text:
\section{Introduction}
 All cells of a multi-cellular organism share the same DNA sequence, yet, depending on location and cell type, display distinct cellular programs as a result of controlled gene expression.
Hence, the expression of genes is regulated by epigenetic factors such as DNA methylation.
In mammals, the methylation of DNA is restricted to the C5 position of cytosine (C) and mostly appears in a CpG di-nucleotide sequence \cite{ehrlich1982amount,feng2010conservation}.
The palindromic nature of CpG positions provides a symmetry which, after DNA replication, allows the stable inheritance of the methylation ``signal''.
Methylation of C to 5-methyl cytosine (5mC) is catalysed by a certain enzyme family,
the DNA methyltransferases (Dnmts) \cite{bestor1983two,okano1998cloning}.
Three conserved and catalytic active family members are associated with the methylation of DNA.
Dnmt1 is mainly responsible for maintenance methylation after DNA replication,
i.e. the enzyme mainly reestablishes the methylation pattern on the newly synthesized daughter strand \cite{hermann2004dnmt1}  according to the inherited information of the parental DNA strand.
The enzymes Dnmt3a and Dnmt3b perform
\emph{de novo} methylation, where new methyl groups are added on unmethylated Cs \cite{okano1999dna}.
However, there is evidence that this separation of tasks is not definite and that all Dnmts may carry out all tasks to a certain extent \cite{arand2012vivo}.

Once established, 5mC can be further modified by oxidation to 5-hydroxyme\-thyl cytosine (5hmC), which  can again be oxidized to 5-formyl cytosine (5fC). Then, 5fC can eventually be converted to 5-carboxy cytosine (5caC).
All these processes are carried out by
the ten-eleven translocation (Tet) enzymes \cite{ito2011tet,tahiliani2009conversion}.
A considerable level of 5hmC can be found in many cells types and its occurrence has been connected to gene regulation as well as genome wide loss of DNA methylation \cite{globisch2010tissue,kriaucionis2009nuclear}.
In contrast, 5fC and 5caC are far less abundant and their particular functions remain more illusive.
Nevertheless, studies suggest that both oxidized cytosine variants  function as intermediates during enzymatic removal of 5mC from the DNA \cite{globisch2010tissue}.

In general, DNA methylation can be removed in two ways.
First, after DNA replication, the absence or blocking of maintenance methylation will result in a passive DNA methylation loss with each cell division  (\emph{passive demethylation}).
Second, generated 5fC or 5caC is enzymatically removed from the DNA and subsequently replaced by unmodified cytosine (\emph{active demethylation})  \cite{cardoso1999dna,hashimoto2012recognition,he2011tet,maiti2011thymine}.

While
 DNA replication  and the associated maintenance methylation happens only once per cell division cycle, \emph{de novo} methylation and the modification processes of 5mC via oxidation, as well as active demethylation, may happen at arbitrary time points.
With purely discrete hidden Markov models (HMM), as used in \cite{arand2012vivo} and   \cite{Giehr2016},
it is difficult to describe multiple instances of \emph{de novo} methylation or other modification events for a given CpG during one cell division cycle.
Here, we introduce a hybrid HMM
to describe  the dynamics of active demethylation.
 It distinguishes between events at fixed time points (cell division and maintenance methylation) and events at random time points (\emph{de novo} methylation, oxidations, active demethylation).
By applying our model to a data set of a single copy gene from mouse embryonic stem cells, we were able to accurately predict the frequency of the observable CpG states and the levels of the hidden states, which correspond to the different modified forms of C.
Furthermore, we show how to estimate the enzymatic
reaction efficiencies using a maximum likelihood approach.

Compared to previous models for describing DNA methylation,
 we here propose an   approach that takes into account both, the rather fixed and deterministic timing of cell division and the random nature of the enzymatic processes.
In this way, we are able to present a   model that realistically describes the dynamics of DNA methylation and improves our understanding of  active demethylation.

The paper is organized as follows:
In Section 2 we give the necessary biological and mathematical background, i.e. we explain   passive and active demethylation in more detail, describe the model and explain the parameter estimation procedure.
In Section 3 the results are discussed and in Section 4 we conclude our findings.

\section{Model}
\subsection{Passive and active demethylation}
One can distinguish between two different ways of losing methylation at cytosines:
After
cell division a new (daughter) strand of the DNA is synthesized.
Initially, all cytosines of the daughter strand
are
unmethylated, while the methylation states of cytosines on the parental strand remain unchanged.
Maintenance methylation, which happens during the replication process, is used to re-establish the methylation
pattern at the newly synthesized strand. However,
the absence or inhibition of maintenance methylation causes a loss of 5mC with each replication step.
This DNA replication dependent loss has been termed \emph{passive demethylation}.

For \emph{active demethylation} it is assumed that   oxidation of 5mC to 5fC or 5caC via 5hmC and a subsequent enzymatic removal of the oxidative cytosine from the DNA occurs (cf. Fig \ref{Fig:MethCycle}).
Since \emph{de novo} methylation as well as \emph{active demethylation} are replication independent, the loop depicted in Fig. \ref{Fig:MethCycle}
can be traversed   multiple times within one cell cycle.
In measurements, we see Cs in all stages, i.e., either methylated, oxidized (5hmC/5fC/5caC) or unmodified.

\begin{figure}[t]
\begin{center}
		\begin{tikzpicture}[minimum size=1cm,scale=0.5,->,>=stealth',thick]

		\node (4) at (34, 3.5) [fill= yellow, circle,draw] {$h$};
		\node (5) at (30,0) [fill= red, circle,draw] {$m$};
		\node (6) at (38,0) [fill=cyan!40, circle,draw] {$f$};
		\node(7) at (34,-3.5) [fill = blue, circle,draw] {$u$};

	\tikzset{LabelStyle/.style={sloped, above,yshift = 0cm, xshift=.0cm}}
	\Edge[label={$\eta$}, style={bend left=30}](5)(4)
	\Edge[label={$\phi$}, style={bend left=30}](4)(6)
	\tikzset{LabelStyle/.style={sloped, below,yshift = -0cm, xshift=.0cm}}
	\Edge[label={$\delta$}, style={bend left=30}](6)(7)
	\Edge[label={$\mu_d$}, style={bend left=30}](7)(5)

	\end{tikzpicture}
\end{center}
\caption{Schematic representation of \emph{de novo} methylation and the active demethylation loop, where we use the following notation for the methylation states: (Unmethylated) C is   denoted by $u$, 5mC by $m$, 5hmC by $h$, and 5fC or 5caC by $f$.
The corresponding enzymatic reaction rates are $\mu_d$ (\emph{de novo} methylation), $\eta$ (oxidation), $\phi$
(formylation), and $\delta$ (demethylation).}
\label{Fig:MethCycle}
\end{figure}

\subsection{Hybrid Markov model}
In this section, we present a model that describes the state changes of a  single CpG over time. It can be seen as a hybrid extension of
previous discrete-time models \cite{arand2012vivo,Giehr2016}.
The (hidden) states of a CpG correspond to the set of all pairs of the four possibilities in Fig.   \ref{Fig:MethCycle}, i.e., $\{u,m,h,f\}^2$, because it contains a C on both strands of the DNA.
We split the transitions of our model into transitions that occur at  fixed times and those that occur at random times.
This results in a mixture of a discrete time Markov chain (DTMC) and a continuous time Markov chain (CTMC).
In the following we will refer to the events or transitions that occur at   fixed time points as \emph{discrete part} of the model, while we refer to the other events or transitions at the random time points as \emph{continuous part} of the model.

We assume that  cells divide after  a fixed time interval (usually every 24 hours). Hence, these events correspond to deterministic transitions at fixed times.
During   cell division one strand is kept as it is (parental strand) and all methylation states and its modifications remain unchanged, while one DNA strand is newly synthesized (daughter strand) and therefore contains only unmethylated cytosine.
Consequently, after cell divison a CpG that was modified on both sides becomes a CpG that is unmodified on one side.
Since the parental strand is chosen at random, the probability for each of the two successor states (corresponding to the state of the CpG in the two daughter cells)   is 0.5.
The full transition probability matrix $\mathbf{D}$ for  cell division  is shown in Tab.~\ref{Tab:D}. Note that the cell division matrix is time independent.

\renewcommand{\arraystretch}{1.25}
\setlength{\tabcolsep}{3pt}
\begin{table}[tb]
\caption{Cell division matrix $\mathbf{D}$.}
\label{Tab:D}
\begin{center}
\begin{tabular}{c|cccccccccccccccc}
~ & $uu$& $um$ & $uh$ & $uf$ & $mu$ & $mm$ & $mh$ & $mf$ & $hu$ & $hm$ & $hh$ & $hf$ & $fu$ & $fm$ & $fh$ & $ff$ \\\hline
$uu$ & 1 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$um$ & 1/2 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$uh$ & 1/2 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$uf$ & 1/2 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$mu$ & 1/2 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$mm$ & 0 & 1/2 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$mh$ & 0 & 0 & 1/2 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$mf$ & 0 & 0 & 0 & 1/2 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$hu$ & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$hm$ & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$hh$ & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$hf$ & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0\\
$fu$ & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0\\
$fm$ & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0\\
$fh$ & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0\\
$ff$ & 0 & 0 & 0 & 1/2 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 1/2 & 0 & 0 & 0\\
\end{tabular}
\end{center}
\end{table}

Maintenance methylation, i.e. methylation events that
occur on hemimethylated CpGs to reestablish methylation patterns, is known to be linked to the replication fork \cite{leonhardt1992targeting}.
We therefore consider   maintenance to occur together with the cell division at the same fixed time points.
Hence, cell division and maintenance can be described by a (discrete-time) Markov chain whose transition probability matrix $\mathbf{P}(t)$ is defined in the sequel in Eq.~\eqref{eq:P}.
Maintenance may happen at the daughter strand if there is a methylated C on the parental strand, i.e. on hemimethylated CpGs ($um$ or $mu$), with probability $\mu_m(t)$.
Since it is reasonable to assume that hemihydroxylated CpGs ($uh$ or $hu$) have different properties compared to hemimethylated CpGs in terms of maintaining existing methylation patterns, we describe the maintenance probability for hemihydroxylated CpGs as follows.
Let $p$ be the probability that 5hmC is recognized as unmethylated by maintenance enzymes, i.e., the enzyme will not perform maintenance of a hemihydroxylated CpG.
Then, the maintenance probability of such a CpG  is  given by $\bar{p}\mu_m(t)$, where $\bar{p}=1-p$.
Note that there is no equivalent probability for $uf$ or $fu$, i.e. we assume that  CpGs with 5fC or 5caC at one strand are not maintained \cite{ji2014effects}.
 The transition probability matrix for maintenance events
 is illustrated in Fig.~\ref{Fig:M}, where we omitted the time dependency of $\mu_m(t)$.
Whenever no transition is possible, i.e. there is only a self loop for this state (omitted in Fig.~\ref{Fig:M}) the corresponding diagonal entry in the matrix is 1.
Where a transition is possible we set the corresponding (off-diagonal) entry in the matrix to its transition probability and the diagonal entry to 1 minus its transition probability.
All other entries in the matrix are 0.
\begin{figure}[tb]
\begin{center}
\includegraphics[scale=0.35]{maint}
\end{center}
\caption{Maintenance methylation events
occur at (fixed) times $t_1,t_2,\ldots,t_n$ and belong therefore to the disrete part of the model.
 Each state is represented by two colored dots, one for each C on the two strands of the DNA. Unmethylated C is blue, 5mC red, 5hmC yellow and 5fmC cyan. The arrows indicate the possible transitions. Note that we omitted the self loops with probability $1$ for states where no transition is possible. The four shown self loops have probability $1-\mu_m$ or $1-\bar{p}\mu_m$ respectively.}
\label{Fig:M}
\end{figure}
One discrete step of the corresponding DTMC corresponds to one cell division, including maintenance methylation.
Hence, its transition probability matrix is defined as
\begin{equation}
\mathbf{P}(t)=\mathbf{D}\cdot\mathbf{M}(t).
\label{eq:P}
\end{equation}

Every other event may occur an arbitrary (unknown) number of times between two cell divisions at random time points and will
be described by  a continuous-time Markov jump process.
These events are \emph{de novo} methylation ($u\rightarrow m$) with rate $\mu_d(t)$, hydroxylation ($m\rightarrow h$) with rate $\eta(t)$,
formylation ($h\rightarrow f$) with rate $\phi(t)$ and active demethylation ($f\rightarrow u$) with rate $\delta(t)$ (cf. Fig~\ref{Fig:MethCycle}).
Note that all these events may happen on both strands, independent of the state on the complementary strand.
All possible reactions are shown in Fig.~\ref{Fig:Q}.
From these transitions the infinitesimal generator matrix $\mathbf{Q}(t)$ of the jump process can easily be inferred.
For the off-diagonal elements, we set the entries to the respective reaction rate if a reaction is possible between two states, indicated by a colored arrow in Fig.~\ref{Fig:Q}, and to $0$ if no reaction is possible.
The diagonal elements are then given by the negative sum of the off-diagonal elements of the respective row.
\begin{figure}[tb]
\begin{center}
\includegraphics[scale=0.45]{QMatrix}
\end{center}
\caption{Possible transitions in the continuous part of the model. Each state is represented by two colored dots, one for each C on the two strands of the DNA. Unmethylated C is blue, 5mC red, 5hmC yellow and 5fmC cyan. The arrows indicate the possible transitions, whereupon the colors indicate the different reactions and their rates, i.e. methylation with rate $\mu_d$ (red), oxidation with rate $\eta$ (yellow), formylation with rate $\phi$ (cyan) and active demethylation with rate $\delta$ (blue).}
\label{Fig:Q}
\end{figure}

 In order to describe the time evolution let us first define the set of time points $T_d=\{t_1,t_2,\ldots,t_n\}$, at which     cell division and maintenance occur.
We assume that there are in total $n$ of these events.
At these time points $t_i$ the time evolution of the probability distribution of the states is given by
\begin{equation}
\pi(t_i+\Delta t)=\pi(t_i)\cdot\mathbf{P}(t_i),
\label{Eq:mult}
\end{equation}
where $\Delta t$ is the duration of cell division and maintenance.
After cell division and maintenance the other events take place at random time points until the time point for the next cell division is reached.
During this interval $[t_i+\Delta t,t_{i+1}]$ the time evolution for $\pi(t)$ is obtained by solving the differential equation
\begin{equation}
\frac{d}{dt}\pi(t)=\pi(t)\cdot\mathbf{Q}(t).
\label{Eq:int}
\end{equation}
Note that since cell division and maintenance methylation occur at a time interval that is much shorter  than the time between two cell divisions, we assume here that these events occur instantaneously, i.e. we let $\Delta t\to 0$, which leads to a jump of the distribution at $t_i$.
It holds that the left-hand and right-hand limit do not coincide (except for the special case of only $uu$ at time $t_i$), i.e.
\begin{equation}
\lim_{t\rightarrow t_i^{-}}\pi(t)=\pi(t_i^-)\neq\pi(t_i^-)\cdot\mathbf{P}(t_i^-)=\pi(t_i^+)=\lim_{t\rightarrow t_i^{+}}\pi(t).
\end{equation}
To resolve the ambiguity we set the value of $\pi$ at time $t_i$ to $\pi(t_i):=\lim_{t\rightarrow t_i^{+}}\pi(t)$, which means that we assume that at time $t_i$ the cell division and maintenance methylation have already happened.
Intuitively, we can obtain the solution for $\pi(t)$ numerically by alternating between multiplication with $\mathbf{P}$ (Eq.~\eqref{Eq:mult}) and integration of Eq.~\eqref{Eq:int}.

\subsection{Efficiencies}
As already discussed in \cite{Giehr2016} the methylation or modification rates may change over time, i.e. with the assumption of constant rates the behavior of the system can not be captured correctly.
We therefore introduce time dependent rates, which we call efficiencies.
The simplest way of making the efficiencies time dependent is to impose a linear form.
Let $r\in \{\mu_m,\mu_d,\eta,\phi,\delta\}$ be one of the reaction rates.
For each reaction we then define
\begin{equation}
r(t):=\alpha_r+\beta_r\cdot t,
\end{equation}
where $\alpha_r$ and $\beta_r$ are some new parameters to characterize the linear efficiency function.
To further compress the notation we write these new parameters into a vector $\vec{v}_r=(\alpha_r,\beta_r)$.

Note that in order to ensure identifiability of the parameters we have to introduce certain constrains.
Obviously since $\mu_m(t)$ and $p$ are probabilities, they have to be bound between zero and one, i.e. $0\leq\mu_m(t)\leq 1$ for all $t\in[0,t_{max}]$ and $0\leq p\leq 1$.
The other efficiencies have to be bound by some upper limit $ub$ since otherwise the methylation cycle may become arbitrarily fast and the optimization algorithm runs into identifiability problems.
We therefore require $0\leq r(t)\leq ub$ for all $t\in[0,t_{max}]$, where $r(t)\neq \mu_m(t)$.
Since typical average turnover times $\mathbb{E}[T_{turnover}]=\mu_d^{-1}+\eta^{-1}+\phi^{-1}+\delta^{-1}$ of the methylation cycle are in the order of $75$ to $120$ minutes in certain promotors of human cells \cite{kangaspeska2008transient,metivier2008cyclical}, a viable choice would be, for example, $ub=12$, i.e. each modification occurs on average not more frequent than 12 times per hour (not faster than every $5$ minutes).

\subsection{Conversion errors}
The actual state of a CpG can not be directly observed.
We therefore have to estimate the hidden states from sequencing experiments.
Since the different modifications of C might lead to the same observable states we perform three different kinds of sequencing experiments:
All sequencing strategies share a bisulfite treatment, which usually converts C and its oxidized variants 5fC and 5caC, summarized to 5fC$^*$, to uracil.
Additionally, in \emph{bisulfite sequencing} (BS) both 5mC and 5hmC remain unconverted
while in {oxidative bisulfite sequencing} (oxBS) only 5mC is
retained as C \cite{booth2012quantitative}.
A combination of both methods can therefore be used to estimate the amount of 5hmC.
In \emph{M. SssI assisted bisulfite sequencing} (MAB-Seq)
at first all Cs in a CpG context are methylated and afterwards BS is applied.
Thus, if all conversions would happen without any errors, only 5fC$^*$ would be converted to T.
In order to capture the methylation pattern of CpG position at both complementary DNA strands, the distinct chemical treatments were combined with hairpin sequencing \cite{giehr2018two,giehr2018hairpin,laird2004hairpin}.
Regular reactions with their respective probabilities are marked with solid black arrows in Fig.~\ref{Fig:ConvErr}, while the possible false reactions are depicted with dashed red arrows.
Since every CpG consists of two Cs (one on each strand) with independent conversion errors, we get the conversion error for CpGs by multiplying the individual conversion errors.
A complete overview of all possible combinations for each of the three methods is shown in Tab.~\ref{Tab:ConvErr}.

\begin{figure}[bt]
\begin{center}
\begin{tikzpicture}[scale=0.90, every node/.style={scale=0.90}]
  \matrix (m) [matrix of math nodes,row sep=3em,column sep=0.1em,minimum width=3em]
  {\text{\textbf{BS}} & ~& ~& ~& \text{\textbf{oxBS}} & ~& ~& ~& \text{\textbf{MAB-Seq}} & ~& ~& ~\\[-2.9em]
  ~& ~& ~& ~& [1em]& ~& ~& ~ & [1em]\text{C} & ~ & ~ &~ \\[-1.5em]
   \text{C} & 5\text{mC} & 5\text{hmC} & 5\text{fC}^* & \text{C} & 5\text{mC} & 5\text{hmC} & 5\text{fC}^* & \text{C} & 5\text{mC} & 5\text{hmC} & 5\text{fC}^* \\
     \text{U} & 5\text{mC} & 5\text{hmC} & 5\text{fU}^* & \text{U} & 5\text{mC} & 5\text{fU} & 5\text{fU}^* & \text{U} & 5\text{mC} & 5\text{hmC} & 5\text{fU}^* \\[-1.5em]
     \text{T} & \text{C} & \text{C} & \text{T} & \text{T} & \text{C} & \text{T} & \text{T} & \text{T} & \text{C} & \text{C} & \text{T} \\};
  \path[-stealth]
    (m-3-1) edge node [left] {$c$} (m-4-1)
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    (m-3-4) edge node [left] {$g$} (m-4-4)
    (m-3-5) edge node [left] {$c$} (m-4-5)
    (m-3-6) edge node [left] {$d$} (m-4-6)
    (m-3-7) edge node [left] {$f$} (m-4-7)
    (m-3-8) edge node [left] {$g$} (m-4-8)
    (m-3-9) edge node [left] {$c$} (m-4-9)
    (m-3-10) edge node [left] {$d$} (m-4-10)
    (m-3-11) edge node [left] {$e$} (m-4-11)
    (m-3-12) edge node [left] {$g$} (m-4-12)

    (m-4-1) edge node [left] {} (m-5-1)
    (m-4-2) edge node [left] {} (m-5-2)
    (m-4-3) edge node [left] {} (m-5-3)
    (m-4-4) edge node [left] {} (m-5-4)
    (m-4-5) edge node [left] {} (m-5-5)
    (m-4-6) edge node [left] {} (m-5-6)
    (m-4-7) edge node [left] {} (m-5-7)
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    (m-4-9) edge node [left] {} (m-5-9)
    (m-4-10) edge node [left] {} (m-5-10)
    (m-4-11) edge node [left] {} (m-5-11)
    (m-4-12) edge node [left] {} (m-5-12)

    (m-3-1) edge [red,dashed] node [right=0.25cm] {}  (m-4-2)
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    (m-3-3) edge [red,dashed] node [right=0.25cm] {}  (m-4-1)
    (m-3-4) edge [red,dashed] node [right=0.25cm] {}  (m-4-2)
    (m-3-5) edge [red,dashed] node [right=0.25cm] {}  (m-4-6)
    (m-3-6) edge [red,dashed] node [right=0.25cm] {}  (m-4-5)
    (m-3-7) edge [red,dashed] node [right=0.25cm] {}  (m-4-6)
    (m-3-8) edge [red,dashed] node [right=0.25cm] {}  (m-4-6)
    (m-3-9) edge [red,dashed] node [right=0.25cm] {}  (m-4-10)
    (m-3-10) edge [red,dashed] node [right=0.25cm] {}  (m-4-9)
    (m-3-11) edge [red,dashed] node [right=0.25cm] {}  (m-4-9)
    (m-3-12) edge [red,dashed] node [right=0.25cm] {}  (m-4-10)

    (m-2-9) edge node [right] {$\mu$} (m-3-10)
    (m-2-9) edge [red,dashed] node [right=0.25cm] {}  (m-3-9);
\end{tikzpicture}
\end{center}
\caption{Cytosine conversions during chemical treatment and sequencing.
The correct reactions with their respective probabilities are marked with black arrows
, while the false reactions are shown with red dashed arrows. The probability for a false reaction is 1-``rate of correct reaction".}
\label{Fig:ConvErr}
\end{figure}

\renewcommand{\arraystretch}{1}
\setlength{\tabcolsep}{0.5pt}
\begin{table}
\begin{center}
\caption{Conversion errors for CpGs, where the rates for single cytosines are defined in Fig.~\ref{Fig:ConvErr}. We define $\bar{x}:=1-x$. Note that for MAB-Seq we also define $j:=\mu d+(1-\mu)(1-c)$.}
\label{Tab:ConvErr}
\begin{tabular}{c|cccc|cccc|cccc}
~ & \multicolumn{4}{|c}{bisulfite seq. (BS)} & \multicolumn{4}{|c}{ox. bisulfite seq. (oxBS)} & \multicolumn{4}{|c}{MAB-Seq}\\
~ & ~~TT~~ & ~~TC~~ & ~~CT~~ & ~~CC~~ & ~~TT~~ & ~~TC~~ & ~~CT~~ & ~~CC~~ & ~~TT~~ & ~~TC~~ & ~~CT~~ & ~~CC~~ \\\hline
$~~uu~~$ & $c^2$ & $c\!\cdot\!\bar{c}$ & $c\!\cdot\!\bar{c}$ & $\bar{c}^2$ & $c^2$ & $c\!\cdot\!\bar{c}$ & $c\!\cdot\!\bar{c}$ & $\bar{c}^2$ & $\bar{j}^2$ & $j\!\cdot\!\bar{j}$ & $j\!\cdot\!\bar{j}$ & $j^2$ \\
$um$ & $c\!\cdot\!\bar{d}$ & $c\!\cdot\!d$ & $\bar{c}\!\cdot\!\bar{d}$ & $\bar{c}\!\cdot\!d$ & $c\!\cdot\!\bar{d}$ & $c\!\cdot\!d$ & $\bar{c}\!\cdot\!\bar{d}$ & $\bar{c}\!\cdot\!d$ & $\bar{j}\!\cdot\!\bar{d}$ & $\bar{j}\!\cdot\!d$ & $j\!\cdot\!\bar{d}$ & $j\!\cdot\!d$ \\
$uh$ & $c\!\cdot\!\bar{e}$ & $c\!\cdot\!e$ & $\bar{c}\!\cdot\!\bar{e}$ & $\bar{c}\!\cdot\!e$ & $c\!\cdot\!f$ & $c\!\cdot\!\bar{f}$ & $\bar{c}\!\cdot\!f$ & $\bar{c}\!\cdot\!\bar{f}$ & $\bar{j}\!\cdot\!\bar{e}$ & $\bar{j}\!\cdot\!e$ & $j\!\cdot\!\bar{e}$ & $j\!\cdot\!e$ \\
$uf$ & $c\!\cdot\!g$ & $c\!\cdot\!\bar{g}$ & $\bar{c}\!\cdot\!g$ & $\bar{c}\!\cdot\!\bar{g}$ & $c\!\cdot\!g$ & $c\!\cdot\!\bar{g}$ & $\bar{c}\!\cdot\!g$ & $\bar{c}\!\cdot\!\bar{g}$ & $\bar{j}\!\cdot\!g$ & $\bar{j}\!\cdot\!\bar{g}$ & $j\!\cdot\!g$ & $j\!\cdot\!\bar{g}$ \\
$mu$ & $c\!\cdot\!\bar{d}$ & $\bar{c}\!\cdot\!\bar{d}$ & $c\!\cdot\!d$ & $\bar{c}\!\cdot\!d$ & $c\!\cdot\!\bar{d}$ & $\bar{c}\!\cdot\!\bar{d}$ & $c\!\cdot\!d$ & $\bar{c}\!\cdot\!d$ & $\bar{j}\!\cdot\!\bar{d}$ & $j\!\cdot\!\bar{d}$ & $\bar{j}\!\cdot\!d$ & $j\!\cdot\!d$ \\
$mm$ & $\bar{d}^2$ & $d\!\cdot\!\bar{d}$ & $d\!\cdot\!\bar{d}$ & $d^2$ & $\bar{d}^2$ & $d\!\cdot\!\bar{d}$ & $d\!\cdot\!\bar{d}$ & $d^2$ & $\bar{d}^2$ & $d\!\cdot\!\bar{d}$ & $d\!\cdot\!\bar{d}$ & $d^2$ \\
$mh$ & $\bar{d}\!\cdot\!\bar{e}$ & $\bar{d}\!\cdot\!e$ & $d\!\cdot\!\bar{e}$ & $d\!\cdot\!e$ & $\bar{d}\!\cdot\!f$ & $\bar{d}\!\cdot\!\bar{f}$ & $d\!\cdot\!f$ & $d\!\cdot\!\bar{f}$ & $\bar{d}\!\cdot\!\bar{e}$ & $\bar{d}\!\cdot\!e$ & $d\!\cdot\!\bar{e}$ & $d\!\cdot\!e$ \\
$mf$ & $\bar{d}\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!g$ & $d\!\cdot\!\bar{g}$ & $\bar{d}\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!g$ & $d\!\cdot\!\bar{g}$ & $\bar{d}\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!g$ & $d\!\cdot\!\bar{g}$ \\
$hu$ & $c\!\cdot\!\bar{e}$ & $\bar{c}\!\cdot\!\bar{e}$ & $c\!\cdot\!e$ & $\bar{c}\!\cdot\!e$ & $c\!\cdot\!f$ & $\bar{c}\!\cdot\!f$ & $c\!\cdot\!\bar{f}$ & $\bar{c}\!\cdot\!\bar{f}$ & $\bar{j}\!\cdot\!\bar{e}$ & $j\!\cdot\!\bar{e}$ & $\bar{j}\!\cdot\!e$ & $j\!\cdot\!e$ \\
$hm$ & $\bar{d}\!\cdot\!\bar{e}$ & $d\!\cdot\!\bar{e}$ & $\bar{d}\!\cdot\!e$ & $d\!\cdot\!e$ & $\bar{d}\!\cdot\!f$ & $d\!\cdot\!f$ & $\bar{d}\!\cdot\!\bar{f}$ & $d\!\cdot\!\bar{f}$ & $\bar{d}\!\cdot\!\bar{e}$ & $d\!\cdot\!\bar{e}$ & $\bar{d}\!\cdot\!e$ & $d\!\cdot\!e$ \\
$hh$ & $\bar{e}^2$ & $e\!\cdot\!\bar{e}$ & $e\!\cdot\!\bar{e}$ & $e^2$ & $f^2$ & $f\!\cdot\!\bar{f}$ & $f\!\cdot\!\bar{f}$ & $\bar{f}^2$ & $\bar{e}^2$ & $e\!\cdot\!\bar{e}$ & $e\!\cdot\!\bar{e}$ & $e^2$ \\
$hf$ & $\bar{e}\!\cdot\!g$ & $\bar{e}\!\cdot\!\bar{g}$ & $e\!\cdot\!g$ & $e\!\cdot\!\bar{g}$ & $f\!\cdot\!g$ & $f\!\cdot\!\bar{g}$ & $\bar{f}\!\cdot\!g$ & $\bar{f}\!\cdot\!\bar{g}$ & $\bar{e}\!\cdot\!g$ & $\bar{e}\!\cdot\!\bar{g}$ & $e\!\cdot\!g$ & $e\!\cdot\!\bar{g}$ \\
$fu$ & $c\!\cdot\!g$ & $\bar{c}\!\cdot\!g$ & $c\!\cdot\!\bar{g}$ & $\bar{c}\!\cdot\!\bar{g}$ & $c\!\cdot\!g$ & $\bar{c}\!\cdot\!g$ & $c\!\cdot\!\bar{g}$ & $\bar{c}\!\cdot\!\bar{g}$ & $\bar{j}\!\cdot\!g$ & $j\!\cdot\!g$ & $\bar{j}\!\cdot\!\bar{g}$ & $j\!\cdot\!\bar{g}$ \\
$fm$ & $\bar{d}\!\cdot\!g$ & $d\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!\bar{g}$ & $\bar{d}\!\cdot\!g$ & $d\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!\bar{g}$ & $\bar{d}\!\cdot\!g$ & $d\!\cdot\!g$ & $\bar{d}\!\cdot\!\bar{g}$ & $d\!\cdot\!\bar{g}$ \\
$fh$ & $\bar{e}\!\cdot\!g$ & $e\!\cdot\!g$ & $\bar{e}\!\cdot\!\bar{g}$ & $e\!\cdot\!\bar{g}$ & $f\!\cdot\!g$ & $\bar{f}\!\cdot\!g$ & $f\!\cdot\!\bar{g}$ & $\bar{f}\!\cdot\!\bar{g}$ & $\bar{e}\!\cdot\!g$ & $e\!\cdot\!g$ & $\bar{e}\!\cdot\!\bar{g}$ & $e\!\cdot\!\bar{g}$ \\
$ff$ & $g^2$ & $g\!\cdot\!\bar{g}$ & $g\!\cdot\!\bar{g}$ & $\bar{g}^2$ & $g^2$ & $g\!\cdot\!\bar{g}$ & $g\!\cdot\!\bar{g}$ & $\bar{g}^2$ & $g^2$ & $g\!\cdot\!\bar{g}$ & $g\!\cdot\!\bar{g}$ & $\bar{g}^2$ \\
\end{tabular}
\end{center}
\end{table}

\subsection{Parameter estimation}

Recall that the set of hidden states is $\mathcal{S}=\{uu, um , uh , uf , mu , mm , mh , mf , hu,$ $ hm , hh , hf , fu , fm , fh , ff \}$.
We now define a hidden Markov model (HMM) based on the
model presented in Section 2.2.
As set of observable states we define $\mathcal{S}_{obs}=\{TT,TC,$ $CT, CC\}$, i.e. we use the results of the sequencing experiments (cf. Fig. \ref{Fig:ConvErr}) on both strands. The conversion errors define the corresponding emission probabilities.
We also define $n_e(j,t)$ as the number of times that state $j\in S_{obs}$ has been observed during independent measurements of sequencing method $e\in E:=\{\text{BS, oxBS, MAB-Seq}\}$.
The probability distribution over all observable states for experiment $e$ is denoted by $\pi_e(t)$, the probability of a state $j\in S_{obs}$ by $\pi_e(j,t)$ and in a similar fashion we denote $\pi(i,t)$ with $i \in S $ for the hidden states, with   probability distribution $\pi(t)$.
The observable and hidden states for all times $t$ are connected via
\begin{equation}
\pi_{e}(t)=\pi(t)\cdot E_{e},
\end{equation}
where $E_e$ is the emission matrix for sequencing method $e$ and is listed in Tab.~\ref{Tab:ConvErr} for each of the three methods.

Our goal is to estimate the efficiencies for the different methylation events given our hybrid HMM and data from the three different experiments at different time points $t\in T_{obs}$ via a maximum likelihood estimator (MLE).
Since an initial distribution over the hidden states, which can not directly be observed, is needed in order to initialize the model, we have to employ the MLE twice:
First we estimate the initial distribution $\pi(0)$ over the hidden states by maximizing
\begin{equation}
\pi(0)^*=\text{argmax}_{\pi(0)}\mathcal{L}_1(\pi(0)),
\end{equation}
under the constrain that $\sum_{i\in S} \pi(i,0)=1$.
The likelihood $\mathcal{L}_1(\pi(0))$ is defined as
\begin{equation}
\mathcal{L}_1(\pi(0))=\prod_{e \in E} \prod_{j \in \mathcal{S}_{obs}} \pi_e(j,0)^{n_e(j,0)}.
\label{Eq:L1}
\end{equation}
Note that Eq.~\eqref{Eq:L1} is independent of the parameters.
Given an initial distribution over the hidden states we can now run our model and apply the MLE
\begin{equation}
\vec{v}^*=\text{argmax}_{\vec{v}}\mathcal{L}_2(\vec{v}),
\end{equation}
a second time in order to estimate the efficiencies, where
\begin{equation}
\mathcal{L}_2(\vec{v})=\prod_{e \in E} \prod_{t \in T_{obs}\backslash \{ 0 \} } \prod_{j \in \mathcal{S}_{obs}} \pi_e(j,t)^{n_e(j,t)}.
\end{equation}
The vector $\vec{v}=(\vec{v}_{\mu_m},\vec{v}_{\mu_d},\vec{v}_{\eta},\vec{v}_{\phi},\vec{v}_{\delta},p)$ contains all unknown parameters for all efficiencies and the probability $p$ of considering a hydroxylated cytosine as unmethylated.
Note that applying the MLE twice and independently leads only to an approximation of the true most likely explanation, since the estimated initial distribution may not lead to the same result in the parameter estimation, as if it would all be done in one estimation.
However, we choose this approach to reduce the computational complexity of the optimization.
In order to estimate the standard deviations of the estimated parameters we use the observed Fisher information matrix \cite{efron1978assessing}.
The Fisher information is defined as $\mathcal{J}(\vec{v}^*)=-\mathcal{H}(\vec{v}^*)$, where $\vec{v}^*$ is the maximum likelihood estimate and $\mathcal{H}(\vec{v})=\nabla\nabla^T\log \mathcal{L}_2(\vec{v})$ the Hessian matrix of the log-likelihood.
The expected Fisher information is then given by $\mathcal{I}(\vec{v}^*)=\mathbb{E}[\mathcal{J}(\vec{v}^*)]$ and its inverse forms a lower bound for the covariance matrix.
Thus, we can approximate the standard deviation of all estimated parameters by $\sigma(\vec{v}^*)=\sqrt{\text{Var}(\vec{v}^*)}\approx\sqrt{\text{diag}(-\mathcal{H}^{-1}(\vec{v}^*))}$.

The implementation of the hybrid HMM and its analysis as explained above has been integrated into the  latest beta version of the H(O)TA tool \cite{Kyriakopoulos2017}. H(O)TA provides results for individual CpGs and also an aggregated profile across all analyzed CpGs.

\section{Results}
In the following, we will discuss the results after applying our model to data
derived from a short region at the single copy gene Afp (alpha fetoprotein), which contains 5 CpGs.
More precisely, we followed the DNA methylation changes during the adjustment of mouse embryonic stem cells (mESCs) towards 2i medium after previous long time cultivation under Serum/LIF conditions \cite{ficz2013fgf,Giehr2016}.
The Serum/ LIF-to-2i shift is a common model system which induces genome wide demethylation in mESCs including the Afp locus.

Here, the individual and aggregated H(O)TA  results  show a very similar behavior. Hence, we only present the aggregated results  in Fig.~\ref{Fig:Afp}.

\begin{figure}[t]
\begin{center}
\includegraphics[scale=0.37]{Afp}
\end{center}
\caption{Results for Afp. (a)-(c) Predicted frequencies (dashed lines) and frequencies obtained from sequencing experiments (solid lines) of the observable states for all three methods. (d) Estimated efficiencies with standard deviation. (e) Probabilities of the hidden states. (f) Detailed distribution of hydroxylated CpGs. (g) Detailed distribution of formalized CpGs.}
\label{Fig:Afp}
\end{figure}

Fig.~\ref{Fig:Afp} (a) - (c) shows a comparison of the actual measurements (solid lines) and the results from the model (dashed lines) of the time evolution for the four observable states for all three sequencing models.
The predictions and measurements are in good agreement.
In BS and oxBS over time the frequency of TT increases, while the frequency of CC decreases.
Moreover, the frequencies of TC and CT
show a temporal increase.
For
MAB-Seq the frequencies of all observable states remain quite constant, except for some small changes in early times.
This behavior can be explained by the changes over time in the efficiencies, which are shown in Fig.~\ref{Fig:Afp} (d).
The maintenance probability $\mu_m$ remains constant at about $0.8$, as well as the probability $p$ of considering 5hmC as unmethylated, which is constant by definition.
The estimation of $p$ gives $p=1$, i.e. 5hmC is always considered to be unmethylated and will not be recognized by Dnmt1 after replication which means that 5hmC leads to an impairment of Dnmt1 activity and a passive loss of DNA methylation with each replication.
The \emph{de novo} efficiency $\mu_d$ decreases over time, while the hydroxylation and
formylation efficiency $\eta$ and $\phi$ increase.
The demethylation efficiency $\delta$ is always 0.
Note, however, that standard deviation becomes very large for later time points due to insufficient data.
The efficiencies explain the behavior of the frequencies of the observable states and is even more evident for the hidden states shown in Fig.~\ref{Fig:Afp} (e):
Over time the probability of being fully or hemimethylated decreases, while the probability of being unmethylated, hydroxylated or formylated increases.
A more detailed look into hydroxylated and formylated states is shown in Fig.~\ref{Fig:Afp} (f) and (g).
Note that the combination of hydroxylation and formylation is only shown in
one of the two subplots, namely in (g).
The observed increase in 5hmC and 5fC/5caC is in accordance with the high oxidation efficiency of Tets in form of hydroxylation and formylation.
Previously, we showed using   a purely discrete HMM, that the presence of 5hmC leads to a block of Dnmt1 activity after replication, which we also observe in the present hybrid model \cite{Giehr2016}.
However, we now also observe an increase in higher oxidized cytosine variants, namely 5fC and 5caC which equally prevents methylation by Dnmt1.
Thus, we reason that the impact of Tet mediated oxidation of 5mC on DNA demethylation in the investigated system plays a much more important role than previously suggested \cite{von2016impairment}.

Considering the rather rapid periodic events of \emph{de novo} methylation and active demethylation, the chosen measurement time points are not ideal.
There is only information available at the end of each cell division cycle (one division within 24h), i.e.,
no information is given for the times between two cell divisions.
With measurements at time points between two cell divisions we would be able to distinguish if a CpG is in a certain state because it initially was, or because it ran through the full cycle, possibly multiple times. We would like to emphasize that with better data, i.e. with sufficiently many measurements between cell divisions, it will be possible to estimate all
 reaction efficiencies with better confidence.

\section{Conclusion}
We proposed a hybrid hidden Markov model which is able to successfully describe both,  events  such as cell division and maintenance methylation that occur at fixed times, and events that occur at random times,
such as \emph{de novo} methylation, oxidizations and active demethylation, according to a continuous-time Markov jump process.
To the best of our knowledge, this is the first model
that describes the dynamics of active demethylation, i.e., the active removal of the methyl group through
several enzymatic steps.
We applied our model to  data from mouse embryonic stem cells, which undergo a gradually loss of DNA methylation over time.
We were able to accurately predict the frequency of the observable states and the levels of the hidden states in all cases.
We were also able to predict the enzymatic reaction efficiencies based on a linear assumption for their time behavior.

As future work  we plan to apply our model to more
informative data such that all efficiencies of the
active demethylation cycle can be estimated with better confidence.
Moreover, we plan to allow different functional forms for the efficiencies since the linear form is  not flexible enough and does not allow to capture complex behaviors.
A suitable choice could be splines of different degrees and with a different number of knots.
However, in this case it is also necessary to perform model selection in order to prevent overfitting.
Another possible extension could be to investigate
potential neighborhood dependencies of the modified cytosines \cite{luck2017stochastic}.

\bibliographystyle{splncs03}

redpajama_set_name: RedPajamaArXiv


Introducing Cartoon Food TV's weird weekends. Every weekend we'll post some of the strangest animations we've found, so if you're into stuff that doesn't fit neatly into the usual categories and is just, well, weird you know where to find us.
Friday funnies continue with this cowboy caper by Tumbleweed animation. Character animation is lovely but look at the textures and compositing too. Great work.
Question: does the Dog wag the tail or does the tail wag the Dog?
Lovingly animated in 2 and a half D by the Talking Animals Studio, this tale of a Dog literally chasing his tail has the answer, sort of! A treat for animation and dog lovers everywhere!
In this cute animation, the adorable knitted bear and duck, know to their fans as U900, rock abbey road with their rendition of the Beatles Twist and Shout. Lovely stop motion animation and quirky visuals as always.
Pub Dog, a very cute animation from the ever brilliant Bird Box Studios. Timing, set-up and character animation are all excellent. Take a couple of minutes to enjoy before breaking off for a quick half!
The original Chipotle commercial pioneered the trend for brands to use groundbreaking animation that we see continuing with the much hyped John Lewis christmas spot. It's use of stop motion gave it a completely unique tone and look. Watch this fascinating behind the scenes video, as it takes us through the variety of techniques from 3D pre-viz to stop motion via 3D printing, to see meticulous process involved in bringing this beautifully crafted animation to the screen.


Tourists visit the Acropolis in Athens. Recent protests and more bad news about Greece's economic crisis are threatening its tourism industry.
Greece is trying to drag itself out of debt, while at the same time imposing an austerity program that is limiting growth. One of the country's best hopes for overcoming this paradox is the tourism industry, which employs about one-fifth of the Greek workforce and accounts for up to 18 percent of gross domestic product.
But media coverage of the Greek economic meltdown and the sometimes violent anti-austerity protests are threatening to keep tourists away. In yet another blow last week, Moody's Investors Service slashed the country's credit rating to junk status.
Nikolaos Kelaiditis runs a small travel agency in central Athens. A faded old poster of Mykonos, a popular island of whitewashed houses, hangs on his wall.
On a recent weekend afternoon, his office is empty until a couple of young Australians wander in looking for a ferry schedule.
"Explore your senses" is the slogan from the glory days of 2007, when Greece spent 40 million euros (nearly $49 million) on a tourism campaign. Three years later, the country is broke. It owes more than 300 billion euros and narrowly avoided defaulting on its debt last month when the International Monetary Fund and the European Union gave the country 110 billion euros in loans.
Kelaiditis says his own business has declined at least 10 percent this year. And some estimates from Greek hotel and hospitality organizations say hotel reservations are down as much as 30 percent nationwide.
Some parts of the country – such as Crete and Rhodes – have been hit especially hard.
In the 24 hours after three people were killed in a May 5 demonstration in central Athens, there were nearly 6,000 cancellations at hotels in the city.
Kelaiditis' clients called him, worried about the violent scenes they saw on TV. "The reality is not exactly like that," he says.
But in the glossy world of travel, perception is reality.
In Greece, there have been scenes of violence, protesters blocking cruise ships and travelers stranded at the airport.
Susan Neal of Clearwater, Fla., says she considered canceling her cruise in Greece after seeing TV news reports about the riots and the deaths.
"We just decided to go ahead and give it a shot, and it's all worked out great," she says.
Any tourism money lost right now hurts Greece at a crucial time, says Jens Bastian, a German economist who has lived in Greece for 13 years. He is a senior research fellow at the Hellenic Foundation for European and Foreign Policy.
"Tourism is the one sector where Greece can regain, most easily, most quickly, competitiveness. You don't do that by preventing people from coming to Athens or visiting the country and sending out a message that you're not welcome," Bastian says.
German politicians are not popular in Greece. Greeks see them as the instigators of austerity measures that will mean years of recession ahead. The German media has also played up the rift between the two countries.
And that seems to be reflected in the number of Germans avoiding holidays here.
Germans usually make up about 15 percent of visitors to Greece. But the Association of Greek Tourism Enterprises estimates that 300,000 of them – or about 12 percent of the Germans who come to Greece annually – will stay away this year. About 16 million travelers visit Greece each year.
So Greece's tourism ministry is trying to restore the country's image in Germany and beyond.
The ministry is promoting Greece with a video that shows whitewashed houses, swimmers in a blue sea, and familiar scenes of Greek dancing and ancient ruins.
These scenes are supposed to remind tourists that Greece hasn't changed, says Giorgos Alimonos of the Greek National Tourism Organization.
"We don't see tourists as clients. We [treat them as if we are] welcoming people into our family. So I think that beside what happened – which was very limited and very isolated – for Greece, tourism is business as usual," Alimonos says.
If Alimonos wants a German couple for the next Greek tourism video, he might try Gerd and Rita Ammereller, who recently visited the popular new Acropolis Museum in Athens.
The Ammerellers are social workers from the city of Bochum. They have visited Greece every May since the 1970s. Some friends tried to talk them out of coming to Greece this year, but Gerd Ammereller says he loves the family-run hotel on the island of Paros where they stay each year.
"It's a little bit like coming home. And they are so friendly and treat us so kindly, and it was a special Greek family atmosphere, and that's what we love," he says.
After the Ammerellers leave the museum, they pass a young busker from Crete named Yiannis Kapetanakis, who is playing a lyra.
"These days, a foreigner sees a crazed Greek, caught up in madness. He should be seeing a happy Greek who is smiling – but genuinely smiling," Kapetanakis says.


text:
Band Gallery
• P R E S S Q U O T E S •
"The Orange Circus Band appear to have been lifted from another time. They've attracted favourable attention from the likes of Bob Harris and Roger Daltrey, and on this showing, the plaudits from such greats of the music world are wholly justified."
Americana UK
"Rooted in Bluegrass and UK Folk Music, they're a wonderful bridge across the Atlantic. "
Whispering Bob Harris, OBE BBC 2 Country
"So much of country music misses the mark, but 'I Miss You' blindsided me...I loved it"
Adam Walton BBC Music Introducing
"Glorious slice of 'O Brother Where Art Thou' style Bluegrass from the UK. Great fiddle action and harmonies take you right to the Appalachians. The circle is still unbroken"
LYRIC MAGAZINE
"They're a brilliant mash-up of old-time Appalachia and British folk traditions, and just a good time party vibe. And they're spectacular live"
Ralph McLean - Country, BBC Radio Ulster
"This is an album that puts the band on the map as one of the leading Americana acts"
– CelebMix
We'll be playing a host of festivals this summer 2022 and have a new single coming out soon...
Enter your email to get a FREE download of 'CARMEN TOWN'
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text:
Places to Stay Near Ricketts Glen State Park in Pennsylvania
Hotels With Indoor Swimming Pools in Lancaster, Pennsylvania
Hotels Near Potato City in Potter County, Pennsylvania
Leah Rendon, Leaf Group
(Photo: Ryan McVay/Photodisc/Getty Images )
Hotel or Motels Near Kinzua State Park in Pennsylvania
Hotels Near the Sight & Sound Theatre in Lancaster, Pennsylvania
In a region ripe with potato fields, it's only fitting that the Potato City Country Inn once served as a sort of monument for the beloved spud. The building opened in 1949 as a laboratory for crossbreeding work and meeting point for farmers, manufacturers and packers of the potato industry. Set along bustling U.S. Route 6, the potato shrine is now a hotel amid a host of other lodging options.
Near and Dear
Potato City Country Inn provides basic motel rooms for rent and a handful of A-frame apartments with full kitchens. The elegant on-site steak and seafood restaurant also has a kids menu. Book a special golf package during your stay which includes 18 holes of play at nearby Coudersport Golf Club, less than 10 minutes from the inn. Sweden Valley Hotel and Cabins is the closest hotel to the Potato City Country Inn. The hotel has rooms and cabins available for rent by the day, week or month. Pets are allowed, and the hotel is attached to a pub.
Bedding Down in Ulysses
Just five minutes east of the Potato City Country Inn, Pine Log Motel is in the small town of Ulysses. Accommodations include rooms with full kitchens and dining areas. Rates are available on a daily or weekly basis. Relax in the spacious lobby at Susquehannock Lodge and Trail Center, home to a library and living room with fireplace. Book a rustic room inside the main lodge, carriage house or barn. Special packages include breakfast and dinner served family-style in the hotel's cozy restaurant, as well as a packed lunch. The hotel is just steps from the 85-mile Susquehannock Trail System, open to hikers and bikers.
Route 'Em
Laurelwood Inn is set right on Route 6. Enjoy a relaxing evening in the hotel's steakhouse and lounge, complete with a bar and fireplace. Free coffee is served all day in the lobby. Pets are allowed and children under age 12 stay free of charge. Millstream Inn is also on bustling Route 6. The hotel provides standard rooms and private cabins, all equipped with microwaves and refrigerators. Complimentary hot breakfast is served each morning. The hotel has picnic tables, pavilions and gas grills as well. Both hotels are a little more than five minutes west of Potato City Country Inn.
Coudersport Centric
Book a room near the center of Coudersport. Five minutes from downtown, Westgate Inn serves free continental breakfast in the morning. Some suites have views of the nearby Allegheny River. Pets weighing 30 pounds or less are allowed in the hotel. Just two minutes from the heart of Coudersport, Lindy Motel and Cottages is spread across 5 acres of property. Nestled along a trout-filled river, the hotel also provides horseshoe pits, a playground and picnic areas with fire pits. Both hotels are approximately 15 minutes from Potato City Country Inn.
Sweden Valley Hotel and Cabins
Susquehannock Lodge and Trail Center
Laurelwood Inn
Millstream Inn
Lindy Motel and Cottages
Visit Pennsylvania: Potato City Country Inn
A travel writer since 2002, Leah Rendon has written countless articles for online pop-culture magazines and various travel-related websites, including Eurobooking and World66. Gosford also has experience in copywriting, having developed a series of articles related to network marketing. She graduated from the University of California, Los Angeles with a degree in theater arts.
Spa Getaways in Pennsylvania
Restaurants in Blakeslee, Pennsylvania
Rustic Cabins in Cook Forest, Pennsylvania
Hotels Halfway Between Pittsburgh, Pennsylvania & Myrtle Beach, South Carolina
Motels and Lodging Near Coatesville, Pennsylvania
Hotel Types»
Hotels in Cities»


Improved street lighting is a form of situational crime prevention that involves increasing the levels of illumination on the street or in other public spaces. It is intended to serve many purposes, including accident prevention, marketing and the reduction of crime. This review covers crime reduction only.
This narrative is primarily based on one systematic review covering 13 studies.
Overall, the evidence suggests that the intervention can reduce crime. Across the 13 studies reviewed, both violent and property crime was reduced by an average of 21 per cent in areas with improved street lighting compared to areas without.
There were no studies for which a statistically significant backfire effect (where crime increased) was reported.
The review provides a description of some of the possible mechanisms through which street lighting might reduce crime, and the testable predictions generated from them.
increasing visibility and the number of people that use the street. This could lead to increased - or more effective - natural surveillance that may deter crime. If this mechanism explained how improved street lighting reduced crime in the studies considered, then the effects of street lighting should be greatest during the hours of darkness. However, in the case of the studies reviewed, for those that only examined the impact of improved street lighting on levels of crime at night (four evaluations), there was no evidence of an impact on crime. In contrast, those studies (9 in total) that examined changes in levels of crime during the day and night found a statistically significant impact on crime in the areas with improved street lighting, which suggests an alternative mechanism (see below).
demonstrating investment in the neighbourhood. This has the potential to improve community conditions that could plausibly lead to residents caring more about their neighbourhood and consequently being more likely to take actions that might reduce crime in them. The finding that improved street lighting did not have an impact on crime solely after dark is consistent with this explanation.
The review discusses the possibility that reductions are likely to be greater if the existing lighting is poor and if the improvement in lighting is considerable.
In the UK, an average of 38 fewer crimes were observed in areas with improved street lighting (based on 5 studies).
In the US, an average of 7 fewer crimes were observed in areas with improved street lighting (based on 8 studies).
For property crime, an average of 17 fewer crimes were observed in areas with improved street lighting (based on 9 studies).
For night-time crime only, no fewer crimes were observed in areas with improved street lighting (based on 4 studies, all conducted in the USA).
If improved street lighting impacts upon crime by influencing community pride, it may be most effective in stable but underinvested communities (see pages 5 and 19 of the review).
Although none of the 13 studies included in the systematic review found a statistically significant backfire effect, it is possible that increased visibility through improved street lighting could increase crime by enabling offenders to make better judgments of the vulnerability and attractiveness of potential targets.
In the overall review, it was not possible to examine geographic displacement or the diffusion of benefits to nearby locations. However, in one study (see page 16 of the review) the findings suggested that crime was statistically significantly reduced in the areas nearby.
The studies in this review did not consider the impact on crime of recent changes in lighting technology such as the introduction of LED lamps, or changes in lighting schedules such as part-night lighting.
Improvements in street lighting levels can be criticised for adversely affecting light pollution, sleep (including of animals) and energy efficiency.
Improved lighting levels might be necessary to the effective deployment of other crime prevention initiatives such as CCTV.
None of the UK studies covered in the review separated daytime crime from night-time crime in assessing effect. It is not, therefore, possible to conclude whether or not it was street lighting improvements that reduced crime, or whether these improvements facilitated other mechanisms such as community cohesion (as is suggested by the four US studies).
Overall, the evidence suggests that improved street lighting can reduce crime. Crime (violent and property) reduced by an average of 21% in treatment areas where street lighting was increased, relative to comparison areas without increased street lighting. Exactly how this effect is achieved remains currently unknown.
Welsh, B., and Farrington, D.F. (2008). Effects of Improved Street Lighting on Crime. Campbell Collaboration Systematic Review. Campbell Collaboration: Norway.


Georgia – a small country of 4.7 million on the Black Sea that receives 2 to 3 million tourists annually (Romania had about 6 million in 2010). This small state existed with it's flag, language, alphabet and form since the 11th century and after it made some rampage in the area it went on and off the charts being attached to various empires, the last being the Soviet Union. Though harassed by it's Russian neighbor (last comflict / invasion being in 2008) this small country is able to advance with big steps and fast, for which I admire it, especially comparing with what you see here in Romania. Home to some of the oldest Orthodox churches with a diversified relief (from cities with a Mediterranean feeling to mountains of 5000m height) and host the Golden Fleece legend this country cannot dissapoint it's visitors.


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The fed ends up investigating Trump Org and sends out money payments
Overdose of Drug Drop in U.S. in the First Time Since 1990
Amazon Prime Day 2019: There are still deals on Chromebooks, gaming laptops and MacBooks
Apollo 11: Neil Armstrong's golden medal that flew to Moon that was sold for over $ 2 million
Home https://server7.kproxy.com/servlet/redirect.srv/sruj/smyrwpoii/p2/ Entertainment https://server7.kproxy.com/servlet/redirect.srv/sruj/smyrwpoii/p2/ Disney Unveils Teaser for Idina Menzel, Kristen Bell's Frozen 2
Disney Unveils Teaser for Idina Menzel, Kristen Bell's Frozen 2
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Image: Disney (YouTube)
Trailer Frenzy A special place to find the newest trailers for movies and TV shows you're craving. ]
Disney has released the first teaser trailer for Frozen 2 bringing back Elsa, Anna, Olaf, and Kristoff in an adventure way outside the chilly comforts of Arendelle. Also, there's ice surfing.
The highly anticipated sequel to 2013's Frozen brings back Kristen Bell and Idina Menzel as Anna and Elsa, two sisters who were torn apart after Elsa's icy gifts were exposed to the world … only to be brought back together through love. According to the synopsis, in this latest film Queen Elsa and her sister will embark on an adventure that takes them to another land, far from the kingdom of Arendelle. Here's a look at the official teaser poster.
It's unclear what adventures lay in front of the sisters and their friends. But I'm wondering if it has to do with finding people with gifts like Elsa's-given how we get a glimpse of a new character out in the leaf-covered wilderness. Let's just hope it does not involve any more of Olaf's frozen adventures.
Frozen 2 arrives in theaters on November 22.
Kate Middleton Breaks the Royal Protocol Again By Working It In Wimbledon


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Revelations of a Strong Woman
by Queen Latifah with Karen Hunter
Pub Date: Jan. 1st, 1999
Publisher: Morrow/HarperCollins
The successful actress and rap singer offers a frank, down-to-earth, but somewhat unsurprising review of the hard life lessons she has learned, about self-respect, love, and independence. There's little here that may be startling ("Too many women look for what's in a man's pocket, not what's in his heart"; "If you're broken, no man is going to fix you"). But Latifah delivers her insights with a biographical frankness (dealing with her struggles with abusive relationships and questions of self-worth) and salty directness that her young female fans (for whom the book seems to be written) are likely to find engaging and convincing. (Author tour)


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NHS faces 'most dangerous situation anyone can remember', Chris Whitty warns
Coronavirus cases across the country are surging to record levels but there are concerns the public aren't following the rules as closely as during the first lockdown last year
New Government TV campaign urges public to stay at home
Aine Fox
England's chief medical officer has warned the NHS faces the "most dangerous situation" in living memory as the pandemic causes record deaths and hospital admissions.
As the country awaits the ramping up of coronavirus testing and vaccinations this week, Chris Whitty has said the only way to prevent avoidable deaths is for the public to stay home wherever possible.
"Hospitals are always busy in winter, but the NHS in some parts of the country is currently facing the most dangerous situation anyone can remember," Prof Whitty wrote in the Sunday Times.
"If the virus continues on this trajectory, hospitals will be in real difficulties, and soon.
"Staff-to-patient ratios - already stretched - will become unacceptable even in intensive care."
The number of patients with Covid-19 in hospital is at a record high in England, while the official coronavirus death toll for the UK passed 80,000 on Saturday and lab-confirmed cases hit more than three million.
The number of intensive care beds for the critically sick has been increased from 60 to 120 at St George's Hospital in London (
UK coronavirus death toll hits 80,000 after 1,035 die in second biggest Saturday rise
Coronavirus will 'never be eliminated and vaccine will be needed every year'
Prof Whitty commended the public for their efforts to stop the spread of Covid-19 and noted the hope offered by various vaccines, but he echoed other experts in saying it would be some weeks before the jabs start to reduce the number of people taken to hospital.
The Department of Health and Social Care (DHSC) has said that expanding the Community Testing Programme to more people without symptoms is "crucial given that around one in three people" who contract Covid-19 are asymptomatic.
DHSC said councils would be encouraged to test those unable to work from home during lockdown - a move likely to include police officers, supermarket workers and taxi drivers.
Prof Chris Whitty is fronting a new ad campaign warning of the dangers of the virus (
London and the South East of England are being hard-hit in the second wave (
Press Association Images)
Lateral flow tests, which can return results in as little as 30 minutes, are at the heart of the programme, the eligibility of which has now been "expanded to cover all 317 local authorities", DHSC said.
Health Secretary Matt Hancock said targeted asymptomatic testing followed by isolation was "highly effective in breaking chains of transmission".
But doctor Angela Raffle, a consultant in public health with the University of Bristol Medical School, said increasing lateral flow testing "is very worrying".
A patient is prepared for transfer from the Acute Dependency Unit to the ICU (
"Any benefit from finding symptomless cases will be outweighed by the many more infectious cases that are missed by these tests," she said.
"Already outbreaks are known to have occurred because people have been falsely reassured by a negative lateral flow result, leading them to attend work whilst having symptoms."
Meanwhile, as the vaccine rollout gathers pace, more than half a million over-80s are due to receive invites this week to sign up to receive a jab.
The NHS is under huge pressure as coronavirus infections surge (
Anadolu Agency via Getty Images)
With the number of coronavirus cases continuing to rise across the country, staff say they are working "to the limit" of their ability (
The first 130,000 invites were due to arrive over the weekend, as the Government strives to meet its target of offering inoculations to almost 14 million vulnerable people in the UK by mid-February.
Boris Johnson said: "There are deeply challenging weeks ahead, but today signals another significant step forward in the race to protect the public, and defeat the virus."
Some experts have branded the current lockdown measures not strict enough, in the face of the more transmissible variant which has spread rapidly in many parts of the country.
NHSHospitalsPoliticsCoronavirus


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Thailand Launches ASEAN's First 5G Smart Hospital
A Joint Launch by Siriraj, NBTC and Huawei
[Bangkok, Thailand, December 17, 2021] Yesterday, Office of The National Broadcasting and Telecommunications Commission (NBTC), Siriraj Hospital, and Huawei Technologies (Thailand) Co., Ltd. jointly launched the "Siriraj World Class 5G Smart Hospital". This is the first and largest 5G smart hospital project in the ASEAN region. It will deliver a more efficient and convenient experience to patients by introducing technologies such as 5G, cloud, and artificial intelligence. Meanwhile, Siriraj Hospital and Huawei will establish a Joint Innovation Lab to incubate over 30 innovative 5G applications that will be promoted nationwide from 2022.
Thailand Prime Minister Prayut Chan-o-cha visits the 5G remote OPD booth at the launch ceremony
General Prayut Chan-o-cha, Prime Minister, addressed the national policy on 5G and digital economy. "Thailand understands the importance of technology, and today is an important first step in the utilization of digital technologies and 5G in the medical field. We are thankful for the long-lasting friendship and collaboration between Thailand and China. We admire Siriraj Hospital and Mahidol University, and would like to thank Huawei, NBTC, and all other partners. We hope the project will act as a blueprint for all smart hospitals in Thailand going forward."
Han Zhiqiang, Ambassador of the People's Republic of China in Thailand, emphasized that China will leverage technology to help Thailand fight the pandemic. "China and Thailand's 5G cooperation has become a model in the region, helping Thailand become the first country in Southeast Asia to launch 5G commercial use. China will continue to support Huawei and other Chinese companies in advancing Smart Hospitals and bringing better lives for Thai and Chinese people."
Prof. Dr. Prasit Watanapa, MD, Dean of Faculty of Medicine, Siriraj Hospital Mahidol University, and Colonel Natee Sukonrat, Ph.D., Vice-Chairman of NBTC emphasized that with the "smart hospitals" model, people in remote areas will have better opportunities to access advanced health care services.
Abel Deng, CEO of Huawei Thailand, said, "This signifies a model for upgrading Thailand's public health industry in the future and contributes to Siriraj's transition to becoming a smart hospital, in line with Huawei's mission to Grow in Thailand, Contribute to Thailand."
Siriraj Hospital and Huawei signed a five-year MoU in December 2020 to accelerate the use of 5G and cloud technologies. In June, 5G unmanned vehicles were introduced for contactless medical supplies delivery. Last year, Siriraj was awarded by CommunicAsia for "Most Innovative 5G Trial in Asia Pacific Region."


Secret Celebrity which was called Kim Seybert Living Tree Skirt- Coastal Theme. Note: the white thin lining is protective only for the beading and will be removed. One Secret Celebrity/ Kim Seybert Ivory/Cream Coastal Themed Tree Skirt.
Edged in a neutral light cocoa color. Skirt is decorated with beaded patterns of a starfish & seashells. I also have 2 matching stockings in this same pattern for sale. Polyester fabric for fire safety. The Kim Seybert Living Collection brings elegant sophistication and exciting glamour to the world of home decor. An authority on luxury lifestyle entertaining Kim Seybert is renowned for her unique fashion inspired style and her. Ornate couture for the table designs featuring intricate beading, embroideries, and embellishments. For vibrant colors and rich textures, Kim creates beautiful pieces to mix, match, and layer to express your personal creativity. Please note: The word Kim Seybert is not on this item.
The item "SC Kim Seybert Ivory Beaded Christmas Tree Skirt Beach Coastal Neutral Pearl 62" is in sale since Thursday, December 6, 2018. This item is in the category "Home & Garden\Holiday & Seasonal Décor\Christmas & Winter\Tree Stands, Skirts & Storage". The seller is "littlebirdlinens2" and is located in Houston, Texas. This item can be shipped to United States, Canada, United Kingdom, Denmark, Romania, Slovakia, Bulgaria, Czech republic, Finland, Hungary, Latvia, Lithuania, Malta, Estonia, Australia, Greece, Portugal, Cyprus, Slovenia, Japan, China, Sweden, South Korea, Indonesia, Taiwan, South africa, Thailand, Belgium, France, Hong Kong, Ireland, Netherlands, Poland, Spain, Italy, Germany, Austria, Bahamas, Israel, Mexico, New Zealand, Philippines, Singapore, Switzerland, Norway, Saudi arabia, Ukraine, United arab emirates, Qatar, Kuwait, Bahrain, Croatia, Malaysia, Brazil, Chile, Colombia, Costa rica, Dominican republic, Panama, Trinidad and tobago, Guatemala, El salvador, Honduras, Jamaica, Antigua and barbuda, Aruba, Belize, Dominica, Grenada, Saint kitts and nevis, Saint lucia, Montserrat, Turks and caicos islands, Barbados, Bangladesh, Bermuda, Brunei darussalam, Bolivia, Ecuador, Egypt, French guiana, Guernsey, Gibraltar, Guadeloupe, Iceland, Jersey, Jordan, Cambodia, Cayman islands, Liechtenstein, Sri lanka, Luxembourg, Monaco, Macao, Martinique, Maldives, Nicaragua, Oman, Peru, Pakistan, Paraguay, Reunion, Viet nam.


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Who Was Featured On The Motown Labels First Gold Record In 1960?
Aun Jafery answered
The Motown labels first ever gold record in 1960 was known as "Shop Around". On its cover it featured four people the singer amongst whom was famous Smokey Robinson. Smokey Robinson's full name was William "Smokey" Robinson Junior. He was born on the 19th of February 1940. He has been active on the music scene since 1955 onwards. Smokey Robinson was born and raised in the city of Detroit in Michigan.
Motown was formed by Smokey Robinson and its record "Shop Around" achieved gold status when sales exceeded a million. Smokey Robinson is still today involved with the Miracles and is the Vice President of Motown Records. Other famous people involved with Motown records include Diana Ross, Marvin Gaye, Little Stevie Wonder, Martha and the Vandella etc.
Bil Nutt commented
Slight correction: Though Smokey Robinson was an important part of Motown, as a performer and songwriter, he didn't "form" the company. That would be Berry Gordy. Motown was one of the first hugely successful companies started by an African American. (It may be THE first, but I'm not positive.)
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I got into my boyfriends facebook by changing his password and the reason why is that there's something he's not telling me, on his messages there is one from a girl and they're saying bad things bout me but that was ages ago but was it bad what Idid?


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Professor held her student's baby during lecture so the mother could take notes
Ryan Prior	CNN Published Friday, September 27, 2019 10:13PM EDT
College professor Ramata Sissoko Cissé teaches an anatomy course with one of her student's children strapped to her back. (Georgia Gwinnett College)
(CNN) -- Last Thursday, when a Georgia college student couldn't find a babysitter, her professor volunteered to hold the student's baby while teaching a three-hour anatomy class.
A photo from that class is showing the world what kindness and compassion look like.
Ramata Sissoko Cissé, an assistant professor of biology at Georgia Gwinnett College in the Atlanta suburb of Lawrenceville, told CNN that one of her students called her late the night before class to let Cissé know that her babysitter was sick. Due to that, the student would have to bring her baby to Cissé's anatomy and physiology class the next day.
The professor, a mother of three, gave her the green light. "For her to trust me made me feel like I had to help," Cissé said. "It's like a moral responsibility."
But in class, the baby kept moving, and it was hard for the student to hold her son and write at the same time.
"I said, 'Hand me the baby,'" Cissé said.
However, Cissé couldn't hold the baby and write on the white board during her anatomy lecture.
The baby became a learning aid
So Cissé improvised. She found a white lab coat and tied the baby to her back.
The baby quickly fell asleep and stayed quiet for the rest of class. Cissé was able to incorporate the baby into the lesson, elaborating on concepts related to the nervous system, brain function and metabolism.
One student asked why he was able to sleep so quietly.
Cissé explained that he was cozy and warm, with her body temperature next to him, and the matching heat made it easier for him to relax.
Later when the baby became hungry, and the baby's bottle was cold, she explained that warming up the milk would help aid the baby's metabolism.
A spokeswoman for Georgia Gwinnett College told CNN that the child's mother asked to not be identified.
'I teach because they need to be prepared for life'
Cissé said the student e-mailed her after class to thank her, and she wrote back, "You're welcome, I'll always be there for you." And the student replied, "I know."
Those two words meant a lot to her.
Many of Cissé's students go on to become nurses or doctors, or work in other health professions. She said teaching them science is only part of her mission to prepare them for life after school.
"Love and compassion are part of the philosophy of my classroom," Cissé said.
She's hoping she can instill those qualities by modeling them in teachable moments like holding the baby during class as being a good health care provider is about more than just studying textbooks.
"I'm hoping they can spread love, take it to other people who need it," she said.
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Fernanda Suzano
My name is Fernanda Suzano, I am a senior technician at Prof Fiona Watt Lab in the Centre for Gene Therapy & Regenerative Medicine. I am originally from Sao Paulo- Brazil, I moved to London in 2012 after finishing my master's degree in Biotechnology of Medical Plants and Microorganisms.
After a challenging master's degree, I decided not to pursue a career in academic research but my passion for science persisted and I wanted to continue to work in the field in a different way. I moved to London partly as an adventure and partly to pursue a career as a laboratory technician, hoping to one day manage a lab. Following this path allows me to stay in the laboratory environment, surrounded by brilliant people, including many great women. Unfortunately, I have encountered challenges progressing in my field as I have only seen British men in higher management positions despite the majority of technicians being women. It can feel as if there is no assistance or interest in developing your career.
At the start of 2022, I moved into the Centre for Gene Therapy and Regenerative Medicine. I have been incredibly lucky to work with amazing women during this time. I take a great deal of inspiration from the head of the Watt Lab, Fiona Watt, in particular. Despite her high standing in the scientific community, she is very humble and ready to help you. She has cultured an understanding and inspiring community, particularly for the women in the lab who she is an advocate for. The scientific community would benefit from more women like her.
Women in Science →
A Photography Collaboration
"If you want science to move forward, you have to share it"
EMBO Director Fiona Watt discusses preprints, data sharing
By Victoria Yan, EMBO
Read full interview →
Fiona Watt & Alice Vickers on Stem Cell Report Podcast
Professor Fiona Watt and recently graduated Alice Vickers joined the Stem Cell Report Podcast with Martin Pera to talk about uncovering the relationship between genetic variants and cellular behaviour. They recently developed a platform to quantify iPSC differentiation propensity to investigate the genetic contribution to phenotypic variability.
next member →
Fiona Watt appointed as EMBO Director
Professor Fiona Watt has announced she will step down from the role as Executive Chair of the Medical Research Council in early 2022 to succeed current EMBO Director Maria Leptin. She has been a member of EMBL's Scientific Advisory Committee since 2016.
Professor Watt announced as government's preferred candidate for Executive Chair of the Medical Research Council (MRC)
Professor Watt has been selected as the government's preferred candidate to be the Executive Chair of the Medical Research Council (MRC) when it becomes a constituent part of UK Research and Innovation (UKRI) in April 2018.
Professor Watt leads the stem cell and cell signalling spoke of Centre for Cellular Signal Patterns (CellPAT)
The National Danish Research Foundation has announced that it will give 61 million DKK (£7 million) to fund the Centre for Cellular Signal Patterns (CellPAT) led by Professor Jorgen Kjems.
Honorary Doctorate Award for Professor Fiona Watt
Professor Watt was delighted to be invited to Spain recently to receive a Doctor Honoris Causa from UAM (Autonomous University of Madrid), an award which also honours their institution.
Professor Fiona Watt to receive Women in Science Award
Professor Watt has been announced as the winner of the 2016 FEBS | EMBO Women in Science Award. She's received the award for uncovering the mechanisms that control mammalian epidermal stem cell renewal and differentiation, and for discovering how these processes are deregulated in cancer, wound healing and inflammatory skin disorders; in addition to recognition for her commitment to gender issues, her leadership qualities and her active mentorship of junior scientists.
Professor Watt encourages international young scientists in London
Professor Watt was delighted to deliver the keynote opening lecture at the 2015 London International Youth Science Forum. The two week residential event held at Imperial College London attracts over 400 of the world's leading young scientists aged 17 - 21 years old from more than 60 participating countries.
Watt Lab on the BBC
Professor Fiona Watt and Dr Inês Sequeira were featured in a piece on the BBC in January, following a visit to our lab from a TV news crew.  Professor Watt was interviewed for the story on "Medical milestones of 2014". Watch the video on BBC online.
Professor Watt links with industry 'down under'
When Professor Watt visited Australia to deliver a keynote speech for the Sydney Cancer Conference, she was also invited by UK Trade & Investment (UKTI) Australia to give a seminar about one of the UK's '8 Great Technologies' - Regenerative Medicine, and opportunities for Australian companies working in this area.
New £2.3m research 'Hub' for regenerative medicine
Professor Fiona Watt will be the Director of a new 'Hub' to address important challenges in translational regenerative medicine.
Royal Society public lecture
Professor Fiona Watt was invited to give a public lecture at the Royal Society's central London headquarters on 10th February. She delivered her talk - "Stem cells and human health" - to a packed auditorium.
Watt Lab paper featured on cover of Nature
Professor Fiona Watt's lab has had a paper published in Nature – and is also featured on the cover.  The paper is about fibroblasts – a type of cell found in the connective tissue of the body's organs, where they produce proteins such as collagen that provide structural support.  It is widely believed that all fibroblasts are the same cell type.
King's College London award for Professor Watt
Professor Fiona Watt's contribution to the promotion of Stem Cell and Regenerative Medicine research was recognised at KCL's annual "King's Awards" ceremony last night.  Fiona was presented with her award for "Most Outstanding Contribution to Public Engagement" by KCL Principal, Professor Rick Trainor.
Professor Watt on Radio 4's "Inside Science"
Professor Fiona Watt was delighted to be invited to take part in BBC Radio 4's "Inside Science" programme on Thursday 12th September.  She spoke to presenter Adam Rutherford about the history of stem cell research and what the future holds for regenerative medicine.
King's appoints stem cell expert to lead new Centre
King's College London announced today that it is set to launch a Centre for Stem Cells & Regenerative Medicine to be led by Professor Fiona Watt, former president of the International Society of Stem Cell Research.


Snake bites, although rare, can be a serious threat in the wilderness. It is important to take all the precautions to not get bit in the first place, but if you do, we can tell you everything you need to know. Below are 2 lists. One list includes some techniques to prevent snake bites and encounters, the second list describes some techniques to apply if you are bitten.
Learn about your environment before entering. You should be fully aware of the types of snakes (and any other dangerous animal, insect, plant, or threat) before spending time camping and hiking through the outdoors. Learn to recognize these threats so when confronted, you can back away and go a different direction.
Always be fully alert with every step and movement you make. Never reach your hand under a log or rock where you can not see. Piles of brush and dead leaves are often home to snakes and other critters. A walking stick can be useful to prod areas before stepping to alert the snake of your presence before your foot gets there.
Do not sleep directly on the ground. Elevation and some type of bounded shelter is ideal.
Try to avoid being in water, but if you have to, move slowly to prevent startling.
Use a light of some kind at the first signs of darkness. Not only will the light help you see snakes but it will also alert them of a presence, making them less likely to attack due to being startled.
Wear snake protective gear like snake boots or gloves designed to prevent a snakes fangs from penetrating.
Contact 911 or help as soon as possible. The faster you can get an antivenin the better.
Keep the bitten area below the level of the heart. Do not elevate.
Do not consume caffiene, energy drinks, alcohol, medications, or any type of drug.
Do not apply a tourniquet or ice.
Remove any constricting clothing or jewelry like watches, necklaces, or tight clothes.
Clean the wound and cover, but try not to rub too much or apply pressure.
Only use a suction technique if you feel it would take too long to reach medical help. This technique often doesn't help, cutting an incision should only be done by professionals.


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Apple MacBook Pro Launch Timeline Tipped, Two Variants Likely
Vinay Patel October 4, 2021
The much-awaited Apple MacBook Pro is could launch sometime in October, if a recently surfaced report is anything to go by. Apple held its launch event last month, where the company unveiled iPhones and iPads. However, some customers have been restlessly waiting to get their hands on the new MacBook Pros. The word on the street is that the long-rumored MacBook Pros will pack a new Apple M1X Silicon.
According to SlashGear, this will enable the tech giant to extend its lead over MacBooks that currently pack Intel chips. Earlier this year, a report suggested that Apple is gearing up to launch new MacBook Pro models later this year. The new Apple MacBook Pro models with Apple M1X silicon were expected to hit the store shelves between September and November. Now, it appears that Apple will be taking the wraps off its hotly-anticipated MacBook Pro later this month.
Apple MacBook Pro Launch Timeline
A new report from MacRumors suggests that the Cupertino-based tech company will launch an Apple M1X MacBook Pro in a few weeks. Furthermore, the new M1X chip is likely to come in two variants featuring 10 CPU cores. Noted Bloomberg analyst Mark Gurman claims that the Pro MacBooks will be unveiled in October. In his Power On newsletter, Gurman said that Apple is gearing up to take the wraps off its MacBook Pros with the new M1X chip within the next month.
Aside from that, Gurman reiterated some of the previously surfaced speculations surrounding the custom Apple silicon. He went on to reveal that the M1X's variants will have a 10-core CPU, eight out of which, will be high-performance cores. The remaining two cores will be high-efficiency cores, Gurman added. As far as the GPU is concerned, Gurman said that the two models will come with 16 and 32 cores. Furthermore, Apple could provide multiple new ports on the new machines. It is likely to bring back an HDMI port and the SD card slot.
Previously Surfaced Rumors
If earlier rumors prove to be true, the new Mac mini will also pack the new M1X chip under the hood. Regrettably, this machine is not likely to arrive in November. It is worth mentioning that Apple is still mum on its rumored launch event and on its new products. Nevertheless, some reports claim that the MacBook Pro will go on sale in a 14-inch and a 16-inch screen size. Moreover, the new MacBooks could support magnetic charging and come in a redesigned chassis.
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MacBook Pro 2021 14-inchMacBook Pro 2021 16-inchMacBook Pro Launch DateMacBook Pro LeaksMacBook Pro Specifications
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Öffentlicher Bereich / Open Forum » Connection issues.
Nexus-Staffel Forum » Bismarck Hangar S0 » Öffentlicher Bereich / Open Forum » Connection issues.
I'm in the Eu and when playing the game I'm connected to the WEU data centre, but in game, I'm having issues or random times where my ping goes wild. going from a steady number like 20 then into hundreds then hitting thousand, only for it to balance out again. does anyone know the reason for this? Before any starts, my internet is stable enough to play Casual it only in ranked where having a slow down then a freeze, it really annoying and only started after the last patch.


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Polyvision was the name given by the French film critic Émile Vuillermoz to a specialized widescreen film format devised exclusively for the filming and projection of Abel Gance's 1927 film Napoleon.

Polyvision involved the simultaneous projection of three reels of silent film arrayed in a horizontal row, making for a total aspect ratio of 4:1 (1.33×3:1). Polyvision's extremely wide aspect ratio was the widest aspect ratio yet seen, even though it is technically just three images side by side. In 1955, the Walt Disney Company developed Circle-Vision 360° for use in Disneyland theme parks which used nine 4:3 35 mm projectors to show an image that completely surrounds the viewer.

This configuration is considered to be a similar precursor to Cinerama, which would debut a quarter of a century later; however, it is unlikely that Polyvision was a direct inspiration for later widescreen techniques, as the triptych sequence of Napoleon was cut from the film by its distributors after only a few screenings and was not seen again until Kevin Brownlow compiled his restorations from the 1970s onwards.

Description
Three film cameras were stacked vertically to shoot the widescreen compositions which would be viewed across all three sections. Gance also used the three strips to create triptych compositions of panels contrasting or simultaneous action, mirrored sides framing the center strip, and perceptual cross-cutting. In this respect, Polyvision can arguably be said to have inspired split screen compositions as well as in-eye edited experiments such as Mike Figgis's Timecode. Gance was unable to eliminate the problem of the two seams dividing the three panels of film as shown on screen, so he avoided the problem by putting three completely different shots together in some of the Polyvision scenes. When Gance viewed Cinerama many years later, he noticed that the widescreen image was still not seamless, that the problem was not entirely fixed.

Polyvision was only used for the final reel of Napoleon, to create a climactic finale. Filming the whole story in Polyvision was impractical as Gance wished for a number of innovative shots, each requiring greater flexibility than was allowed by three interlocked cameras. When the film was severely re-cut by the distributors very early on during exhibition, the new version only retained the center strip in order to allow projection in standard single-projector cinemas. Brownlow's restored version, first seen on 31 August 1979 at the Telluride Film Festival, in Telluride, Colorado, finishes with a flourish intended by Gance: it uses red and blue tinted film on the left and right panels to create le tricolore—the flag of Napoleon's triumphant army.

Difficulties in mounting a full screening of Napoleon with three simultaneous projectors mean that a true Polyvision presentation is rarely seen, with recent exhibitions of Napoleon using Polyvision having been in December 2004 and November 2013 at the Royal Festival Hall, in December 2009 at Cité de la Musique, and in March 2012 at the Paramount Theatre in Oakland, California.

Gance continued to tinker with the system with Parvo camera designer Andre Debrie for several decades afterward, and by 1956, it evolved into a system called Magirama very similar to the later Cinemiracle format. Magirama used three 35 mm film cameras at Academy format with the two side cameras shooting into mirrors; the projectors then used mirrors in an identical configuration in order to properly reverse the side images. This system was only used on a limited number of shots.

See also
List of film formats

References

Cinematic techniques
Motion picture film formats
Multi-screen film

redpajama_set_name: RedPajamaWikipedia


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Charlotte Cunningham 21 August 2021 https://www.fwi.co.uk
Careers and personal development
How share-milking can provide a route into dairy farming
Charlotte Cunningham 17 August 2021
© Otago Daily Times
Gregor and Lynne Ramsay credit share-milking with allowing them to get a foot firmly in the dairying door.
Born and raised in the small rural village of Biggar, Lanarkshire, Mr Ramsay says a career in farming was always on the agenda, despite his non-farming background.
After studying agriculture and business management at Aberdeen University and working as a herd manager at a local dairy farm, he travelled to New Zealand in 2010 for a working holiday and fell in love with the country.
See also: Why share farming can be the fairest joint venture option
Gregor and Lynne Ramsay with their first son, George © Otago Daily Times
He returned home, but the pull of New Zealand was too great, so he and his then-girlfriend, Lynne, moved to South Island in 2015, where they both found jobs on dairy farms.
Mr Ramsay says: "I was working as a farm manager on a 250-cow farm in Southland, but an opportunity came up to join Fonterra [the country's biggest milk processor] and I ended up becoming the area manager for Otago, which helped broaden my knowledge and experience across the whole sector."
The couple say the goal was to always have their own herd, and after marrying in 2018 and receiving their official New Zealand residency permits a year later – a pivotal requirement for being able to borrow money from the bank – they began looking for opportunities.
The one they chose was a 50/50 share-milking venture with the Wilson family on a farm on the Taieri Plains, just outside of Dunedin in South Otago, where they milk 140 Jersey cows on a 40ha (99 acre) grazing platform, with 20ha (49 acres) of support land.
Farm facts
Milking 140 Jersey cows
Farming 60ha (148 acres)
Averaging 4,843 litres a cow a year at 5.27% fat and 4.06% protein
Supplying Fonterra
Breed 40 replacement heifers annually and sell surplus for breeding
How the agreement works
Costs and profits are split between the two parties (see table "Income and expenditure split"). The Wilson family owns the land, while the Ramsays own the cows and machinery.
The couple initially bought stock from a nearby dispersal herd, selecting cows based on health, fertility and quality.
"We chose Jerseys for their milk production efficiency, the value of fat, and the saleable bull calf value," says Mr Ramsay.
The couple also had to source machinery and equipment and run a very basic fleet consisting of a Massey Ferguson 5455 with a loader, a silage wagon, a mower and two quad bikes.
They also invested in calf rearing equipment and fencing supplies.
Income and expenditure split
Farm owner
Share-milker
Fonterra dividend
Stock sales
Fonterra shares
Rates/water supply etc
Farm infrastructure
Capital fertiliser
Bought in feed
Nitrogen fertiliser
Power/machinery/fuel
Silage made on the farm
Note: Capital fertiliser is a fertiliser application done on top of maintenance requirements to increase nutrient status in the soil
Last season, the farm averaged 452kg milk solids (MS) a cow (above the New Zealand average of 385kg) and average fat and protein percentages were 5.27% and 4.06%, respectively.
Total milk solids supplied last year came in at 63,302kg – up 17% on the previous year – which equates to about 1,620kg MS/ha – well above the New Zealand average of 1,096kg MS/ha.
Maximising profits
Keeping costs low is key to achieving good profits. Less is more when it comes to technology, and the cows are milked in a 14-a-side herringbone parlour.
In true New Zealand style, the ethos is to get as much milk as possible from grass. The cows are outside all year round and are outwintered on kale and silage bales.
In the milking season, cows are fed a diet of grass, silage, rolled barley and brewers' grain, fed out in the paddock.
The stocking rate runs at 3.5 cows a hectare (1.4 cows an acre), with the whole herd run as one mob and youngstock grazed on the support block.
The small scale of the business means it is just Mr and Mrs Ramsay labour-wise – although Mrs Ramsay is currently on maternity leave after giving birth to their second son, Hamish.
Last season, costs averaged NZ$2.79/kg MS (£1.42 or 13p/litre equivalent) with a farm economic surplus of NZ$1.23/kg MS (63p or 6p/litre equivalent).
Challenges and successes
Mr Ramsay says the low cost of production of the share-milking model makes entry into the country's dairy industry much easier.
"This gives New Zealand a competitive advantage globally, but also allows the next generation to get on the ladder, with limited capital requirements."
© Gregor Ramsay
Despite the many advantages of the system, fluctuations in global markets present a big challenge, he adds. "With share-milking, you want to be getting as big a milk cheque as possible to ensure profitability. However, in New Zealand, 95% of milk products are exported, which means prices can be very volatile.
"Over the past 10 years, the milk price has swung from the highs of NZ$8.40/kg MS (£4.25) to the lows of NZ$3.90/kg MS (£1.98).
"Thankfully, the price has been relatively stable for the past three years and also at a reasonable level."
The family are in the final year of their current share-milking contract and are considering the next step. They would like to increase their herd size to about 250.
Mr Ramsay says share-milking would be possible in the UK, but warns that it needs real support from everyone involved to ensure it remains profitable.
"One area where share-milking can be beneficial is succession planning. It allows for a clear and defined pathway for the relinquishing of responsibility and assets from the farm owners to the share-milker, with the eventual goal of the share-milker having the desired equity and performance history to purchase the farm.
Grass is supplemented with silage, rolled barley and brewers' grain © Gregor Ramsay
"Share-milking is rather like a marriage. It takes both parties to make it work and this is achieved through good communication, alignment of goals and respect.
"Both parties have significant investment involved, and as such should be striving for the best returns.
"The share-milking system is well developed in New Zealand and has been the pathway to success for many to realise their dreams and ambitions."
Keep up with the Ramsays' progress on Instagram @Ramsay_Dairies