Im Glenner

Die letzte Woche war bestimmt von Jetlag und Vorbereitungen für mein erstes SNF Projekt. Zum Glück hat mir Mami geholfen. So konnte ich alles reibungslos erledigen während sie meine Wäsche wusch und auf Jacoby schaute.

 

Gestern ging es dann auf Laichfischfang. Es war streng und sehr schön. Der Glenner floss wild zwischen den Valser Steinen und das Herbstwetter sorgte für eine wundervolle Stimmung. Die Forellen liessen nicht auf sich warten. Auch im Ferrera Bach fanden wir später viele.

 

Ich möchte mich hiermit bei all den Fischern und den Wildhütern Arnold Caminada, Moritz Schmid, Gieri Derungs, Gion Bundi, Pirmina Caminada, Patric Ragettli, Thomas Durschei und Martin Cavegn für ihre grosse Hilfe bedanken. Und natürlich Roland, der wichtigste von allen! Wir werden noch viele gemeinsame Stunden verbringen…

 

Bei meinen Recherchen habe ich noch diese Webseite über Pirmina gefunden:

https://www.graubuenden.ch/de/regionen-entdecken/geschichten/pirmina-caminada-erste-wildhueterin-graubuendens

Es war mir eine grosse Freude, Pirmina gestern kennenzulernen!

 

forelle4 forelle2 forelle1 img_6521 img_6518 img_6513 img_6488

First Fieldwork in California

L.A. is huge. There is no Swiss comparison. Zürich or Geneva are little villages compared to it. San Francisco and the whole Bay area are also bigger than Switzerland and overpopulated. When you drive during the night it seems like the whole world is artificially illuminated. Thousands of cars, houses and signs are flashing. It is hard to believe that 150 years ago none of these people were here. This was a wild place with whales, sharks, condors, mountain lions and even golden bears. If you want to get a glimpse of the past you should go to the Angelo Coast Range Reserve. This is a reserve that is run by the University of California, Berkeley.

http://angelo.berkeley.edu/

We drove up the coast the night before fieldwork and slept at Fort Bragg. We had dinner with the locals. A bar full of sailors, butchers and carpenters – us in the middle with the kids. And all these rough men turned soft and playful. Challenging Linnea in dominos and watching Donny feed Jacoby. Anyways, early in the morning we drove into the reserve and I helped Suzanne collect some data for her doctoral thesis. Donny stayed at the station during the day and I followed Suzanne into the woods. Climbing up the creeks against the flow and catching rainbow trout until it got dark. The mountain lions saw us but we never saw them. Suzanne caught her fish with electro shocks. While she administers current underwater we were trying to catch all the stunned critters with hand nets. We caught lots of salamanders (Dicamptodon spp.), frogs (Foothill yellow-legged frog / Rana boylei), crabs, and even a lamprey (Lampetra tridentate). And lots of rainbow trout, maybe some steelheads. Suzanne collected scales, tissue, wrote down sizes, weights, and stuck little PIT tags into the fish. She reminded me of myself working on my PhD. She is great and it seems she has it all under control. I am looking forward to collaborate with her next summer. We want to collect lots of juveniles and sample their gut microbes and at the same time we decided we should also have a look at the gut contents.

Linnean’s name for the rainbow trout is O. mykiss. In the South Fork Eel river watershed, where I went to visit Suzanne, O. mykiss individuals exhibit two different life-forms. Some individuals stay for their whole life in the river pools. These are the ‘actual rainbow trout’. Their counterparts swim down the river as juveniles and live for part of their lives in the Pacific Ocean. They come back as much bigger fish to spawn. These individuals are called ‘steelheads’. I want to find out if the gut microbes of juvenile rainbow trout and steelheads (before they leave) from the same river pools differ. My hypothesis is that their symbiont bacteria are different. I think that bacteria help the steelheads during the process of smolting – when they prepare to swim away.

Suzanne made me familiar with the typical fieldwork that is required to sample wild O. mykiss. We were crawling up and down the creeks with all the material. I really enjoyed the reserve and its wilderness. Most of the sampling techniques (sampling tissues and scales, measuring the fish) I already knew well. I was impressed how easy it seems to tag the fish with PIT tags and then how to scan them and recognize individuals. My task before next summer is to learn how to get stomach content samples from wild fish without hurting them. I am also thinking about getting ‘stool’ samples simultaneously.

Lucky, as I am, I joined the Eisen group right after the annual STAMPS course.

https://stamps.mbl.edu/index.php/Main_Page

Holly and Guillaume took that course this summer and devoted the last three lab meetings on summarizing the most important bits from the course and sharing it with the rest of the group. I got all the course slides about the latest advancements and conclusions how to analyze microbial genomic data. It feels like I made the right choice of groups for my project. With Suzanne and the other people at ESPM I was deeply impressed about their expertise in ecology. In Jonathan’s group I met a bunch of people who are totally specialized on the analysis of microbial communities from different angles. Needless to say again that the Lake Arrowhead conference just blew me away. Hence, both aspects of my project, the host system and its symbionts are nicely covered.

img_1714

into the wild

img_1719

yellow legged frog

img_1736

our van and home

img_4935

giant salamander

img_5001

giant rainbow trout…

img_5017

giant lamprey

img_1799 img_4925 img_4963 img_4978 img_5010 img_5014 img_5025 img_5026

Bacteria on naturally spawned trout eggs and regionally increasing temperatures

Freely available online!!!

www.nature.com/articles/srep17084

Here we published a correlative study of bacterial assemblages on naturally-spawned brown trout (Salmo trutta) eggs. During my first year as a PhD student I collected brown trout eggs within a big river system in the canton of Berne. I joined Joachim Guthruf, a passionate brown trout specialist, who monitored natural spawning in the main river Aare and its tributaries. He studied the fish for days in order to see them spawning and mark the burial sites of the eggs. About three weeks later he would go back and dig out the eggs, count them and analyze the spawning success at the different spawning sites. I had the opportunity to join and help him during most his trips. While I helped him with his data acquisition, I also collected eggs at the late-eyed developmental stage for my own studies. I was interested in investigating the spatial pattern of host-associated bacterial communities in brown trout eggs across a rivers system. Recent experiments with salmonid embryos established important principles of microbial colonization, maternal transmission of bacteria, bacterial virulence factors, and host genetic responses to bacterial infections. This progress stands in sharp contrast to what is known about the diversity of host-associated bacteria of fish in their natural environment.

IMG_0601

Salmo trutta

IMG_8918

For this study we characterized bacteria at nine different locations. Eight locations were within the river Aare system and one location was in Sils/Maria, within a non-connected river system of a different river, the river Inn. Bacterial communities on brown trout eggs differed markedly from the composition of bacteria found in their water environment. However, they were strikingly similar across different habitats and rivers regardless of (i) geographic distance among spawning places (isolation-by-distance) and (ii) host genetic and morphological differentiation. These findings strongly suggest that brown trout have egg-specific microbiomes. In the paper we describe the trout egg-associated microbiomes in detail and report evidence that bacterial diversity increases with water temperature.

Aeschenfeld2 Forellen_Graben IMG_3518 IMG_3584I worked a very long time on this manuscript and I learned some very important conceptual points about working with environmental sampling of bacterial communities. First, I think it is extremely important to collect water samples or any kind of environmental samples when characterizing host-associated bacteria in their natural environment. This allows the discrimination of bacteria that are specifically associated with a host and bacteria that seem to be omnipresent in their environment. Second, I find it important to sample blank samples and sequence them. It is an illusion to think that we can work under sterile conditions in the field. So why not capturing all kinds of contaminations that we collect in the course of the study and subtract this from the real samples? We already followed this approach in our Aquatic Sciences paper earlier this year (blog post about it). Recently, Noah Fierer also tweeted about the importance of this approach and I must admit that I was very proud I already have published some articles where we took care of this issue. Third, we were dealing with some pretty low sample sizes for some locations. As we had to find a way to optimize the rather expensive sequencing technique (454 Pyrosequencing, Roche), we ended up pooling all the different sites where brown trout bury their eggs for each spawning location so the comparisons between locations could not be based anymore on variance estimates. Consequently, our data only allowed for (i) correlations of water temperature with bacterial community diversity, (ii) correlations of geographic and host genetic distance among spawning places with phylogenetic distance of bacterial communities, (iii) characterizations of core bacterial communities on all brown trout eggs, and (iv) comparisons of egg samples and water samples with regard to bacterial composition and its putative function. With regard to the comparison between the bacterial communities of the main river Aare with its tributaries we added a bootstrapping approach that is similar to a power analysis. Here we increased the number of samples for the main river Aare and its tributaries sequentially, using simulations, and investigated how many samples would be needed to find a significant difference in bacterial composition given the observed distribution of bacteria in our dataset. With regard to relationships between phylogenetic distance of bacterial communities among spawning places with geographic distance and host genetic distance respectively, we also analyzed the effect of reducing the number of pairwise comparisons on slope estimates and discussed this in the light of low sample sizes.

pipeline

At this point I would like to thank Joachim for his time in the field with me, his patience and his generosity, sharing all this insider information about the local brown trout populations. I am also grateful to Claus and Luca for letting me follow my own curiosity and helping me a great deal with the writing for this manuscript. Aude, my first Master student at the University of Lausanne helped me a lot with the wet lab work for this manuscript, which was very time consuming and also under time pressure as I was expecting my daughter Linnea at that time. Finally, Frédéric supported me with the statistical analysis after the first revision by two very competent Scientific Reports reviewers. He backed up my statistical approaches and pointed out some weaknesses. I am very happy that this article is out now!

Paper out!

Here is the Link!

I guess I should keep this going and write a little story for everybody about each of my manuscripts that some nice editors decide to accept.

This one here is about gene expression in whitefish embryos. Regarding the blog I wrote about the Aquatic Science paper, this could be interpreted as a continuation, a different perspective of our study system. Here we collected sperm and unfertilized eggs from whitefish that can be found in lake Geneva (Coregonus palaea). We fertilized the eggs in vitro and in a full-factorial breeding design. That means that we took the eggs of 4 females and the sperm of 4 males and then crossed them in all possible combinations. This gave us 16 little Tupperware buckets with freshly fertilized eggs. These we brought to the University where we have climate chambers at a constant temperature of 6.5° Celsius. All eggs were filled into individual wells with 2ml of standardized water. In their little microcosmos they grew and developed into embryos. At the late-eyed developmental stage when the blood circulation system is fully functional, we infected 13 replicates of each family with a nasty bacterial pathogen that can be found across many Swiss lakes and rivers Pseudomonas fluorescens. We also added some nutrients that these bacteria like. As a control we added only the nutrients without the pathogen to another 13 replicates of the same families. We already knew from previous studies that at this stage of development it matters who the father of the embryos was with regard to mortality and performance of the embryos under stress. Since the father only provided sperm and no paternal care, father effects can be interpreted as genetic effects. That means, he contributes genes to the embryos only. Once the embryo is old enough it starts expressing these genes and they have an impact on how it will perform under stress. However, what we did not know yet was which genes might be involved. So my goal for this study was to give these significant genetic effects a name.

Whitefish, an economically important fish species for the gastronomy in Switzerland

Whitefish, an economically important fish species for the gastronomy in Switzerland

my husband waiting for me outside the cold climate chamber

my husband waiting for me outside the cold climate chamber

Wells where embryos are raised.

Wells where embryos are raised.

Accordingly two days after treatment, we collected 3 embryos from each family and treatment and extracted all the gene expression products in their whole body, the so-called RNA. These are the gene products in all animals and plants that will be translated into proteins, which make up what we are. With the use of next-generation sequencing, we digitalized all this information. That means, that only messenger RNAs are filtered out, tagged according to which individual they belong to, pooled and then translated into letters. For this process we collaborated with a Swiss company in Geneva Fasteris. Months later, I received a huuuuuge text file that I could then use to satisfy my hunger for learning bioinformatics tools. With the help of a very friendly guy at Fasteris I found most overlapping sequences of text and aligned everything to a collection of longer text segments (contigs). These were then compared to an online reference of gene expression reads. The amount of data we produced was extremely big. Since I was only interested in genes that are differentially expressed between embryos in our treatment and control group, I decided to quantify the reads first and only compare the ones to a reference, that are also different between the two groups. Many of the reads did not result in a match, however, 1096 did and those could be characterized further. They told us which functional pathways are already active at this early stage of fish development and they gave us some insight into what defense mechanisms these embryos already have.

Whitefish embryos at the late-eyed developmental stage

Whitefish embryos at the late-eyed developmental stage

There is some background information to this paper. First, I must admit that I convinced my PhD supervisor to conduct this experiment because I wanted to get the chance to learn more about bioinformatics. I was the one responsible for the experimental treatment, the laboratory analysis of extracting RNA, and the one who did all the bioinformatics and data analyses. It was also the first manuscript that I wrote mostly on my own. It is not a very ground-breaking story. I would call it a lesson.

This project was always a side project during my PhD. We started with the fieldwork in 2010 when I just began my doctoral studies in Lausanne. The lab work was done in the end of 2011. I had serious troubles getting RNA of good quality from these embryos. We did not extract RNA directly but froze the embryos first for a couple of months at -80° Celsius. I would not recommend that to anybody. I was very busy working on different projects, some of them I considered my main projects at that time, and therefore this side project had to wait several times.

From the 16 families I managed to get a rather high amount of good quality RNA from 4 families. I made sure that I would have gene products from the same mother, crossed with 4 different fathers. I would say that this is the heart of this study. We could reduce variation in gene expression due to maternal effects. Different genotypes from the fathers were investigated against a constant background of the same mother. The whitefish external breeding system allowed us to control for host genotypes and contrast environment-induced changes in gene expression. I am discussing this strength of the study in the paper and encourage other scientists to use the same host system of fish with an external breeding system to investigate gene expression due to different treatments. It does not only present a way to reduce variation in expression due to maternal and environmental effects, it also provides the possibility to study gene expression in natural populations and their ecologically-relevant context.

FSI_blog2I also would like to add that it was very pleasant to work with Laurent Farinelli at Fasteris. He is one of the founders of the Illumina sequencing methodology and he had the balls to start his own company. Now he is collaborating on many very exciting projects and he delivers high quality data and an exceptional service. I enjoyed my few meetings in Geneva at his company.

To end the story of this paper I have to mention that I finally sent the extracted RNA for sequencing in summer 2012. I received the data during my maternity leave and already started playing with it. In 2013 I managed to assemble all reads. I had to digest quite a bit of theory about partly assembled gene expression reads (transcriptomes) and I learned how to use a high-performance computing system (clusters at the SIB). In 2014 I did the differential gene expression analysis and compared reads of interest to different online reference databanks. Here I could rely on the theory about differential gene expression in lung cancer datasets that I was exposed to during my internship at Novartis. This project would not have been possible without the help of several co-workers at UNIL, such as Oksana Riba, Kate Ridout, Paris Veltsos and my co-author Emily Clark. At this point I would like to thank them again for their help and advice.

At the moment I am supervising a Master student who is applying the same experimental set-up in grayling (Thymallus thymallus). He is looking at sex-specific gene expression of grayling embryos under estrogen stress. In this project we also did all the steps from fieldwork until bioinformatics ourselves. However, he can rely on several collaborators who are specialized in the different aspects of the project and as a Master student he concentrates on only one project at a time. I am very excited to see him advancing so fast.

Post for ESEB 15 Poster

Table 1: The influence of treatment, maternal, paternal, maternal x paternal effects, and bacterial diversity on brown trout embryo survival.

a = Five main logistic mixed effect models were compared to a reference model (in bold) to test if treatment ‘T’, dam ‘D’, sire ‘S’, and bacterial alpha diversity (phylogenetic distance, for its calculation see Material and methods) on the eggs explain a significant part of the variance in embryo mortality of brown trout. Alpha diversity was measured on the eggs before fertilization ‘A1’ or 14 days after treatment with nutrient broth ‘A2’. Two additional models were fitted to investigate the interaction of treatment with dam and sire effects. These models were fitted using the combined data of all three treatments (controls, nutrient broth at a dilution of 1:1000 and 1:500 in the wells).

b = The effects of the two different bacterial alpha diversity measures, random sire and random dam effects on embryo survival were also investigated within treatments.
survival_poster

Still tired

In the morning I get up early. This is so untypically me. Strong coffee needed. Riding my bike to the train station. Buying more coffee at Segafredo. Vegging out in the train to Lausanne. Fribourg is mysterious with all the fog and a golden autumn sun in the morning. Usually I work on my private defense in the train. My presentation is still too long.

I have experienced quite a bit of instability lately. After I handed in my thesis I did not experience a magic happiness as I had been expecting. On the contrary, I got extremely exhausted. I felt heavy and got pulled down. At the same time I was shaken by existential thoughts. Big questions about life. Feeling lonely and more intensively. Convinced that I have no future I started worrying about the people in Gaza, and in the end I mostly cried.  It is difficult to slow down if you have been driving in the sixth gear for too long.

It is good to write about it now. For several days I could not understand what is going on with me. I repeated often that everything is too much to me. It felt like a huge wave that was falling down on me. I suddenly realized that it had been too much. I finally reached a point in my life where I can truly say, I did too much. With all my honesty, it is hard to admit that I actually do have limits. It is no nice experience to feel your limit. That moment when you are enjoying a fast drive with your SAAB. Faster than ever. Totally hyper. And then the road bends and for a little second you lose control. This is how it feels like. During this little second something triggers a reaction, a little cut.

Now it heals. It feels like a wound that needs to heal. I will let it heal. I just handed in my PhD thesis and I am not allowed to take a step back and look at it with pride. While I was convinced during the last couple of days that I have no future and that I did not achieve anything, my husband and our daughter convinced me otherwise. My future started yesterday and I will have to cope with it. I discovered my limits and the people working with me need to respect them.

I do not expect that I am able to change much. I am 30 years old and the last 10 years I developed my own surviving strategies. However, I am hurting and I do not want this to happen again. Taking care of yourself is much more difficult for me than I would like to admit.

Here are some simple rules:

1)   Sleep enough. Take those fucking naps if you do not sleep at night.

2)   Try to eat healthy. Even if it has to be cute little calves and innocent fish.

3)   Stay away from nicotine.

4)   Do not think that you will stop drinking alcohol or coffee one day. It is too tempting and my brain got used to it.

5)   Given that I have an awesome husband and the cutest and smartest daughter in the world, I have to pull the emergency break at the moment when I come home and feel unable to enjoy them. My little family is the most precious thing I have in my life and I want to always prioritize them. They will give me the strength to endure injustice and immoral behaviors at work.

6)   I try to challenge myself and reach new goals every day at work. However, sometimes I realize that my contribution to science does not matter at all. Reminding myself about the feedback I get after presenting my work helps. I see my job as a way to learn how to solve problems and answer questions. While I feel pushed and forced in different directions right now, I should remember that one day I will be able to decide myself which problems I want to solve.

 

 

 

 

 

 

 

 

Appendix: When darkness falls and surrounds you, when you fall down, when you’re scared and you’re lost – be brave.

I am coming to hold you. When all your strength has gone and you feel wrong like your life has slipped away – follow me. You can follow me and I will not desert you now. I will keep you safe. You can trust me. I will protect you, my love.

Is there a glass ceiling for women in science?

While I am waiting for my dropbox to synchronize I will do a little brainstorm about women in science. Recently I discussed with Donny about the ‚glass ceiling‘.

 

What is the “glass ceiling”? – There appear to be two main bottlenecks for women in Science:

1)   Getting permanent positions

2)   Getting awards and invitations

 

Tasks where we are good at take us away from the final place. We seem to diverge away from the goal. That is mainly because we are good at it, because women say “yes” and are more cooperative than men in general. Also, women are asked more frequently than men. Because there still are fewer women than men it makes sense that women are asked more often, not only because of their gender.

 

–> We need to reveal these facts and make people in the individual departments aware of it.

 

Take home message:

A)   TRANSPARENCY

B)   VALIDATE WHAT YOU SAY YES TO

 

If you have already too many tasks and you are asked to take on more give a positive “no” as answer. Make your own work visible. Suggest another task that you can take over. Set time limits, re-evaluate, and help to find a replacement when the time limit is met. Allow other people to mess tasks up. Do not step in to help. Take over graduate school work. In the end of a PhD you can use “finishing up” as a good excuse not to take another task.

 

What remains an open question: What is your goal? You need to define your goal before you can evaluate what to say “yes” to. The tasks you need to do to become a full professor differ among universities, even among departments. In the UK, for example, there are criteria what you need to achieve in order to be able to step up.

 

Generally we evaluate people on their self-promotion. You need to think you are the best before you can get a position. This selects for alpha-males and we lose a lot of good researchers. There should be a place for any kind of character in science, as long as he or she is a brilliant researcher.

 

General take home messages:

–       ask people to nominate you for awards

–       define your goals

–       find a mentor

–       preferably at another university

–       email to people and start groups

–       be present

–       be seen

–       ask for stuff

–       volunteer at hosting symposia

–       build a network during grad school

–       be your own personality

Working from home

With my job I can work from home. The fact that this is possible makes me very happy. Usually it involves a great deal of self-discipline, organization and cooperation from husband and daughter or other family members. In my case it works out fine.

The following points are important to make it work:

 

1) trust your partner that he/she is able to watch your kid.

Even if you can hear your kid crying, screaming, yelling, complaining – it is your partner’s responsibility now.

–> the same applies to other family members / babysitters

 

2) find a room in your appartment where you can close the door and concentrate on your work

This might involve ‚Boze‘ noise-killing headphones. I put up several Campus maps of UC Davis and Stanford to motivate myself. I also put up some sand from Carmel and Seaside at Monterey. In the middle there is a little toy boat that my father carved. When I see that stuff it signals to my brain that it is time to work and give the responsibility for my daughter to somebody else.

 

3) Make sure that you have a functioning internet connection wherever you are

I end up using it all the time to get access to papers and look up how different R-scripts work.

 

4) If your babysitter or kid is sick, make sure that there is a corner in your working room where the kid can hang out and spend some hours on its own.

Linnea is totally fascinated by the following:

– cat food

– fish food

– cute baby dolls with cute faces that she can put down in a toy bed

– clothes to put on her dolls

– counting real money

– sorting playing cards

– sorting tablets for the dishwasher

– eating a joghurt on her own

– cleaning everything with a wet wash rag

– painting on herself and on the walls

 

Linnea plays in the background with her dolls and baby beds

Linnea plays in the background with her dolls and baby beds

From time to time she wants me to acknowledge her work and give her a hug. When I am too absorbed with my work she does not even try to approach me.

 

If she does not want to play on her own anymore make her useful to do some work for you.

 

5) When you get to a point where you are not productive anymore let yourself be distracted and do some housework

 

insulating the house

insulating the house

insulating 2 insulating 3

You are at home. Undone housework can be distracting. Do not fight against it. Little breaks from writing a manuscript can be very helpful to come up with new thoughts and ways for phrasing. Big projects such as insulating an old house or painting shutters are very rewarding projects to distract from work for a while. They make you feel useful and successful and this makes you work better when you are back working on research.

 

6) At home there is no 8h-days.

Working at nights is very efficient. It makes me feel good because I am at peace and I know that my family is happily asleep very close to me. Work whenever it is possible and you will anyway end up with more than 8h every day.

 

Photo booth – pictures from the last 18 months:

early working from home

early working from home

answering emails

answering emails

getting distracted

getting distracted

somebody woke up

somebody woke up

 

back in H-Town

Back in Houston, life is normal again. Lots of excitement in California. All three of us fell in love with that place. Today I looked at some ‚old‘ pending manuscripts and finished a discussion. It amazed me how free I felt in my mind. I could just sit down, think about what I want to write and then write it down. I guess this is a good strategy. Put it away for a while and then come back to it. Full of joy and good thoughts. Even all my collaborators had answered by the time we came back to Houston. I know I am very impatient.

Our second visit to Berkeley was very different. I met Stephanie Carlson and her whole group. They all took some time to talk to me and show me around. We had lunch all together and dinner with Séb and his family. Linnea was so super happy to see kids. She hugged Vadim again and again and I could not complain about some very Swiss Spaghetti. Ben showed us his favorite Baseball team and Linnea fell asleep…

Papa was soooo proud

Papa was soooo proud

SF skyline

SF skyline

Linnea fell in love with a donkey in downtown Berkeley

Linnea fell in love with a donkey in downtown Berkeley

Talking with Stephanie was the highlight. I felt immediately comfortable and my mind was open. Brainstorming. Thinking about cool projects and suggesting what I would love to do. Because I also care a lot about Davis and Jonathan I was totally overwhelmed when Stephanie suggested to collaborate with people in Davis because we do not have a wet lab at Berkeley. I am totally happy. There is even a train running from UC Davis to UC Berkeley.

UC Davis love

UC Davis love

The last day we had breakfast with Beni. I felt heavy from all these good discussions about science, possible projects and hearing what these nice and friendly people do for their research. UC Berkeley was overwhelming, and so was UC Davis, the human environment and all the natural beauties. California love. I will end this post with some more pics about the last days of our Cali trip.

 

Sleeping out in nowhere Napa

Sleeping out in nowhere Napa

For my post about the Big Sur you still need to be patient. I did not finish Kerouac’s book yet…

mysterious