Novel enEbCas12a protein shows potential promise as gene-editing tool to one day treat disease
Researchers have developed a novel version of a key CRISPR gene-editing protein that shows efficient editing activity and is small enough to be packaged within a non-pathogenic virus that can deliver it to target cells. Hongjian Wang and colleagues at Wuhan University, China, present these findings May 30th in the open-access journal PLOS Biology.
Recent years have seen an explosion of research attempting to harness CRISPR gene-editing systems – which are found naturally in many bacteria as a defence against viruses – so they can be used as potential treatments for human disease. These systems rely on so-called CRISPR-associated (Cas) proteins, with Cas9 and Cas12a being the two most widely used types, each with their own quirks and strengths.
One promising idea is to package CRISPR proteins within a non-pathogenic virus, which could then deliver the proteins to target cells; there, they would modify specifically targeted DNA sequences to treat disease. However, the commonly used adeno-associated virus is small, and while some Cas9 proteins can fit inside, Cas12a proteins are typically too large.
Now, Wang and colleagues have identified a relatively small version of Cas12a, termed EbCas12a, that occurs naturally in a species of the Erysipelotrichia class of bacteria. By deliberately switching out one of the amino acid building blocks of the protein for another, they boosted its gene-editing efficiency. When applied to mammalian cells in a dish in the lab, this modified protein—dubbed enEbCas12a—shows gene-editing efficiency comparable to that of two other Cas12a proteins known for highly accurate gene editing.
The research team then demonstrated that enEbCas12a is small enough to be used for adeno-associated virus-based gene therapy. They modified enEbCas12a to target a specific cholesterol-associated gene, packaged it within the virus, and administered the virus to mice with high cholesterol. One month later, they found a significant reduction of blood cholesterol levels in the treated mice, compared to mice that did not receive the virus.
More research will be needed to determine if enEbCas12a could one day be used to address human disease. Nonetheless, these findings suggest it could be possible to use adeno-associated virus to deliver Cas12a proteins for gene therapy.
The authors add, “The novel compact enEbCas12a, along with its crRNA, can be packaged into an all-in-one AAV system for convenient gene editing in vitro and in vivo with high-fidelity, which can be very beneficial for future clinical applications and more tool developments including all-in-one AAV- based multi-gene editing, base editing, primer editing, etc.”
Negating an adjective by placing ‘not’ in front of it affects the way our brains interpret its meaning, mitigating but not entirely inverting our interpretation of its definition. In a study published May 30th in the open-access journal PLOS Biology, Arianna Zuanazzi at New York University, US, and colleagues offer insight into how the brain represents changes of meaning over time and offer new methods for further linguistic research.
The way the brain processes negated adjectives – ‘not bad’ or ‘not good’ – is not understood. Previous studies suggest that negated phrases are processed more slowly and with more errors than their affirmative counterparts. Cutting-edge artificial neural networks appear to be largely insensitive to the contextual impacts of negation, leading many researchers to wonder how negation operates.
In lab-based experiments, 78 participants were asked to read affirmative or negated adjective phrases, good/bad, not good/not bad, happy/sad, not happy/not sad etc. on a screen and rate their meaning on a scale of one (really really bad/really really sad) to ten (really really good/really really happy). Answers took longer for negated adjectives and interpreted meaning was more varied. Cursor tracking showed that people are slower to interpret them, first understanding them to be affirmative before modifying towards their opposite meaning.
In a second experiment, participants rated affirmative or negated phrases on a scale. Meanwhile, magnetic fields generated by the electrical activity of their brains were captured by magnetoencephalography (MEG). Zuanazzi and colleagues again saw slower reaction times for negated adjectives. The brain activity shows that initial interpretations and early neural representations of negated adjectives are similar to that of affirmative adjectives, but are weakened, backing up the previous suggestion of a mitigated effect.
The analysis contributes to the debate as to how negation operates. The ability to characterize the subtle changes of linguistic meaning through negation in the brain using imaging methods could help to tease apart understanding of other linguistic processes beyond the sum of the processing of individual word meanings.
The authors add, “The study of negation offers a compelling linguistic framework to understand how the human brain builds meaning through combinatoric processes. Our time-resolved behavioural and neurophysiological data show that, in a sentence like ‘your coffee is not hot’, negation (‘not’) mitigates rather than inverts the representations of a scalar adjective (‘hot’). In other words, negation reduces the temperature of your coffee, though it does not make it cold.”
Our willingness to help others is governed by a specific brain region pinpointed by researchers in a study of patients with brain damage to that region.
Learning about where in the brain ‘helping’ decisions are made is important for understanding how people might be motivated to tackle large global challenges, such as climate change, infectious disease and international conflict. It is also essential for finding new approaches to treating disorders of social interactions.
The study, published in Nature Human Behaviour, was carried out by researchers at the University of Birmingham and the University of Oxford, and shows for the first time how a region called the ventromedial prefrontal cortex (vmPFC) has a critical role in helping, or ‘prosocial’ behaviours.
Lead author Professor Patricia Lockwood said: “Prosocial behaviours are essential for addressing global challenges. Yet helping others is often effortful and humans are averse to effort. Understanding how effortful helping decisions are processed in the brain is extremely important.”
In the study, the researchers focused on the vmPFC, a region located right at the front of the brain, which is known to be important for decision-making and other executive functions. Previous studies using magnetic resonance imaging (MRI scanning) have linked the vmPFC to choices that involve a trade-off between the rewards available and the effort required to obtain rewards. However, these techniques cannot show whether a part of the brain is essential for these functions.
Three groups of participants were recruited for the study. 25 patients had vmPFC damage, 15 patients had damage elsewhere in the brain, and 40 people were healthy age and gender-matched control participants. These groups allowed the researchers to test the impact of damage to vmPFC specifically.
Each participant attended an experiment where they met another person anonymously. They then completed a decision-making task that measured how willing they were to exert physical effort (squeezing a grip force device) to earn rewards (bonus money) for themselves and for the other person.
By enabling participants to meet – but not see – the person they were ‘working’ for in advance, researchers were able to convey the sense that participants’ efforts would have real consequences, but hide any information about the other person that could affect decision-making.
Each choice the participants made varied in how much bonus money for them or the other person was available, and how much force they would have to exert to obtain the reward. This allowed the researchers to measure the impact of reward and effort separately, and to use advanced mathematical modelling to precisely quantify people’s motivation.
The results of the study clearly showed that the vmPFC was necessary for motivation to help others. Patients with vmPFC damage were less willing to choose to help others, exerted less force on even after they did decide to help, and earned less money to help others compared to the control groups.
In a further step, the researchers used a technique called lesion symptom mapping which enabled them to identify even more specific subregions of the vmPFC where damage made people particularly antisocial and unwilling to exert effort for the other person. Surprisingly, damage to a nearby but different subregion made people relatively more willing to help.
Co-lead author Dr Jo Cutler said: “As well as better understanding prosocial motivation, this study could also help us to develop new treatments for clinical disorders such as psychopathy, where understanding the underlying neural mechanisms can give us new insights into how to treat these conditions.”
“This region of the brain is particularly interesting because we know that it undergoes late development in teenagers, and also changes as we get older,” added Professor Lockwood. “It will be really interesting to see whether this area of the brain can also be influenced by education – can we learn to be better at helping others?”
Several political parties have pledged to plug shortages of healthcare staff at government hospitals and clinics by training more health workers. They’re right to be concerned with understaffing, but are they putting the right solutions on the table? Jesse Copelyn investigates.
As the election approaches, one message seems ubiquitous among opposition parties: there is a severe shortage of health workers at government hospitals and clinics. Manifestoes of the DA, EFF, MK, IFP, ActionSA, UDM, Rise Mzansi and the ACDP all make some reference to the issue or simply state they would increase the number of health workers in the system if they were in power.
But why are so many parties from across the political spectrum pointing to this particular problem, and are they proposing realistic solutions?
Government health facilities are shedding staff
Various sources of data show that public health facilities are indeed heavily understaffed, giving weight to parties’ concerns. For instance, in March, the National Health Department revealed that appointments for a number of key clinical posts across the country have not been made. In some of the worst-performing provinces – the Free State, North West and Limpopo – more than 20% of posts for medical officers (i.e. non-specialised doctors) were unfilled.
Additionally, in the North West, almost 2 out of 5 nursing posts were vacant, while half of all positions for psychiatrists were unstaffed. Meanwhile in the Free State, a mere 3 out of 5 posts were filled for physiotherapists and occupational therapists.
These health worker shortages appear to be getting worse. The 2030 Human Resources for Health strategy document, which was published by the National Department of Health, estimated that in 2019, we required about 186 000 primary healthcare workers in the public sector. This would ensure that every person that relies on government services had access to a basket of primary healthcare services that matches the country’s needs. Yet at the time, we only had about 115 000, meaning that we were short by about 71 000 workers. And by 2025, that gap was projected to widen to over 87 000. This is because it was assumed that the number of clinical staff would remain the same over time, while the overall population (and thus the number of patients) would increase.
In reality, this actually understates the problem, Dr Donnella Besada, a health economist who was involved in that research, tells Spotlight. Rather than remaining the same, the number of health personnel in the public sector probably will have declined by 2025.
“The workforce is likely to go down over time as a result of the freezing of posts, retirement, illness and death,” she explains.
Indeed this was a trend that had already begun in the 2010s when total government spending on health began to stagnate in real terms, and irregular expenditure ballooned. Thus, government health facilities didn’t have the money to hire more staff, and between 2012 and 2016, the total number of people employed by provincial health departments actually declined.
The extent of the problem is perhaps most acutely seen in the area of specialist care, as the Human Resources for Health strategy document shows. Take anaesthesiologists – the doctors who put you to sleep before an operation and monitor your vital signs. Researchers estimated that given factors like the age of the population and the types of diseases that are prevalent, South Africa should have about 50 anaesthesiologists for every million people. In the private sector, we’re well over the bar, with nearly double that targeted ratio. In government health facilities, however, we’re way under, at about 6 anaesthesiologists for every million patients.
Right problem, wrong solution?
Clearly, politicians are onto something when they talk about the need to increase the number of health workers in public hospitals and clinics. But how do parties propose that we do this?
While solutions vary, one of the most common proposals that has been put forward both in party manifestos, and in interview responses to questions by Spotlight, is that we should invest more in training of health workers. For instance, the EFF manifesto states that the party would establish “at least one health care training facility per province and [ensure] that there is no province without a health sciences campus, inclusive of nursing school and medical school [sic]”. Similarly, the newly established MK party states that it would “expand the capacity and intake of medical schools”.
Manifestoes by ActionSA and RiseMzansi also state that they would train more health workers, while the UDM and ACDP told Spotlight that they would invest more in nursing colleges, along with other measures.
What unites these approaches is the belief that a central reason for understaffing is that we aren’t training enough health workers, and we have to find ways of boosting this capacity. However, two senior managers in the public health system that spoke to Spotlight provide a very different take. They argue that the most fundamental reason for understaffing is budgetary – facilities simply cannot afford to appoint more health workers even though there are often qualified people available for hire.
For instance, a former CEO of a public hospital in the Western Cape, who would prefer to remain anonymous, explains to Spotlight that the reason their hospital was unable to plug shortages is simply due to “affordability in terms of the budget received from the national government”.
In this context, more campuses and colleges would do little to solve the problem. “[T]oo many training institutions mean that once they graduate there are too few posts for internships or community service”, the former CEO says, referring to the positions that medical students must take up at government hospitals and clinics after graduating. He elaborates: “Once [the internship and community service] is done, there are no posts for permanent positions”.
All the way on the other side of the country, a senior manager at a government hospital in KwaZulu-Natal, who also wanted to remain anonymous, says much the same. He tells Spotlight that “understaffing has been a problem for some time”, and that the shortage of nurses is currently the most significant obstacle. Asked about the causes, he says “financial reasons” are almost always to blame (though he did feel that we needed to train more specialists). He elaborates “this year the budget has been cut compared to last financial year, so [the shortages are] a bit severe now”.
Asked whether more training would solve the shortage of nurses and medical officers, he is doubtful. “[M]any of the already-qualified people were not able to be employed, so training more? I don’t think this is a solution… for now the focus should be on employing the unemployed people”, he says.
This sentiment is also largely echoed by the National Department of Health, which in April stated that there were over 2000 unfunded posts for medical doctors in the country. An additional R2.4 billion was needed to fill them, according to the department, which has also been battling accusations from the South African Medical Association that over 800 qualified doctors cannot find work. In response, the department claimed that the majority of them had only just finished their training.
Training capacity has already hit its ceiling
What one might not realise from reading party manifestos is that the country has already substantially boosted the training of doctors over the last decade. As I have previously written for Bhekisisa, it is partially because of this that the public health system is increasingly struggling to absorb new medical graduates entering the system.
Professor Shabir Madhi of Wits University. Photo: Wits University.
For instance, Professor Shabir Madhi, the dean of the health faculty at the University of Witwatersrand (WITS), tells Spotlight that universities began to increase the intake of medical students (ie, those training to be doctors) some time ago, partly due to state pressure. Over a similar time period, the government expanded the Nelson Mandela Fidel Castro programme, which educates medical students in Cuba. As a result, while there were fewer than 1500 medical graduates that were available to be placed for internships in 2017, there were over 2100 in 2024.
The opposite trends have nonetheless taken place for some other health worker categories. For instance, in 2017, there were over 21 000 student nurses and midwives, and this dropped to below 15 000 in 2022. As Spotlight previously reported, this decline is at least in part due to disruptions related to how nurse training is accredited in South Africa.
According to Madhi, we’re still not training enough health workers to meet the needs of the country, but further expanding student intake wouldn’t address the current understaffing crisis, as the government is unable to employ the health workers that we’re already producing. Instead of training more health science students, he says, the health department needs to focus on “incorporating existing and newly graduating healthcare workers into the public sector”.
Additionally, even if we resolved our budgetary problems, there are hard limits on how many more students we can currently train, says Madhi, who laughs off campaign promises about building more medical campuses and scaling up student intake. “[M]ost of the training of health workers takes place outside of the classroom in our healthcare facilities,” he says, adding that “there are only so many healthcare facilities that have the right type of personnel to be involved in training, and their ability to absorb more trainee healthcare workers is fairly limited”.
While universities have increased the intake of medical students over the years, the ceiling has now been reached, argues Madhi, who notes that the number of trainee doctors that WITS is sending to its academic hospitals is “already exceeding the capacity that they can accommodate”. As a result, the university now sends students “to other hospitals which weren’t necessarily designed, and are not necessarily equipped or resourced, to undertake training”. He notes that these problems don’t just apply to trainee doctors, but also “occupational therapists, physiotherapists, oral hygienists and dentists”.
Madhi concludes: “Unfortunately, politicians are somewhat naive of what is required to establish training programmes in the health sciences”.
Toxic chemicals used to flame-proof plastic materials can be absorbed into the body through skin, via contact with microplastics, new research shows. The study offers the first experimental evidence that chemicals present as additives in microplastics can leach into human sweat, and then be absorbed through the skin, into the bloodstream.
Many chemicals used as flame retardants and plasticisers have already been banned, due to evidence of adverse health effects including damage to the liver or nervous system, cancer, and risks to reproductive health. However, these chemicals are still present in the environment in older electronics, furniture, carpets, and building materials.
While the harm caused by microplastics is not fully understood, there is increasing concern over their role as conduits of human exposure to toxic chemicals.
The research team demonstrated in a study published last year, that chemicals were leached from microplastics into human sweat. The current study now shows that those chemicals can also be absorbed from sweat across the skin barrier into the body.
In their experiments, the team used innovative 3D human skin models as alternatives to laboratory animals and excised human tissues. The models were exposed over a 24-hour period to two common forms of microplastics containing polybrominated diphenyl ethers (PBDEs), a chemical group commonly used to flame retard plastics.
The results, published in Environment International, showed that as much as 8% of the chemical exposed could be taken up by the skin, with more hydrated – or ‘sweatier’ – skin absorbing higher levels of chemical. The study provides the first experimental evidence into how this process contributes to levels of toxic chemicals found in the body.
Dr Ovokeroye Abafe, now at Brunel University, carried out the research while at the University of Birmingham. He said: “Microplastics are everywhere in the environment and yet we still know relatively little about the health problems that they can cause. Our research shows that they play a role as ‘carriers’ of harmful chemicals, which can get into our bloodstream through the skin. These chemicals are persistent, so with continuous or regular exposure to them, there will be a gradual accumulation to the point where they start to cause harm.”
Dr Mohamed Abdallah, Associate Professor of Environmental Sciences at the University of Birmingham, and principal investigator for the project, said: “These findings provide important evidence for regulators and policymakers to improve legislation around microplastics and safeguard public health against harmful exposure.”
Professor Stuart Harrad, co-author of the paper, added “the study provides an important step forward in understanding the risks of exposure to microplastics on our health. Building on our results, more research is required to fully understand the different pathways of human exposure to microplastics and how to mitigate the risk from such exposure.”
In future research, the team plan to investigate other routes through which microplastics could be responsible for toxic chemicals entering the body, including inhalation and ingestion.
A certain biological pathway involving interleukin-17 drives the inflammation seen in the skin disease psoriasis, according to a new study published in the journal Immunity. The work could lead to improved therapies for all inflammatory skin diseases, including atopic and allergic dermatitis and a type of boil called hidradenitis suppurativa, say the study authors.
Led by researchers at NYU Langone Health, the new study found that the interleukin-17 (IL-17) pathway, whose activity is blocked by existing anti-inflammatory drugs, activates a protein called hypoxia inducible factor 1-alpha (HIF-1-alpha) in psoriasis. Researchers say that IL-17 has long been known to be active in inflammation, but the role of HIF-1-alpha has until now been unclear.
The research team also found that HIF-1-alpha let inflamed skin cells more actively break down sugar for energy, supporting their metabolism and leading to the production of a waste product called lactate. When consumed by inflammatory T cells, lactate triggered production of IL-17, fuelling even more inflammation.
The findings show that in human skin tissue samples from psoriatic patients, measures of gene activity around IL-17 and HIF-1-alpha were similar, suggesting that these factors are interconnected. Experiments in mice treated to develop psoriasis found that subsequent treatment with an experimental drug that blocks the action of HIF-1-alpha, called BAY-87-2243, resolved inflammatory skin lesions.
Further, skin samples from 10 patients successfully treated with anti-inflammatory drug etanercept showed diminished activity for both IL-17 and HIF-1-alpha, suggesting to researchers that when IL-17 is blocked, so is HIF-1-alpha.
“Our study results broadly show that activation of HIF-1-alpha is at the crux of metabolic dysfunction observed in psoriasis and that its action is triggered by IL-17, another key inflammatory-signaling molecule,” said corresponding study author Shruti Naik, PhD, associate professor at NYU Grossman School of Medicine.
Further experiments were performed on skin samples from five patients with psoriasis whose healthy and inflamed skin was separately treated with either BAY-87-2243 or an existing combination of topical drugs (calcipotriene and betamethasone dipropionate). Researchers then compared differences in inflammatory gene activity as a measure of impact and found that the HIF-1-alpha inhibitor had a greater effect than existing topical drugs. Specifically, skin samples that responded to HIF-1-alpha therapy had 2,698 genes that were expressed differently, while standard-of-care-treated samples had 147 differently expressed genes.
Genetic analysis of skin samples from another 24 psoriatic patients treated with the IL-17A-blocking drug secukinumab showed only decreased, not heightened, gene activity connected to HIF-1-alpha when compared to HIF-1-alpha gene activity in nine healthy patients with no psoriatic disease. Researchers say this indicates HIF-1-alpha’s blocked action was codependent on blockage of IL-17.
Additional experiments in mice showed that blocking glucose uptake in the skin slowed psoriatic disease growth by limiting glucose metabolism, or glycolysis. Both the number of immune T cells tied to inflammation and the cell levels of IL-17 also decreased. The researchers found further that levels of lactate, the main byproduct of glycolysis, in psoriatic skin cell cultures dropped once exposed to the glycolysis-inhibiting drug 2-DG.
Directly targeting lactate production in psoriatic mice using a topical skin cream containing lactate dehydrogenase, which breaks down lactate, also slowed disease progression in the skin, with reduced numbers of inflammatory gamma-delta T cells and reduced IL-17 activity. Gamma-delta T cells were shown to take up lactate and use it to produce IL-17.
“Evidence of HIF-1-alpha’s depressed action, or downregulation, could also serve as a biomarker, or molecular sign, that other anti-inflammatory therapies are working,” said study co-senior investigator Jose U. Scher, MD, professor at NYU Grossman School of Medicine.
Scher, who also serves as director of NYU Langone’s Psoriatic Arthritis Center and the Judith and Stewart Colton Center for Autoimmunity, says the team plans to develop experimental drugs that can block HIF-1-alpha and lactate action in the skin “to end the underlying vicious cycle of IL-17-driven inflammation in skin disease. Our research fundamentally expands the scope of feasible therapeutic options.”
Naik points out that while many available therapies for psoriasis, including steroids and immunosuppressive drugs, reduce inflammation and symptoms, they do not cure the disease. She said further experiments are needed to refine which experimental drug works best, with respect to HIA-1-alpha inhibition, before clinical trials could start.
