Artificial intelligence yields new antibiotic

Using a machine-learning algorithm, MIT researchers have identified a powerful new antibiotic compound. In laboratory tests, the drug killed many of the world's most problematic disease-causing bacteria, including some strains that are resistant to all known antibiotics. It also cleared infections in two different mouse models.

The computer model, which can screen more than a hundred million chemical compounds in a matter of days, is designed to pick out potential antibiotics that kill bacteria using different mechanisms than those of existing drugs.

"We wanted to develop a platform that would allow us to harness the power of artificial intelligence to usher in a new age of antibiotic drug discovery," says James Collins, the Termeer Professor of Medical Engineering and Science in MIT's Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering. "Our approach revealed this amazing molecule which is arguably one of the more powerful antibiotics that has been discovered."

In their new study, the researchers also identified several other promising antibiotic candidates, which they plan to test further. They believe the model could also be used to design new drugs, based on what it has learned about chemical structures that enable drugs to kill bacteria.

"The machine learning model can explore, in silico, large chemical spaces that can be prohibitively expensive for traditional experimental approaches," says Regina Barzilay, the Delta Electronics Professor of Electrical Engineering and Computer Science in MIT's Computer Science and Artificial Intelligence Laboratory (CSAIL).

Barzilay and Collins, who are faculty co-leads for MIT's Abdul Latif Jameel Clinic for Machine Learning in Health, are the senior authors of the study, which appears today in Cell. The first author of the paper is Jonathan Stokes, a postdoc at MIT and the Broad Institute of MIT and Harvard.

A new pipeline

Over the past few decades, very few new antibiotics have been developed, and most of those newly approved antibiotics are slightly different variants of existing drugs. Current methods for screening new antibiotics are often prohibitively costly, require a significant time investment, and are usually limited to a narrow spectrum of chemical diversity.

"We're facing a growing crisis around antibiotic resistance, and this situation is being generated by both an increasing number of pathogens becoming resistant to existing antibiotics, and an anemic pipeline in the biotech and pharmaceutical industries for new antibiotics," Collins says.

To try to find completely novel compounds, he teamed up with Barzilay, Professor Tommi Jaakkola, and their students Kevin Yang, Kyle Swanson, and Wengong Jin, who have previously developed machine-learning computer models that can be trained to analyze the molecular structures of compounds and correlate them with particular traits, such as the ability to kill bacteria.

The idea of using predictive computer models for "in silico" screening is not new, but until now, these models were not sufficiently accurate to transform drug discovery. Previously, molecules were represented as vectors reflecting the presence or absence of certain chemical groups. However, the new neural networks can learn these representations automatically, mapping molecules into continuous vectors which are subsequently used to predict their properties.

In this case, the researchers designed their model to look for chemical features that make molecules effective at killing E. coli. To do so, they trained the model on about 2,500 molecules, including about 1,700 FDA-approved drugs and a set of 800 natural products with diverse structures and a wide range of bioactivities.

Once the model was trained, the researchers tested it on the Broad Institute's Drug Repurposing Hub, a library of about 6,000 compounds. The model picked out one molecule that was predicted to have strong antibacterial activity and had a chemical structure different from any existing antibiotics. Using a different machine-learning model, the researchers also showed that this molecule would likely have low toxicity to human cells.

This molecule, which the researchers decided to call halicin, after the fictional artificial intelligence system from "2001: A Space Odyssey," has been previously investigated as possible diabetes drug. The researchers tested it against dozens of bacterial strains isolated from patients and grown in lab dishes, and found that it was able to kill many that are resistant to treatment, including Clostridium difficile, Acinetobacter baumannii, and Mycobacterium tuberculosis. The drug worked against every species that they tested, with the exception of Pseudomonas aeruginosa, a difficult-to-treat lung pathogen.

To test halicin's effectiveness in living animals, the researchers used it to treat mice infected with A. baumannii, a bacterium that has infected many U.S. soldiers stationed in Iraq and Afghanistan. The strain of A. baumannii that they used is resistant to all known antibiotics, but application of a halicin-containing ointment completely cleared the infections within 24 hours.

Preliminary studies suggest that halicin kills bacteria by disrupting their ability to maintain an electrochemical gradient across their cell membranes. This gradient is necessary, among other functions, to produce ATP (molecules that cells use to store energy), so if the gradient breaks down, the cells die. This type of killing mechanism could be difficult for bacteria to develop resistance to, the researchers say.

"When you're dealing with a molecule that likely associates with membrane components, a cell can't necessarily acquire a single mutation or a couple of mutations to change the chemistry of the outer membrane. Mutations like that tend to be far more complex to acquire evolutionarily," Stokes says.

In this study, the researchers found that E. coli did not develop any resistance to halicin during a 30-day treatment period. In contrast, the bacteria started to develop resistance to the antibiotic ciprofloxacin within one to three days, and after 30 days, the bacteria were about 200 times more resistant to ciprofloxacin than they were at the beginning of the experiment.

The researchers plan to pursue further studies of halicin, working with a pharmaceutical company or nonprofit organization, in hopes of developing it for use in humans.

Optimized molecules

After identifying halicin, the researchers also used their model to screen more than 100 million molecules selected from the ZINC15 database, an online collection of about 1.5 billion chemical compounds. This screen, which took only three days, identified 23 candidates that were structurally dissimilar from existing antibiotics and predicted to be nontoxic to human cells.

In laboratory tests against five species of bacteria, the researchers found that eight of the molecules showed antibacterial activity, and two were particularly powerful. The researchers now plan to test these molecules further, and also to screen more of the ZINC15 database.

The researchers also plan to use their model to design new antibiotics and to optimize existing molecules. For example, they could train the model to add features that would make a particular antibiotic target only certain bacteria, preventing it from killing beneficial bacteria in a patient's digestive tract.

CRISPR gene cuts may offer new way to chart human genome

Gene manipulation concept photo illustration

In search of new ways to sequence human genomes and read critical alterations in DNA, researchers at Johns Hopkins Medicine say they have successfully used the gene cutting tool CRISPR to make cuts in DNA around lengthy tumor genes, which can be used to collect sequence information.

A report on the proof-of-principle experiments using genomes from human breast cancer cells and tissue appears in the Feb. 10 issue of Nature Biotechnology.

The researchers say that pairing CRISPR with tools that sequence the DNA components of human cancer tissue is a technique that could, one day, enable fast, relatively cheap sequencing of patients' tumors, streamlining the selection and use of treatments that target highly specific and personal genetic alterations.

"For tumor sequencing in cancer patients, you don't necessarily need to sequence the whole cancer genome," says Winston Timp, Ph.D., assistant professor of biomedical engineering and molecular biology and genetics at the Johns Hopkins University School of Medicine. "Deep sequencing of particular areas of genetic interest can be very informative."

In conventional genome sequencing, scientists have to make many copies of the DNA at issue, randomly break the DNA into segments, and feed the broken segments through a computerized machine that reads the string of chemical compounds called nucleic acids, made up of the four "bases" that form DNA, and are lettered A, C, G and T. Then, scientists look for overlapping regions of the broken segments and fit them together like tiles on a roof to form long regions of DNA that make up a gene.

In their experiments, Timp and M.D./Ph.D. student Timothy Gilpatrick were able to skip the DNA-copying part of conventional sequencing by using CRISPR to make targeted cuts in DNA isolated from a sliver of tissue taken from a patient's breast cancer tumor.

Then, the scientists glued so-called "sequencing adaptors" to the CRISPR-snipped ends of the DNA sections. The adaptors serve as a kind of handle that guide DNA to tiny holes or "nanopores" which read the sequence.

By passing DNA through the narrow hole, a sequencer can build a read-out of DNA letters based on the unique electrical current that occurs when each chemical code "letter" slides through the hole.

Among 10 breast cancer genes the team focused on, the Johns Hopkins scientists were able to use nanopore sequencing on breast cancer cell lines and tissue samples to detect a type of DNA alteration called methylation, where chemicals called methyl groups are added to DNA around genes that affect how genes are read.

The researchers found a location of decreased DNA methylation in a gene called keratin 19 (KRT19), which is important in cell structure and scaffolding. Previous studies have shown that a decrease in DNA methylation in KRT19 is associated with tumor spread.

In the breast cancer cell lines they studied, the Johns Hopkins team was able to generate an average of 400 "reads" per basepair, a reading "depth" hundreds of times better than some conventional sequencing tools.

Among their samples of human breast cancer tumor tissue taken at biopsies, the team was able to produce an average of 100 reads per region. "This is certainly less than what we can do with cell lines, but we have to be more gentle with DNA from human tissue samples because it's been frozen and thawed several times," says Timp.

In addition to their studies of DNA methylation and small mutations, Timp and Gilpatrick sequenced the gene commonly associated with breast cancer: BRCA1, which spans a region on the genome more than 80,000 bases long. "This gene is really long, and we were able to collect sequencing reads which went all the way through this large and complex region," says Gilpatrick.

"Because we can use this technique to sequence really long genes, we may be able to catch big missing blocks of DNA we wouldn't be able to find with more conventional sequencing tools," says Timp.

In addition to its potential to guide treatment for patients, Timp says the combination of CRISPR technology and nanopore sequencing provides such depth that it may help scientists find new disease-linked gene alterations specific to one allele (inherited from one parent) and not another.

Timp and Gilpatrick plan to continue refining the CRISPR/nanopore sequencing technique and testing its capabilities in other tumor types.

Funding for the research was provided by the National Institutes of Health's National Human Genome Research Institute (R01 HG009190).

In addition to Timp and Gilpatrick, other scientists who contributed to the research include Isac Lee from Johns Hopkins University; Bradley Downs and Saraswati Sukumar from the Johns Hopkins Kimmel Cancer Center; James E. Graham, Etienne Raimondeau, Rebecca Bowen and Andrew Heron from Oxford Nanopore Technologies; and Fritz Sedlazeck from Baylor College of Medicine.

Under a licensing agreement between Oxford Nanopore Technologies and The Johns Hopkins University, Timp is entitled to a share of royalty payments. This arrangement has been reviewed by The Johns Hopkins University in accordance with its conflict of interest policies.

New study allows brain and artificial neurons to link up over the web

Brain-digital interface concept illustration

Brain functions are made possible by circuits of spiking neurons, connected together by microscopic, but highly complex links called synapses. In this new study, published in the scientific journal Nature Scientific Reports, the scientists created a hybrid neural network where biological and artificial neurons in different parts of the world were able to communicate with each other over the internet through a hub of artificial synapses made using cutting-edge nanotechnology. This is the first time the three components have come together in a unified network.

During the study, researchers based at the University of Padova in Italy cultivated rat neurons in their laboratory, whilst partners from the University of Zurich and ETH Zurich created artificial neurons on Silicon microchips. The virtual laboratory was brought together via an elaborate setup controlling nanoelectronic synapses developed at the University of Southampton. These synaptic devices are known as memristors.

The Southampton based researchers captured spiking events being sent over the internet from the biological neurons in Italy and then distributed them to the memristive synapses. Responses were then sent onward to the artificial neurons in Zurich also in the form of spiking activity. The process simultaneously works in reverse too; from Zurich to Padova. Thus, artificial and biological neurons were able to communicate bidirectionally and in real time.

Themis Prodromakis, Professor of Nanotechnology and Director of the Centre for Electronics Frontiers at the University of Southampton said "One of the biggest challenges in conducting research of this kind and at this level has been integrating such distinct cutting edge technologies and specialist expertise that are not typically found under one roof. By creating a virtual lab we have been able to achieve this."

The researchers now anticipate that their approach will ignite interest from a range of scientific disciplines and accelerate the pace of innovation and scientific advancement in the field of neural interfaces research. In particular, the ability to seamlessly connect disparate technologies across the globe is a step towards the democratisation of these technologies, removing a significant barrier to collaboration.

Professor Prodromakis added "We are very excited with this new development. On one side it sets the basis for a novel scenario that was never encountered during natural evolution, where biological and artificial neurons are linked together and communicate across global networks; laying the foundations for the Internet of Neuro-electronics. On the other hand, it brings new prospects to neuroprosthetic technologies, paving the way towards research into replacing dysfunctional parts of the brain with AI chips."

The research was funded by the EU Future and Emerging Technologies programme as well as the Engineering and Physical Sciences Research Council in the UK. Professor Prodromakis also holds a Royal Academy of Engineering Chair in Emerging Technologies with a focus on developing energy-efficient AI Hardware solutions.

How resident microbes restructure body chemistry

Lab mouse 

The makeup of our microbiomes -- the unique communities of bacteria, viruses and other microbes that live in and on us -- have been linked, with varying degrees of evidence, to everything from inflammatory bowel disease to athletic performance.

But exactly how could such tiny organisms have such immense influences on a person?

University of California San Diego researchers have created the first-ever map of all the molecules in every organ of a mouse and the ways in which they are modified by microbes. In one surprising example, they discovered that microbes control the structure of bile acids in both mice and people.

The study, published February 26, 2020 in Nature, was led by Pieter Dorrestein, PhD, professor and director of the Collaborative Mass Spectrometry Innovation Center in the Skaggs School of Pharmacy and Pharmaceutical Sciences at UC San Diego, and Robert Quinn, PhD, assistant professor at Michigan State University.

When you change the structure of molecules, such as bile acids, you could change how cells talk to one another and which genes are turned "on" or "off" at a given time, Dorrestein said. And that might have huge consequences for body function and the development of disease.

"We hear a lot about how our own human genes influence our health and behaviors, so it may come as a shock to think that we could have molecules in the body that look and act the way they do not because of our genes, but because of another living organism," Dorrestein said.

Mapping molecules and microbes in mice

The team compared germ-free (sterile) mice and mice with normal microbes. They used a laboratory technique called mass spectrometry to characterize the non-living molecules in every mouse organ. They identified as many molecules as possible by comparing them to reference structures in the GNPS database, a crowdsourced mass spectrometry repository developed by Dorrestein and collaborators. They also determined which living microbes co-locate with these molecules by sequencing a specific genetic region that acts as a barcode for bacterial types.

In total, they analyzed 768 samples from 96 sites of 29 different organs from four germ-free mice and four mice with normal microbes. The result was a map of all of the molecules found throughout the body of a normal mouse with microbes, and a map of molecules throughout a mouse without microbes.

A comparison of the maps revealed that as much as 70 percent of a mouse's gut chemistry is determined by its gut microbiome. Even in distant organs, such as the uterus or the brain, approximately 20 percent of molecules were different in the mice with gut microbes.

Bacteria modify bile acids

After constructing these maps, the researchers homed in on one particular family of molecules that appeared to be significantly different when microbes were present: bile acids. Bile acids are primarily produced by the mouse or human liver, and they help digest fats and oils. They can also carry messages throughout the body.

The team discovered bile acids with previously unknown structures in mice with normal microbiomes, but not in germ-free mice. It's long been known that host liver enzymes add amino acids to bile acids, specifically the amino acids glycine and taurine. But in mice with normal microbiomes, the team found that bacteria are tagging bile acids with other amino acids -- phenylalanine, tyrosine and leucine.

"More than 42,000 research papers have been published about bile acids over the course of 170 years," Quinn said. "And yet these modifications had been overlooked."

Influence on human health

Curious if the same types of microbe-modified bile acids are found in humans, the researchers used a tool they created, the Mass Spectrometry Search Tool (MASST), to search 1,004 public datasets of samples analyzed with mass spectrometry. They also analyzed by mass spectrometry approximately 3,000 fecal samples submitted to the American Gut Project, a large citizen science effort based at UC San Diego School of Medicine.

Here's what they found: The unique microbial-modified bile acids the researchers saw in mice were also present in up to 25.3 percent of all human samples in the datasets. These novel bile acids were more abundant in infants and patients with inflammatory bowel disease or cystic fibrosis.

One way bile acids can deliver messages from the gut to other parts of the body is through specific gut receptors called farnesoid X receptors. Bile acids bind and activate the receptors, which then inhibit genes responsible for making more bile acids. The receptors also help regulate liver triglyceride levels and fluid regulation in the intestines, making them important in liver disease and possibly obesity. Several drugs are currently being developed to treat liver disease by activating farnesoid X receptors.

Sure enough, in mice and human cells grown in the lab, Dorrestein, Quinn and team found that the newly discovered, microbe-modified bile acids strongly stimulate farnesoid X receptors, reducing expression of genes responsible for bile acid production in the liver.

The study raises many questions about the role microbes might play in driving liver and other diseases, and in influencing the activity of therapeutics, such as drugs that target farnesoid X receptors.

"This study provides a clear example of how microbes can influence the expression of human genes," Dorrestein said. "What we still don't know is the downstream consequences this could have, or how we might be able to intervene to improve human health."

How to Change a Habit With the Four Quadrants of Change

Quitting smoking is the easiest thing in the world. Some people quit smoking a thousand times in their lives! Everyone knows someone with this mindset.

But this type of change is superficial. It doesn’t last. For real, lasting change to take place, we need to consider the quadrants of change.

Real change, the change that is fundamental, consistent, and longitudinal (lasting over time) has to happen in four quadrants of your life.

It doesn’t have to be quitting smoking; it can be any habit you want to break — drinking, biting your nails, overeating, playing video games, shopping, and more.

Most experts focus on only one area of change, some focus on two areas, but almost none focus on all four quadrants of change. That’s why much of change management fails.

Whether it is in the personal life of a single individual through actions and habits, or in a corporate environment, regarding the way they conduct their business, current change management strategies are lacking.

It all stems from ignoring at least one part of the equation.

So, today, we will cover all four quadrants of change and learn the formula for how to change fundamentally and never go back to your “old self.”

A word of warning: this is simple to do, but it’s not easy. Anyone who tells you that change is easy is either trying to sell you something, or they have no idea what they’re talking about.

Those who want an overnight solution have left the article now, so that leaves you, me, and the real process of change.

TABLE OF CONTENTS
The Four Quadrants of Change
First Quadrant -- Internal Individual
Second Quadrant -- External Individual
Third Quadrant -- Internal Collective
Fourth Quadrant -- External Collective
Putting It All Together
Read More on Making Changes
The Four Quadrants of Change
There are four areas, or quadrants, in which you need to make a change in order for it to stick. If you miss or ignore a single one of these, your change won’t stick, and you will go back to your previous behavior.

The four quadrants are:

Internal individual – mindset
External individual – behavior
Internal collective – culture/support system
External collective – laws, rules, regulations, teams, systems, states
All four of these quadrants of change may sound like they could carry change all by themselves, but they can’t. So, be sure to implement your change in all four quadrants. Otherwise, it will all be in vain.

First Quadrant — Internal Individual
This quadrant focuses on the internal world of an individual, and it concerns itself with the mindset of a person.

Our actions stem from our thoughts (most of the time), and if we change our mindset toward something, we will begin to process of changing the way we act.

People who use the law of attraction fall into this category, where they’ve recognized the strength of thoughts and how they make us change ourselves.

Even Lao Tzu had a great saying regarding this:

“Watch your thoughts. They become words. Watch your words. They become deeds. Watch your deeds. They become habits. Watch your habits. They become character. Character is everything.”

One of the most impactful ways you can make a change in this quadrant is to implement what James Clear calls identity-based habits.

Instead of prioritizing the outcome of a change (ex.: I want to lose 20 pounds), you prioritize your identity as a person (I want to become/remain a healthy person).

Here are a couple of examples for you to see the strength of this kind of resolution:

I want to watch many movies = I am a cinema lover
I want to clean my apartment = I am a clean person
I want to harvest my crops = I am a harvester (farmer)
I want to swim = I am a swimmer

This quadrant is about changing the identity you attach to a certain action. Once you re-frame your thinking in this way, you will have completed the first of the quadrants of change.

Second Quadrant — External Individual
This quadrant focuses on the external world of an individual and concerns itself with the behavior of a person.

This is where people like Darren Hardy, the author of the Compound Effect reside. Hardy is about doing small, consistent actions that will create change in the long run (the compound effect).

You want to lose 30 pounds? Start by eating just 150 calories (approximately two slices of bread) less a day, and in two and a half years, you will have lost 30 pounds.

The same rules apply to business, investing, sports, and multiple other areas. Small, consistent actions can create big changes.

This works — I’ve read 20 extra pages a day for the past two years, and it accumulated into 90 books read in two years. [3]

Here, you have two ways of dealing with change behaviorally: negative environmental design and positive environmental design.

Negative Environmental Design
This is when you eliminate the things from your environment that revert you to the old behavior. If you want to stop eating ice cream, you don’t keep it in your freezer.

If you want to stop watching TV, you remove the batteries from the remote and put them on the other side of the house (it works!).

Positive Environmental Design
This is when you put the things that you want to do withing reach — literally!

You want to learn how to play guitar? Put your guitar right next to your sofa. You want to head to the gym? Put the gym clothes in a backpack and put it on top of your shoes.

You want to read more books? Have a book on your nightstand, your kitchen table, and on the sofa.

You can even combine this last trick with my early advice about removing the batteries from your remote control, combining the negative and positive environmental designs for maximum effect.

Two Sides of the Same Coin
If you just change your behavior and leave your intentions (thoughts) intact, your discipline will fail you and the real change won’t happen.

You will simply revert back to the previous behavior because you haven’t changed the fundamental root of why this problem occurs in the first place.

That is why you need to create change both in the first quadrant (internal individual — mindset) and the second quadrant (external individual — behavior). These quadrants of change are two sides of the same coin.

Most change management would stop here, and that’s why most change management fails.

No matter how much you focus on yourself, there are things that affect our lives that are happening outside of us. That is the focus of the two remaining quadrants.

Third Quadrant — Internal Collective
This quadrant focuses on the internal world of the collective where the individual resides, and it concerns itself with the culture of that collective.

There are two different distinctions here: the Inner Ring and the Outer Ring.

The Inner Ring
These are your friends and your family. The Inner Ring is the place where the social and cultural norms of your friends and family rule.

So, if everyone in your family is overweight and every lunch is 1,000 calories per person, then you can say goodbye to your idea of becoming healthy.

In this case, the culture of your group, the inner norms that guide the decisions, actions, thoughts, ideas, and patterns of behaviors are all focused on eating as much as possible. [4]

You need to have the support of your Inner Ring if you want to achieve change. If you don’t have this support, the the best way to proceed is by either changing your entire Inner Ring or distancing yourself from it.

Beware — most Inner Rings won’t accept the fact that you want to change and will undermine you on many occasions — some out of habit, some due to jealousy, and some because supporting you would mean that they have to change, too.

You don’t have to cut ties with people, but you can consciously decide to spend less time with them.

The Outer Ring
The Outer Ring consists of the culture of your company, community, county, region, and country. For example, it’s quite hard to be an open-minded person in North Nigeria, no matter how you, your friends, and your family think.

The Outer Ring is the reason why young people move to the places that share their value systems instead of staying in their current city, county, or country.

Sometimes, you need to change your Outer Ring as well because its culture is preventing you from changing.

I see this every single day in my country, where the culture can be so toxic that it doesn’t matter how great of a job you have or how great your life currently looks — the culture will change you, inch by inch, until you become like it.

Fourth Quadrant — External Collective
This quadrant focuses on the external world of the collective where the individual resides, and it concerns itself with the systems, teams, laws, and rules of that collective.

This quadrant is about the external manifestations of the collective culture. If the majority of the environment thinks in a certain way, they will create institutions that will implement that way of thinking.

The same rules apply to companies.

One example for companies would be those managers who think that employees are lazy, lack responsibility, and need constant supervision (or what is called Theory X in management).

Then, those managers implement systems that reflect that kind of culture– no flexible work hours, strict rules about logging work, no remote work, etc.

Your thoughts, however, may be different. You might believe that people want responsibility, that they are capable of self-direction, that they can make good decisions, and that managers don’t need to stand on their necks if they want something done (this is called Theory Y in management).

Then, you would want to have flexible working hours, different ways of measuring your productivity (for example, not time on the job but work produced), and remote work, if possible for your profession.

This is when you enter into a conflict with the external collective quadrant. Here, you have four options: leave, persevere, neglect, and voice.

Leave
You can simply leave the company/organization/community/country and go to a different place. Most people decide to do this.

Persevere
This is when you see that the situation isn’t good, but you decide to stick at it and wait for the perfect time (or position) where you can implement change.

Neglect
This is where you give up on the change you want to see and just go with the flow, doing the minimal work necessary to keep the status quo.

These are the people who are disengaged at work and are doing just the bare minimum necessary (which, in the U.S. is around 65% of the workforce).

I did this only once, and it’s probably the only thing I regret doing in my life.

Voice
This is where you actively work on changing the situation, and the people in charge know that you want to create a change.

It doesn’t matter if it’s your company, community, or your country; you are actively calling for a change and will not stop until it’s implemented.

Putting It All Together
When you take it all into account, change is simple, in theory, but it isn’t easy to execute. It takes work in all four quadrants:

Internal individual — mindset
External individual — behavior
Internal collective — culture/support system
External collective — laws, rules, regulations, teams, systems, states
Some will require more work, some less, but you will need to create a change in all four of them.

But don’t let that discourage you because change is possible, and many people have done this. The best time to start changing was yesterday, but the second best time is today.