consolation.  I’m writing to request that you “randomly” pick me to win the Virus Book contest from episode 468, as I’m always looking for a good read outside my comfort zone in my limited free time, which is why I picked up Principles of Virology, as well as multiple other books that you all have picked over past episodes.  

1) First off, here in Northern Kentucky (exactly 7.5 miles from CVG on a heading of 180), at 02:52Z, it is a misty 41 deg F (5.6 deg Canadian)  Winds are from 310 at 8, visibility 10, skies clear, temp 6, dewpoint 3, Altimeter 30.16

2) Happy Birthday to Kathy!!

3) My greatest contribution to science (so far) is my daughter Hannah who is in her 3rd year of a fully funded MD/PhD at UIC – we’re watching for great things from her in pediatric oncology research. We’re also keeping a keen eye on the pending tax bill with the greatest interest due to the possible tax consequences for her.

4) Vincent had mentioned bourbon in a few recent episodes, if you all are ever in NKY with time on your hands, I would love to take you on a tour of any of Kentucky’s amazing distilleries (especially Bulleit) and our beautiful horse country.

5) More importantly, if any of you would consider presenting or even just meeting briefly to inspire middle school science students at my wife’s school during a layover at or near CVG, I would happily facilitate any logistics.

6) Transcribing of the episodes had been mentioned early in the TWIV series, but not so much recently.  If that is still of interest to you, I would be happy to volunteer to start working on that if you feel that it could still be helpful.

7) My suggestion for a pick of the week :  (summary from the site : the Sagan series is a collection of tribute videos dedicated to the late, great Carl Sagan.)   My parents purchased the Cosmos hardback for me when the series was first airing and I read that book to death… Leonard Nimoy’s “In Search Of” was a close second – where are the shows of that nature today amidst the shark week pablum the “science” channel spews?     

8) Vincent, please be nice to Dickson.

In closing, if I ever get the opportunity to meet any of you in person, please don’t be offended if I ask you speak at 1.5 speed so I can tell who is who !!

Thanks – Scott

Paul writes:



Getting colder

Trudy writes:

Could I squeeze this in as a pick of the week before Christmas?

If scientists wrote Christmas songs… so funny. And the part about Celsius made me lol!

Speaking of Celsius and snow, not sure if you guys heard about this but we got a serious snowfall last Friday!

Here’s a picture of my daughter building a snowman in our backyard last weekend.

Happy almost TWiV day!

Aria Snow