Author Archives: Patrick James Hibbert

Scientists Find New Technique to Defeat Antibiotic-Resistant Bacteria

Petri Dish Bacteria
Photo by Andrian Lange/Unsplash

Stress often causes bacteria to form biofilms. The stress can be in the form of a physical barrier, ultraviolet light, or a toxic substance such as antibiotics. These biofilms take from hours to days to form and can be of various shapes, sizes, colors, and textures depending on the species of bacteria involved.

Being in the state of a biofilm protects them from hazardous substances in their environment — biofilms have a unique outer wall, with different physical and chemical properties than their individual cells. They can coordinate metabolically, slow their growth, and even form an impenetrable barrier of wrinkles and folds.

This is one way they achieve high antibiotic resistance. Researchers from the United Kingdom recently studied the bacteria B. Sultilis transition from a free-moving swarm to a biofilm as a defense mechanism and published what they did to combat its antibiotic resistive properties in eLife.

Photo by Clemencedg/CC BY-SA 3.0/Wikimedia

To determine if their test strain behaves as others do, they recreated first performed stress tests on them. They tested the bacteria’s response to a physical barrier, ultraviolet light, and an antibiotic. The addition of a physical barrier led to a single-to-multi-layer transition of the bacteria, followed by an increase in cell density and the formation of multilayer islands near the barrier. Later, wrinkles developed on the islands near the barrier in the area the islands had started to appear initially.

When they applied ultraviolet light to the swarm, they again observed a drop in cell speed and an increase in density. And after the scientists added a large dose of the antibiotic kanamycin the bacterial cells formed a biofilm. The researchers then devised a strategy to tackle this bacteria biofilm.

They added kanamycin to the environment of a new batch of swarming bacterial cells and watched as a biofilm began to take shape. They then re-administered the antibiotic in a much larger dose than the first one, just before the completion of the biofilm’s formation. The breakdown of the partially formed biofilm and the death of the bacterial cells occurred as a result.

This shows that antibiotic-resistant bacteria lose their resistance to antibiotics when they undergo a phase transition, right before transitioning to a biofilm, where they would become much more resilient. So with proper timing of the administering of antibiotics, bacteria can be attacked in their most vulnerable state and eliminated. Researchersbelieve similar swarm-to-biofilm transitions occur in other bacterial species too.

Their research could pave the way to finding more effective ways of managing clinically relevant bacteria. Such as Salmonella enterica which spreads to the bloodstream and is transmitted by contaminated food. Or the multidrug-resistant Pseudomonas aeruginosa which causes infections in the blood, lungs (pneumonia), and other parts of the body after surgery and is spread in hospitals.

Scientists Find That Social Distancing Reduces COVID-19’s Infection Rate by Approximately 1% per Day

All states in the U.S. initiated social distancing measures between March 10 and March 25, 2020. Researchers predicted that this type of intervention will prevent a rapid, overwhelming epidemic according to modeling studies. Governments also enacted physical distancing measures in prior pandemics including the 1918 influenza pandemic with moderate success.

Prior to the coronavirus pandemic, there wasn’t much evidence about the net benefits of imposing statewide social distancing measures to reduce the transmission of viral infections. Because of this, a team of researchers from the United States, South Africa, and the United Kingdom conducted a study on it. They wanted to know what the COVID-19 case growth rate was, before and after social distancing measures where enacted. And, what the public health impacts of government-mandated non-pharmacological interventions were after they started and before they ended.

Data Collection

A search of government websites and third-party sources provided nationwide social distancing measures implemented between January 21 and May 1, 2020. These sources included the New York Times COVID-19 database from which they obtained daily state-specific reported COVID-19 cases and deaths. The measures included cancellations of public events, restrictions on internal movement, and closures of schools, workplaces, and state borders. Researchers also sought for state orders to shelter-in-place, also referred to as lockdowns, and categorized them as restrictions on internal movement.

What researchers found was:

  • Beginning 4 days after social distancing, the case growth rate declined by ~ 1% per day.
  • Beginning 7 days after social distancing, the mortality growth rate decreased by 2% per day.

However, they did not observe a major difference in the average daily case growth rate, before versus after, statewide restrictions on internal movement were enacted. Though separating their close connection with one another did prove difficult.

The authors’ findings imply that social distancing reduced the total number of COVID-19 cases substantially every week:

  • 1st week by approximately 1,600.
  • 2nd week by approximately 55,000
  • 3rd week by approximately 600,000

Nanobodies On The Road To Curing Parkinson’s Disease

Image Credits: Pixabay

study published in the journal BMC Biology by researchers from the United Kingdom, China, and Germany revealed the discovery of nanobodies akin to each other that inhibit the fibril formation of alpha-synuclein, the protein responsible for Parkinson’s Disease (PD).

Parkinson’s Disease, the degenerating movement disorder where nerve cells become impaired and die, disrupts most aspects of life for those stricken with it. The NIH describes this affliction as sporadic, progressive, and chronic. It is thought to result from genetic mutation and environmental factors.

The telltale sign indicating its presence is easily discernible: the presence of Lewy bodies in neurons. Difficulty walking, tremors, and rigidity are early symptoms. Later stages include severe cognitive decline. Presently, PD has no cure.

How PD works

Alpha-synuclein proteins form Lewy bodies then conjoin into cytotoxic oligomers, and finally, aggregate into fibrils (small fibers).

[accordion style=”info”][accordion_item title=”What are those?”]Oligomers are long chemical strands, made up monomers. Unlike plastics, which are polymers and can theoretically be any length, oligomers only contain a few monomers, i.e. they are shorter. “Cytotoxic” means these substances are toxic to life.[/accordion_item][/accordion]

They kill dopamine-producing neurons near the base of the brain, resulting in a loss of this chemical messenger molecule — among others, it handles signaling between the substantia nigra and the corpus striatum. This loss leads to neurons with abnormal firing patterns and uncoordinated movement. To illustrate the severity of this illness, most people with Parkinson’s have a noticeable absence of dopamine-producing cells in their substantia nigra. PD victims lose 60-80% of these cells.

A neuron. Image Credits: Wikipedia Commons

Scientists seeking a remedy for this immutable aberration tried a new pair of nanobodies on alpha-synuclein.  Derived from the antibodies of camels, they work remarkably in the laboratory. Their names: NBSyn2 and NBSyn87.

They exceed the abilities of previously studied antibodies and nanobodies. Whereas those took a while to cause sufficient changes to oligomer concentrations, these carry out a rapid conformational conversion. This is great news, as passive immunization using antibodies targeting alpha-synuclein has shown promise in several clinical trials.

After studying them, scientists outlined each of their strengths and shared esoteric knowledge on the nanobodies. To allude to them, NBSyn2 is better at reducing cytotoxicity, they explain, while NBSyn87 slows fibril formation to a greater degree. They break it down further, stating NBSyn2 attributes its ability to its lower positive charge and decreased interaction with the cell membrane. NBSyn87 achieves its feat by binding with a higher affinity, closer to the folding region of the alpha-synuclein protein, thereby slowing fibril formation with steric hindrance.

Lewy bodies. Image Credits: Suraj Rajan / Wikipedia Commons

Working in tandem, these nanobodies do three things: First, they prevent alpha-synuclein from aggregating into Lewy bodies; second, they hinder it from propagating in a prion-like manner; third, they destabilize cytotoxic oligomers through conformational conversion, antagonistically altering its final make-up and stopping its maturity.

Nanobodies got their name, unsurprisingly, from being miniature in size. They’re almost 10 times smaller than normal antibodies. And, being comprised of only heavy chains, they can easily cross the blood-brain barrier and bind to a variety of “hard to reach” epitopes.

[accordion style=”info”][accordion_item title=”Epitope”]An epitope is the part of a molecule to which an antibody ties — this bonding is how they ‘cure things’.[/accordion_item][/accordion]

Only 0.3% of brain synapses use dopamine; however, it has an important role to play in movement, pleasure-seeking, avoiding bad situations, and addictive behaviors, among others. Clinically, dopamine is used to dilate the renal artery and increase cardiac output. It’s in the same family of substances as epinephrine, norepinephrine, histamine, and serotonin — a family called catecholamines.

Mostly affecting those 60 years old and older, an estimated 7 to 10 million people worldwide have PD. That’s 0.09% to 0.13% globally, with the current population standing at 7.6 billion people. In 2009, the highest prevalence of this disease was in the Northeastern United States’ Amish community.

Years of research aimed at developing a quick-acting treatment for PD, using full-length antibodies and antibody fragments, targeted against different regions and different species of alpha-synuclein, led to this study. Still afoot, this undertaking is going somewhere. Now, researchers await the animal testing phase of these nanobodies with anticipation. “This find has the potential to form the basis of a new therapeutic strategy to combat PD and related protein misfolding conditions,” state researchers.

In time, other major protein misfolding diseases that these nanobodies might develop into an antidote to treat include Alzheimer’s Disease, Huntington’s disease, Creutzfeldt-Jakob disease, Cystic Fibrosis, and Gaucher’s Disease.

Slowing Down Cancer by Activating the Circadian Clock

 

Humans have genes that, when working properly, prevent cancer, the uncontrolled division of abnormal cells in the body. They are called clock genes and they control our body’s circadian rhythms, which hold power over our cortisol hormones, blood pressure, melatonin, growth hormones, testosterone, prolactin, temperature, and cell replication. “In the majority of cancer cell lines, circadian rhythms are absent or poorer compared to normal cells,” said Dr. Nicolas Cermakian, a researcher at McGill University.

The circadian clock consists of two clock genes, called per and cry and they interact with each other to generate oscillations of gene expressions (BMAL1 and CLOCK) called transcription factors. They operate on an auto-regulatory feedback loop, where transcription factor products negatively feedback on their own expression.

study done in 2010 showed how the clock gene transcription factor BMAL1 played a role in regulating tumor cell apoptosis, cell cycle progression, DNA damage response, and homeostasis regulation. Scientists from McGill University, the University of California, and the University of Montreal conducted a similar study in 2017 that resulted in the discovery of the cellular mechanisms involved between activating tumor clock genes, restoring circadian rhythms, and slowing down the replication of cells in tumors.

The tumors of mice were examined before and after circadian clock activation. Image Credits: Pixabay

The 2017 study used mice to investigate the role of the tumor cell’s circadian clock in relation to cell proliferation and tumor growth using Dexamethasone (DEX), a glucocorticoid that is both a clock gene activator and synchronizer. Taking into account circadian rhythms display oscillations of about 24 hours, they were able to calculate circadian variations based on data from their experiments. Researchers examined levels of gene and protein expressions in tumor cells before and after clock gene activation to understand the roles each played in the cell cycle in response to the treatment. They did this by, extracting and incubated cells with antibodies and then analyzing the cell suspensions with flow cytometry for cell cycle genes – c-MYC, Cyclin E, CDK2, and p21 – and the clock gene transcription factor BMAL1.

Each gene has a specific function, c-MYC activates Cyclin E, Cyclin E complexes with CDK2 to start the cell cycle, and p21 inhibits CDK2. Transcription factors BMAL1 and CLOCK complex together to represses Cyclin E, which causes cell replication to slow down. They found that treatment with DEX returned cell cycle genes and BMAL1 generation to a 24-hour rhythm.

Afterward, they tested DEX’s effect on the distribution of melanoma cells at several cell cycle stages. The tests were conducted by collecting, staining, and analyzing cells at different points in time with flow cytometry. It was found that fewer cells entered the DNA replication phase, 24 hours after treatment. And that a single DEX treatment significantly reduced cell numbers after 50 hours, and a second treatment further reduced the cell count after 96 hours.

“It is hard to tell whether the treatment reduced the proliferation speed down to normal speeds because we did not have a control cell type, B16 (non-cancerous metastasizing tumor cells) were used, and different cell types have different division times,” said Dr. Cermakian.

They were able to verify the activation of the tumor clock by DEX, however, by two means. First, by subcutaneously injecting the tumors of mice with DEX every 48 hours and then conducting an Immunohistochemistry test on the tumor slices, which revealed a significant rhythm of BMAL1 protein levels in DEX-treated tumors.

Then they harvested melanoma cells from mice and knocked down their BMAL1 clock transcription factor with RNA using a lentiviral vector. Researchers then subcutaneously injected those cells back into the mice, now lacking a functional clock, and administered DEX. They discovered that the knockdown of the circadian clock transcription factor prevented the induction of circadian rhythms by DEX in the tumors of the mice.

Once researchers found that the treatment produced circadian rhythmicity, the efficiency and specificity of the intra-tumoral injections were also evaluated by injecting methylene blue into the tumors. The injected fluid spanned the whole tumor tissue 6 hours after injection but was absent in surrounding tissues.

In order to rule out that reduced tumor growth, after DEX treatment, was caused by an immune response to the drug, they repeated the experiment in immune-deficient mice, which lack T cells, B cells, and natural killer cells. Their results showed an immune response was not the cause of the reduction in tumor growth but it was, in fact, the DEX treatment at work.  To test DEX’s effect on human cells, the scientists treated human colon carcinoma cells with DEX and found that they too exhibited rhythmic clock gene expression after treatment.

How do circadian clocks malfunction when clock genes are not mutated? “One possibility is that the factors in the cancer cells that are either absent, mutant or over-expressed act in some way on the clock mechanisms to alter their rhythms,” said Dr. Cermakian.

The researchers raised the temperature of tumor cells to activate their circadian clock. Image Credits: Pixabay

The other clock gene activators they studied were serum shock, heat shock – exposing cells to a temperature of 109.4 degrees Fahrenheit for 30 minutes – and forskolin (FSK) treatment. They found that they all reset cellular clocks, induced transcript oscillations in tumor cells slowed down tumor cell proliferation, and each used a different signaling pathway to do so.

“Each of the treatments we have used had similar magnitudes of effects both on the restoration of the clock and on tumor cell proliferation,” said Dr. Cermakian. “With each treatment, the cells remained rhythmic for 2-4 cycles.”

“We are not proposing the use of glucocorticoids, forskolin, etc, to treat cancer though. More specific drugs targeted to the clock, that some companies are currently trying to design, might be used for this in the future,” says Dr. Cermakian.

Individual circadian genes are already being targeted by drug companies, a 2014 study highlights the popular cancer drug called Alimta that targets four circadian genes. Created by the pharmaceutical company Eli Lilly and Company, it’s used to treat mesothelioma and non-small cell lung cancer.